Science.gov

Sample records for activin membrane-bound inhibitor

  1. The maturation-inducing hormone 17a-20b-dihydroxy-4pregnen-3-one regulates gene expression of inhibin A and bambi (bone morphogenetic protein and activin membrane bound inhibitor) in the rainbow trout ovary

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Transforming growth factor-beta (TGFb) superfamily members are important paracrine and autocrine regulators of ovarian development and steroidogenesis in mammals and birds, but their reproductive roles in fish are not well understood. The activin system, Tgfb, and bone morphogenetic protein 15 (Bmp...

  2. Membrane bound O-acyltransferases and their inhibitors.

    PubMed

    Masumoto, Naoko; Lanyon-Hogg, Thomas; Rodgers, Ursula R; Konitsiotis, Antonios D; Magee, Anthony I; Tate, Edward W

    2015-04-01

    Since the identification of the membrane-bound O-acyltransferase (MBOATs) protein family in the early 2000s, three distinct members [porcupine (PORCN), hedgehog (Hh) acyltransferase (HHAT) and ghrelin O-acyltransferase (GOAT)] have been shown to acylate specific proteins or peptides. In this review, topology determination, development of assays to measure enzymatic activities and discovery of small molecule inhibitors are compared and discussed for each of these enzymes. PMID:25849925

  3. Specific activin receptor-like kinase 3 inhibitors enhance liver regeneration.

    PubMed

    Tsugawa, Daisuke; Oya, Yuki; Masuzaki, Ryota; Ray, Kevin; Engers, Darren W; Dib, Martin; Do, Nhue; Kuramitsu, Kaori; Ho, Karen; Frist, Audrey; Yu, Paul B; Bloch, Kenneth D; Lindsley, Craig W; Hopkins, Corey R; Hong, Charles C; Karp, Seth J

    2014-12-01

    Pharmacologic agents to enhance liver regeneration after injury would have wide therapeutic application. Based on previous work suggesting inhibition of bone morphogenetic protein (BMP) signaling stimulates liver regeneration, we tested known and novel BMP inhibitors for their ability to accelerate regeneration in a partial hepatectomy (PH) model. Compounds were produced based on the 3,6-disubstituted pyrazolo[1,5-a] pyrimidine core of the BMP antagonist dorsomorphin and evaluated for their ability to inhibit BMP signaling and enhance liver regeneration. Antagonists of the BMP receptor activin receptor-like kinase 3 (ALK3), including LDN-193189 (LDN; 4-[6-[4-(1-piperazinyl)phenyl]pyrazolo[1,5-a]pyrimidin-3-yl]-quinoline), DMH2 (4-(2-(4-(3-(quinolin-4-yl)pyrazolo[1,5-a]pyrimidin-6-yl)phenoxy)ethyl)morpholine; VU0364849), and the novel compound VU0465350 (7-(4-isopropoxyphenyl)-3-(1H-pyrazol-4-yl)imidazo[1,2-a]pyridine; VU5350), blocked SMAD phosphorylation in vitro and in vivo, and enhanced liver regeneration after PH. In contrast, an antagonist of the BMP receptor ALK2, VU0469381 (5-(6-(4-methoxyphenyl)pyrazolo[1,5-a]pyrimidin-3-yl)quinolone; 1LWY), did not affect liver regeneration. LDN did not affect liver synthetic or metabolic function. Mechanistically, LDN increased serum interleukin-6 levels and signal transducer and activator of transcription 3 phosphorylation in the liver, and modulated other factors known to be important for liver regeneration, including suppressor of cytokine signaling 3 and p53. These findings suggest that inhibition of ALK3 may be part of a therapeutic strategy for treating human liver disease.

  4. Specific activin receptor-like kinase 3 inhibitors enhance liver regeneration.

    PubMed

    Tsugawa, Daisuke; Oya, Yuki; Masuzaki, Ryota; Ray, Kevin; Engers, Darren W; Dib, Martin; Do, Nhue; Kuramitsu, Kaori; Ho, Karen; Frist, Audrey; Yu, Paul B; Bloch, Kenneth D; Lindsley, Craig W; Hopkins, Corey R; Hong, Charles C; Karp, Seth J

    2014-12-01

    Pharmacologic agents to enhance liver regeneration after injury would have wide therapeutic application. Based on previous work suggesting inhibition of bone morphogenetic protein (BMP) signaling stimulates liver regeneration, we tested known and novel BMP inhibitors for their ability to accelerate regeneration in a partial hepatectomy (PH) model. Compounds were produced based on the 3,6-disubstituted pyrazolo[1,5-a] pyrimidine core of the BMP antagonist dorsomorphin and evaluated for their ability to inhibit BMP signaling and enhance liver regeneration. Antagonists of the BMP receptor activin receptor-like kinase 3 (ALK3), including LDN-193189 (LDN; 4-[6-[4-(1-piperazinyl)phenyl]pyrazolo[1,5-a]pyrimidin-3-yl]-quinoline), DMH2 (4-(2-(4-(3-(quinolin-4-yl)pyrazolo[1,5-a]pyrimidin-6-yl)phenoxy)ethyl)morpholine; VU0364849), and the novel compound VU0465350 (7-(4-isopropoxyphenyl)-3-(1H-pyrazol-4-yl)imidazo[1,2-a]pyridine; VU5350), blocked SMAD phosphorylation in vitro and in vivo, and enhanced liver regeneration after PH. In contrast, an antagonist of the BMP receptor ALK2, VU0469381 (5-(6-(4-methoxyphenyl)pyrazolo[1,5-a]pyrimidin-3-yl)quinolone; 1LWY), did not affect liver regeneration. LDN did not affect liver synthetic or metabolic function. Mechanistically, LDN increased serum interleukin-6 levels and signal transducer and activator of transcription 3 phosphorylation in the liver, and modulated other factors known to be important for liver regeneration, including suppressor of cytokine signaling 3 and p53. These findings suggest that inhibition of ALK3 may be part of a therapeutic strategy for treating human liver disease. PMID:25271257

  5. Membrane-bound respiratory of Spirillum itersonii.

    PubMed Central

    Dailey, H A

    1976-01-01

    The membrane-bound respiratory system of the gram-negative bacterium Spirillum itersonii was investigated. It contains cytochromes b (558), c (550), and o (558) and beta-dihydro-nicotinamide adenine dinucleotide (NADH) and succinate oxidase activities under all growth conditions. It is also capable of producing D-lactate and alpha-glycerophosphate dehydrogenases when grown with lactate or glycerol as sole carbon source. Membrane-bound malate dehydrogenase was not detectable under any conditions, although there is high activity of soluble nicotinamide adenine dinucleotide: malate dehydrogenase. When grown with oxygen as the sole terminal electron acceptor, approximately 60% of the total b-type cytochrome is present as cytochrome o, whereas only 40% is present as cytochrome o in cells grown with nitrate in the presence of oxygen. Both NADH and succinate oxidase are inhibited by azide, cyanide, antimycin A, and 2-n-heptyl-4-hydroxyquinoline-N-oxidase at low concentrations. The ability of these inhibitors to completely inhibit oxidase activity at low concentrations and their effects upon the aerobic steady-state reduction levels of b- and c-type cytochromes as well as the aerobic steady-state reduction levels obtained with NADH, succinate, and ascorbate-dichlorophenolindophenol suggest that presence of an unbranched respiratory chain in S. itersonii with the order ubiquinone leads to b leads to c leads to c leads to oxygen. PMID:182674

  6. Membrane bound pyrophosphatase and P-type adenosine triphosphatase of Leishmania donovani as possible chemotherapeutic targets: similarities and differences in inhibitor sensitivities.

    PubMed

    Sen, S S; Bhuyan, N R; Lakshman, K; Roy, A K; Chakraborty, B; Bera, T

    2009-12-01

    The activities of inorganic pyrophosphatase (PPase) and adenosine triphosphatase (ATPase) were studied in the plasma membrane of Leishmania donovani promastigotes and amastigotes. It was shown that the specific activity of PPase was greater than that of ATPase in the promastigote plasma membrane. We characterized H+-PPase present in the plasma membrane of L. donovani and investigated its possible role in the survival of promastigote and amastigote. PPase activity was stimulated by K+ and sodium orthovanadate and inhibited by pyrophosphate analogs (imidodiphosphate and alendronate), KF, N,N'-dicyclohexylcarbodiimide (DCCD), thiol reagents (p-chloromercuribenzenesulfonate (PCMBS), N-ethylmaleimide (NEM), and phenylarsine oxide (PAO)), the ABC superfamily transport modulator verapamil, and also by the F(1)F(o)-ATPase inhibitor quercetin. ATPase activity was stimulated by K+ and verapamil, inhibited by DCCD, PCMBS, NEM, sodium azide, sodium orthovanadate, and quercetin, and was unaffected by PAO. We conclude that there are significant differences within promastigote, amastigote, and mammalian host in cytosolic pH homeostasis to merit the inclusion of PPase transporter as a putative target for rational drug design. PMID:19961421

  7. Thermoreversible gel for delivery of activin receptor-like kinase 5 inhibitor SB-505124 for glaucoma filtration surgery.

    PubMed

    Sutariya, Vijaykumar; Miladore, Nicholas; Geldenhuys, Werner; Bhatia, Deepak; Wehrung, Daniel; Nakamura, Hiroshi

    2013-01-01

    The purpose of this study is to investigate a thermoreversible gel using Pluronic F-127 to deliver an activin receptor-like kinase 5 (ALK-5) inhibitor SB-505124 in glaucoma filtration surgery (GFS). The gel was characterized for in vitro drug release and viscosity studies. Cytotoxicity of Pluronic F-127 was examined by MTT assay using cultured rabbit subconjunctival fibroblasts. In addition, Pluronic F-127 gel (18% w/v) containing 5 mg of SB-505124 was applied at the surgical site in an in vivo rabbit GFS model. In the in vitro viscosity study, the gel showed a change in viscosity (from 1000 cps to 45,000 cps) from low temperature (10°C) to body temperature (37°C). The in vitro drug release study demonstrated 100% drug release within 12 h. The gel did not show cytotoxicity to the cultured rabbit subconjunctival cells by MTT assay. In the in vivo rabbit GFS model, the drug was successfully delivered by injection and no severe post-surgical complications were observed. A thermoreversible gel system with SB-505124 was successfully prepared and delivered for the rabbit GFS model, and it may provide a novel delivery system in GFS.

  8. Activins and activin antagonists in hepatocellular carcinoma

    PubMed Central

    Deli, Alev; Kreidl, Emanuel; Santifaller, Stefan; Trotter, Barbara; Seir, Katja; Berger, Walter; Schulte-Hermann, Rolf; Rodgarkia-Dara, Chantal; Grusch, Michael

    2008-01-01

    In many parts of the world hepatocellular carcinoma (HCC) is among the leading causes of cancer-related mortality but the underlying molecular pathology is still insufficiently understood. There is increasing evidence that activins, which are members of the transforming growth factor β (TGFβ) superfamily of growth and differentiation factors, could play important roles in liver carcinogenesis. Activins are disulphide-linked homo- or heterodimers formed from four different β subunits termed βA, βB, βC, and βE, respectively. Activin A, the dimer of two βA subunits, is critically involved in the regulation of cell growth, apoptosis, and tissue architecture in the liver, while the hepatic function of other activins is largely unexplored so far. Negative regulators of activin signals include antagonists in the extracellular space like the binding proteins follistatin and FLRG, and at the cell membrane antagonistic co-receptors like Cripto or BAMBI. Additionally, in the intracellular space inhibitory Smads can modulate and control activin activity. Accumulating data suggest that deregulation of activin signals contributes to pathologic conditions such as chronic inflammation, fibrosis and development of cancer. The current article reviews the alterations in components of the activin signaling pathway that have been observed in HCC and discusses their potential significance for liver tumorigenesis. PMID:18350601

  9. Conformational phases of membrane bound cytoskeletal filaments

    NASA Astrophysics Data System (ADS)

    Quint, David A.; Grason, Gregory; Gopinathan, Ajay

    2013-03-01

    Membrane bound cytoskeletal filaments found in living cells are employed to carry out many types of activities including cellular division, rigidity and transport. When these biopolymers are bound to a membrane surface they may take on highly non-trivial conformations as compared to when they are not bound. This leads to the natural question; What are the important interactions which drive these polymers to particular conformations when they are bound to a surface? Assuming that there are binding domains along the polymer which follow a periodic helical structure set by the natural monomeric handedness, these bound conformations must arise from the interplay of the intrinsic monomeric helicity and membrane binding. To probe this question, we study a continuous model of an elastic filament with intrinsic helicity and map out the conformational phases of this filament for various mechanical and structural parameters in our model, such as elastic stiffness and intrinsic twist of the filament. Our model allows us to gain insight into the possible mechanisms which drive real biopolymers such as actin and tubulin in eukaryotes and their prokaryotic cousins MreB and FtsZ to take on their functional conformations within living cells.

  10. FOXL2-induced follistatin attenuates activin A-stimulated cell proliferation in human granulosa cell tumors

    SciTech Connect

    Cheng, Jung-Chien; Chang, Hsun-Ming; Qiu, Xin; Fang, Lanlan; Leung, Peter C.K.

    2014-01-10

    Highlights: •Activin A stimulates cell proliferation in KGN human granulosa cell tumor-derived cell line. •Cyclin D2 mediates activin A-induced KGN cell proliferation. •FOXL2 induces follistatin expression in KGN cells. •FOXL2-induced follistatin attenuates activin A-stimulated KGN cell proliferation. -- Abstract: Human granulosa cell tumors (GCTs) are rare, and their etiology remains largely unknown. Recently, the FOXL2 402C > G (C134W) mutation was found to be specifically expressed in human adult-type GCTs; however, its function in the development of human GCTs is not fully understood. Activins are members of the transforming growth factor-beta superfamily, which has been shown to stimulate normal granulosa cell proliferation; however, little is known regarding the function of activins in human GCTs. In this study, we examined the effect of activin A on cell proliferation in the human GCT-derived cell line KGN. We show that activin A treatment stimulates KGN cell proliferation. Treatment with the activin type I receptor inhibitor SB431542 blocks activin A-stimulated cell proliferation. In addition, our results show that cyclin D2 is induced by treatment with activin A and is involved in activin A-stimulated cell proliferation. Moreover, the activation of Smad signaling is required for activin A-induced cyclin D2 expression. Finally, we show that the overexpression of the wild-type FOXL2 but not the C134W mutant FOXL2 induced follistatin production. Treatment with exogenous follistatin blocks activin A-stimulated cell proliferation, and the overexpression of wild-type FOXL2 attenuates activin A-stimulated cell proliferation. These results suggest that FOXL2 may act as a tumor suppressor in human adult-type GCTs by inducing follistatin expression, which subsequently inhibits activin-stimulated cell proliferation.

  11. Uric acid: a modulator of prostate cells and activin sensitivity.

    PubMed

    Sangkop, Febbie; Singh, Geeta; Rodrigues, Ely; Gold, Elspeth; Bahn, Andrew

    2016-03-01

    Elevated serum uric acid (SUA) or urate is associated with inflammation and gout. Recent evidence has linked urate to cancers, but little is known about urate effects in prostate cancer. Activins are inflammatory cytokines and negative growth regulators in the prostate. A hallmark of prostate cancer progression is activin insensitivity; however, mechanisms underlying this are unclear. We propose that elevated SUA is associated with prostate cancer counteracting the growth inhibitory effects of activins. The expression of activins A and B, urate transporter GLUT9 and tissue urate levels were examined in human prostate disease. Intracellular and secreted urate and GLUT9 expression were assessed in human prostate cancer cell lines. Furthermore, the effects of urate and probenecid, a known urate transport inhibitor, were determined in combination with activin A. Activin A expression was increased in low-grade prostate cancer, whereas activin B expression was reduced in high-grade prostate cancer. Intracellular urate levels decreased in all prostate pathologies, while GLUT9 expression decreased in benign prostatic hyperplasia, prostatitis and high-grade prostate cancer. Activin responsive LNCaP cells had higher intracellular and lower secreted urate levels than activin-insensitive PC3 cells. GLUT9 expression in prostate cancer cells was progressively lower than in prostate epithelial cells. Elevated extracellular urate was growth promoting in vitro, which was abolished by the gout medication probenecid, and it antagonized the growth inhibitory effects of activins. This study shows for the first time that a change in plasma or intracellular urate levels, possibly involving GLUT9 and a urate efflux transporter, has an impact on prostate cancer cell growth, and that lowering SUA levels in prostate cancer is likely to be therapeutically beneficial. PMID:26910779

  12. Membrane-bounded nucleoid in the eubacterium Gemmatata obscuriglobus.

    PubMed Central

    Fuerst, J A; Webb, R I

    1991-01-01

    The freshwater budding eubacterium Gemmata obscuriglobus possesses a DNA-containing nuclear region that is bounded by two nuclear membranes. The membrane-bounded nature of the nucleoid in this bacterium was shown by thin sectioning of chemically fixed cells, thin sectioning of freeze-substituted cells, and freeze-fracture/freeze-etch. The fibrillar nucleoid was surrounded by electron-dense granules that were in turn enveloped by two nuclear membranes separated by an electron-transparent space. Immunogold labeling of thin sections of conventionally fixed cells with anti-double-stranded DNA antibody demonstrated double-stranded DNA associated with fibrillar material within the membrane boundary. The occurrence of a membrane-bounded nucleoid in a eubacterial prokaryote is a significant exception to the evidence supporting the prokaryote/eukaryote dichotomous classification of cell structure. Images PMID:11607213

  13. Membrane-bound complement regulatory proteins as biomarkers and potential therapeutic targets for SLE.

    PubMed

    Das, Nibhriti; Biswas, Bintili; Khera, Rohan

    2013-01-01

    For the last two decades, there had been remarkable advancement in understanding the role of complement regulatory proteins in autoimmune disorders and importance of complement inhibitors as therapeutics. Systemic lupus erythematosus is a prototype of systemic autoimmune disorders. The disease, though rare, is potentially fatal and afflicts women at their reproductive age. It is a complex disease with multiorgan involvement, and each patient presents with a different set of symptoms. The diagnosis is often difficult and is based on the diagnostic criteria set by the American Rheumatology Association. Presence of antinuclear antibodies and more specifically antidouble-stranded DNA indicates SLE. Since the disease is multifactorial and its phenotypes are highly heterogeneous, there is a need to identify multiple noninvasive biomarkers for SLE. Lack of validated biomarkers for SLE disease activity or response to treatment is a barrier to the efficient management of the disease, drug discovery, as well as development of new therapeutics. Recent studies with gene knockout mice have suggested that membrane-bound complement regulatory proteins (CRPs) may critically determine the sensitivity of host tissues to complement injury in autoimmune and inflammatory disorders. Case-controlled and followup studies carried out in our laboratory suggest an intimate relation between the level of DAF, MCP, CR1, and CD59 transcripts and the disease activity in SLE. Based on comparative evaluation of our data on these four membrane-bound complement regulatory proteins, we envisaged CR1 and MCP transcripts as putative noninvasive disease activity markers and the respective proteins as therapeutic targets for SLE. Following is a brief appraisal on membrane-bound complement regulatory proteins DAF, MCP, CR1, and CD59 as biomarkers and therapeutic targets for SLE. PMID:23402019

  14. Membrane-Bound ATPase Contributes to Hop Resistance of Lactobacillus brevis

    PubMed Central

    Sakamoto, Kanta; van Veen, H. W.; Saito, Hiromi; Kobayashi, Hiroshi; Konings, Wil N.

    2002-01-01

    The activity of the membrane-bound H+-ATPase of the beer spoilage bacterium Lactobacillus brevis ABBC45 increased upon adaptation to bacteriostatic hop compounds. The ATPase activity was optimal around pH 5.6 and increased up to fourfold when L. brevis was exposed to 666 μM hop compounds. The extent of activation depended on the concentration of hop compounds and was maximal at the highest concentration tested. The ATPase activity was strongly inhibited by N,N′-dicyclohexylcarbodiimide, a known inhibitor of FoF1-ATPase. Western blots of membrane proteins of L. brevis with antisera raised against the α- and β-subunits of FoF1-ATPase from Enterococcus hirae showed that there was increased expression of the ATPase after hop adaptation. The expression levels, as well as the ATPase activity, decreased to the initial nonadapted levels when the hop-adapted cells were cultured further without hop compounds. These observations strongly indicate that proton pumping by the membrane-bound ATPase contributes considerably to the resistance of L. brevis to hop compounds. PMID:12406727

  15. Purification and characterization of the membrane-bound quinoprotein glucose dehydrogenase of Gluconacetobacter diazotrophicus PAL 5.

    PubMed

    Sará-Páez, Martin; Contreras-Zentella, Martha; Gómez-Manzo, Saúl; González-Valdez, Alejandra Abigail; Gasca-Licea, Rolando; Mendoza-Hernández, Guillermo; Escamilla, José Edgardo; Reyes-Vivas, Horacio

    2015-02-01

    Acetic acid bacteria oxidize a great number of substrates, such as alcohols and sugars, using different enzymes that are anchored to the membrane. In particular, Gluconacetobacter diazotrophicus is distinguished for its N2-fixing activity under high-aeration conditions. Ga. diazotrophicus is a true endophyte that also has membrane-bound enzymes to oxidize sugars and alcohols. Here we reported the purification and characterization of the membrane-bound glucose dehydrogenase (GDHm), an oxidoreductase of Ga. diazotrophicus. GDHm was solubilized and purified by chromatographic methods. Purified GDHm was monomeric, with a molecular mass of 86 kDa. We identified the prosthetic group as pyrroloquinoline quinone, whose redox state was reduced. GDHm showed an optimum pH of 7.2, and its isoelectric point was 6.0. This enzyme preferentially oxidized D-glucose, 2-deoxy-D-glucose, D-galactose and D-xylose; its affinity towards glucose was ten times greater than that of E. coli GDHm. Finally, Ga. diazotrophicus GDHm was capable of reducing quinones such as Q 1, Q 2, and decylubiquinone; this activity was entirely abolished in the presence of micromolar concentrations of the inhibitor, myxothiazol. Hence, our purification method yielded a highly purified GDHm whose molecular and kinetic parameters were determined. The possible implications of GDHm activity in the mechanism for reducing competitor microorganisms, as well as its participation in the respiratory system of Ga. diazotrophicus, are discussed.

  16. Purification and characterization of the membrane-bound quinoprotein glucose dehydrogenase of Gluconacetobacter diazotrophicus PAL 5.

    PubMed

    Sará-Páez, Martin; Contreras-Zentella, Martha; Gómez-Manzo, Saúl; González-Valdez, Alejandra Abigail; Gasca-Licea, Rolando; Mendoza-Hernández, Guillermo; Escamilla, José Edgardo; Reyes-Vivas, Horacio

    2015-02-01

    Acetic acid bacteria oxidize a great number of substrates, such as alcohols and sugars, using different enzymes that are anchored to the membrane. In particular, Gluconacetobacter diazotrophicus is distinguished for its N2-fixing activity under high-aeration conditions. Ga. diazotrophicus is a true endophyte that also has membrane-bound enzymes to oxidize sugars and alcohols. Here we reported the purification and characterization of the membrane-bound glucose dehydrogenase (GDHm), an oxidoreductase of Ga. diazotrophicus. GDHm was solubilized and purified by chromatographic methods. Purified GDHm was monomeric, with a molecular mass of 86 kDa. We identified the prosthetic group as pyrroloquinoline quinone, whose redox state was reduced. GDHm showed an optimum pH of 7.2, and its isoelectric point was 6.0. This enzyme preferentially oxidized D-glucose, 2-deoxy-D-glucose, D-galactose and D-xylose; its affinity towards glucose was ten times greater than that of E. coli GDHm. Finally, Ga. diazotrophicus GDHm was capable of reducing quinones such as Q 1, Q 2, and decylubiquinone; this activity was entirely abolished in the presence of micromolar concentrations of the inhibitor, myxothiazol. Hence, our purification method yielded a highly purified GDHm whose molecular and kinetic parameters were determined. The possible implications of GDHm activity in the mechanism for reducing competitor microorganisms, as well as its participation in the respiratory system of Ga. diazotrophicus, are discussed. PMID:25576305

  17. Tunable Tensor Voting Improves Grouping of Membrane-Bound Macromolecules

    SciTech Connect

    Loss, Leandro A.; Bebis, George; Parvin, Bahram

    2009-04-15

    Membrane-bound macromolecules are responsible for structural support and mediation of cell-cell adhesion in tissues. Quantitative analysis of these macromolecules provides morphological indices for damage or loss of tissue, for example as a result of exogenous stimuli. From an optical point of view, a membrane signal may have nonuniform intensity around the cell boundary, be punctate or diffused, and may even be perceptual at certain locations along the boundary. In this paper, a method for the detection and grouping of punctate, diffuse curvilinear signals is proposed. Our work builds upon the tensor voting and the iterative voting frameworks to propose an efficient method to detect and refine perceptually interesting curvilinear structures in images. The novelty of our method lies on the idea of iteratively tuning the tensor voting fields, which allows the concentration of the votes only over areas of interest. We validate the utility of our system with synthetic and annotated real data. The effectiveness of the tunable tensor voting is demonstrated on complex phenotypic signals that are representative of membrane-bound macromolecular structures.

  18. Activin C Antagonizes Activin A in Vitro and Overexpression Leads to Pathologies in Vivo

    PubMed Central

    Gold, Elspeth; Jetly, Niti; O'Bryan, Moira K.; Meachem, Sarah; Srinivasan, Deepa; Behuria, Supreeti; Sanchez-Partida, L. Gabriel; Woodruff, Teresa; Hedwards, Shelley; Wang, Hong; McDougall, Helen; Casey, Victoria; Niranjan, Birunthi; Patella, Shane; Risbridger, Gail

    2009-01-01

    Activin A is a potent growth and differentiation factor whose synthesis and bioactivity are tightly regulated. Both follistatin binding and inhibin subunit heterodimerization block access to the activin receptor and/or receptor activation. We postulated that the activin-βC subunit provides another mechanism regulating activin bioactivity. To test our hypothesis, we examined the biological effects of activin C and produced mice that overexpress activin-βC. Activin C reduced activin A bioactivity in vitro; in LNCaP cells, activin C abrogated both activin A-induced Smad signaling and growth inhibition, and in LβT2 cells, activin C antagonized activin A-mediated activity of an follicle-stimulating hormone-β promoter. Transgenic mice that overexpress activin-βC exhibited disease in testis, liver, and prostate. Male infertility was caused by both reduced sperm production and impaired sperm motility. The livers of the transgenic mice were enlarged because of an imbalance between hepatocyte proliferation and apoptosis. Transgenic prostates showed evidence of hypertrophy and epithelial cell hyperplasia. Additionally, there was decreased evidence of nuclear Smad-2 localization in the testis, liver, and prostate, indicating that overexpression of activin-βC antagonized Smad signaling in vivo. Underlying the significance of these findings, human testis, liver, and prostate cancers expressed increased activin-βC immunoreactivity. This study provides evidence that activin-βC is an antagonist of activin A and supplies an impetus to examine its role in development and disease. PMID:19095948

  19. Development of Novel Activin-Targeted Therapeutics

    PubMed Central

    Chen, Justin L; Walton, Kelly L; Al-Musawi, Sara L; Kelly, Emily K; Qian, Hongwei; La, Mylinh; Lu, Louis; Lovrecz, George; Ziemann, Mark; Lazarus, Ross; El-Osta, Assam; Gregorevic, Paul; Harrison, Craig A

    2015-01-01

    Soluble activin type II receptors (ActRIIA/ActRIIB), via binding to diverse TGF-β proteins, can increase muscle and bone mass, correct anemia or protect against diet-induced obesity. While exciting, these multiple actions of soluble ActRIIA/IIB limit their therapeutic potential and highlight the need for new reagents that target specific ActRIIA/IIB ligands. Here, we modified the activin A and activin B prodomains, regions required for mature growth factor synthesis, to generate specific activin antagonists. Initially, the prodomains were fused to the Fc region of mouse IgG2A antibody and, subsequently, “fastener” residues (Lys45, Tyr96, His97, and Ala98; activin A numbering) that confer latency to other TGF-β proteins were incorporated. For the activin A prodomain, these modifications generated a reagent that potently (IC50 5 nmol/l) and specifically inhibited activin A signaling in vitro, and activin A-induced muscle wasting in vivo. Interestingly, the modified activin B prodomain inhibited both activin A and B signaling in vitro (IC50 ~2 nmol/l) and in vivo, suggesting it could serve as a general activin antagonist. Importantly, unlike soluble ActRIIA/IIB, the modified prodomains did not inhibit myostatin or GDF-11 activity. To underscore the therapeutic utility of specifically antagonising activin signaling, we demonstrate that the modified activin prodomains promote significant increases in muscle mass. PMID:25399825

  20. Photochemical energy conversion by membrane-bound photoredox systems

    SciTech Connect

    Tollin, G.

    1992-03-01

    Most of our effort during the past grant period has been directed towards investigating electron transfer processes involving redox proteins at lipid bilayer/aqueous interfaces. This theme, as was noted in our previous three year renewal proposal, is consistent with our goal of developing biomimetic solar energy conversion systems which utilize the unique properties of biological electron transfer molecules. Thus, small redox proteins such as cytochrome c, plastocyanin and ferredoxin function is biological photosynthesis as mediators of electron flow between the photochemical systems localized in the membrane, and more complex soluble or membrane-bound redox proteins which are designed to carry out specific biological tasks such as transbilayer proton gradient formation, dinitrogen fixation, ATP synthesis, dihydrogen synthesis, generation of strong reductants, etc. In these studies, we have utilized two principal experimental techniques, laser flash photolysis and cyclic voltammetry, both of which permit direct measurements of electron transfer processes.

  1. Photochemical energy conversion by membrane-bound photoredox systems

    NASA Astrophysics Data System (ADS)

    Tollin, G.

    1992-03-01

    Most of our effort during the past grant period has been directed towards investigating electron transfer processes involving redox proteins at lipid bilayer/aqueous interfaces. This theme, as was noted in our previous three year renewal proposal, is consistent with our goal of developing biomimetic solar energy conversion systems which utilize the unique properties of biological electron transfer molecules. Thus, small redox proteins such as cytochrome c, plastocyanin and ferredoxin function in biological photosynthesis as mediators of electron flow between the photochemical systems localized in the membrane, and more complex soluble or membrane bound redox proteins which are designed to carry out specific biological tasks such as transbilayer proton gradient formation, dinitrogen fixation, ATP synthesis, dihydrogen synthesis, generation of strong reductants, etc. In these studies, we have utilized two principal experimental techniques, laser flash photolysis and cyclic voltammetry, both of which permit direct measurements of electron transfer processes.

  2. Sialic Acid Is Required for Neuronal Inhibition by Soluble MAG but not for Membrane Bound MAG

    PubMed Central

    Al-Bashir, Najat; Mellado, Wilfredo; Filbin, Marie T.

    2016-01-01

    Myelin-Associated Glycoprotein (MAG), a major inhibitor of axonal growth, is a member of the immunoglobulin (Ig) super-family. Importantly, MAG (also known as Siglec-4) is a member of the Siglec family of proteins (sialic acid-binding, immunoglobulin-like lectins), MAG binds to complex gangliosides, specifically GD1a and/or GT1b. Therefore, it has been proposed as neuronal receptors for MAG inhibitory effect of axonal growth. Previously, we showed that MAG binds sialic acid through domain 1 at Arg118 and is able to inhibit axonal growth through domain 5. We developed a neurite outgrowth (NOG) assay, in which both wild type MAG and mutated MAG (MAG Arg118) are expressed on cells. In addition we also developed a soluble form NOG in which we utilized soluble MAG-Fc and mutated MAG (Arg118-Fc). Only MAG-Fc is able to inhibit NOG, but not mutated MAG (Arg118)-Fc that has been mutated at its sialic acid binding site. However, both forms of membrane bound MAG- and MAG (Arg118)- expressing cells still inhibit NOG. Here, we review various results from different groups regarding MAG’s inhibition of axonal growth. Also, we propose a model in which the sialic acid binding is not necessary for the inhibition induced by the membrane form of MAG, but it is necessary for the soluble form of MAG. This finding highlights the importance of understanding the different mechanisms by which MAG inhibits NOG in both the soluble fragmented form and the membrane-bound form in myelin debris following CNS damage. PMID:27065798

  3. Structural analysis of membrane-bound retrovirus capsid proteins.

    PubMed Central

    Barklis, E; McDermott, J; Wilkens, S; Schabtach, E; Schmid, M F; Fuller, S; Karanjia, S; Love, Z; Jones, R; Rui, Y; Zhao, X; Thompson, D

    1997-01-01

    We have developed a system for analysis of histidine-tagged (His-tagged) retrovirus core (Gag) proteins, assembled in vitro on lipid monolayers consisting of egg phosphatidylcholine (PC) plus the novel lipid DHGN. DHGN was shown to chelate nickel by atomic absorption spectrometry, and DHGN-containing monolayers specifically bound gold conjugates of His-tagged proteins. Using PC + DHGN monolayers, we examined membrane-bound arrays of an N-terminal His-tagged Moloney murine leukemia virus (M-MuLV) capsid (CA) protein, His-MoCA, and in vivo studies suggest that in vitro-derived His-MoCA arrays reflect some of the Gag protein interactions which occur in assembling virus particles. The His-MoCA proteins formed extensive two-dimensional (2D) protein crystals, with reflections out to 9.5 A resolution. The image-analyzed 2D projection of His-MoCA arrays revealed a distinct cage-like network. The asymmetry of the individual building blocks of the network led to the formation of two types of hexamer rings, surrounding protein-free cage holes. These results predict that Gag hexamers constitute a retrovirus core substructure, and that cage hole sizes define an exclusion limit for entry of retrovirus envelope proteins, or other plasma membrane proteins, into virus particles. We believe that the 2D crystallization method will permit the detailed analysis of retroviral Gag proteins and other His-tagged proteins. PMID:9135137

  4. Platelets induce apoptosis via membrane-bound FasL

    PubMed Central

    Schleicher, Rebecca I.; Reichenbach, Frank; Kraft, Peter; Kumar, Anil; Lescan, Mario; Todt, Franziska; Göbel, Kerstin; Hilgendorf, Ingo; Geisler, Tobias; Bauer, Axel; Olbrich, Marcus; Schaller, Martin; Wesselborg, Sebastian; O’Reilly, Lorraine; Meuth, Sven G.; Schulze-Osthoff, Klaus; Gawaz, Meinrad; Li, Xuri; Kleinschnitz, Christoph; Edlich, Frank

    2015-01-01

    After tissue injury, both wound sealing and apoptosis contribute to restoration of tissue integrity and functionality. Although the role of platelets (PLTs) for wound closure and induction of regenerative processes is well established, the knowledge about their contribution to apoptosis is incomplete. Here, we show that PLTs present the death receptor Fas ligand (FasL) on their surface after activation. Activated PLTs as well as the isolated membrane fraction of activated PLTs but not of resting PLTs induced apoptosis in a dose-dependent manner in primary murine neuronal cells, human neuroblastoma cells, and mouse embryonic fibroblasts. Membrane protein from PLTs lacking membrane-bound FasL (FasL△m/△m) failed to induce apoptosis. Bax/Bak-mediated mitochondrial apoptosis signaling in target cells was not required for PLT-induced cell death, but increased the apoptotic response to PLT-induced Fas signaling. In vivo, PLT depletion significantly reduced apoptosis in a stroke model and an inflammation-independent model of N-methyl-d-aspartic acid-induced retinal apoptosis. Furthermore, experiments using PLT-specific PF4Cre+ FasLfl/fl mice demonstrated a role of PLT-derived FasL for tissue apoptosis. Because apoptosis secondary to injury prevents inflammation, our findings describe a novel mechanism on how PLTs contribute to tissue homeostasis. PMID:26232171

  5. Platelets induce apoptosis via membrane-bound FasL.

    PubMed

    Schleicher, Rebecca I; Reichenbach, Frank; Kraft, Peter; Kumar, Anil; Lescan, Mario; Todt, Franziska; Göbel, Kerstin; Hilgendorf, Ingo; Geisler, Tobias; Bauer, Axel; Olbrich, Marcus; Schaller, Martin; Wesselborg, Sebastian; O'Reilly, Lorraine; Meuth, Sven G; Schulze-Osthoff, Klaus; Gawaz, Meinrad; Li, Xuri; Kleinschnitz, Christoph; Edlich, Frank; Langer, Harald F

    2015-09-17

    After tissue injury, both wound sealing and apoptosis contribute to restoration of tissue integrity and functionality. Although the role of platelets (PLTs) for wound closure and induction of regenerative processes is well established, the knowledge about their contribution to apoptosis is incomplete. Here, we show that PLTs present the death receptor Fas ligand (FasL) on their surface after activation. Activated PLTs as well as the isolated membrane fraction of activated PLTs but not of resting PLTs induced apoptosis in a dose-dependent manner in primary murine neuronal cells, human neuroblastoma cells, and mouse embryonic fibroblasts. Membrane protein from PLTs lacking membrane-bound FasL (FasL(△m/△m)) failed to induce apoptosis. Bax/Bak-mediated mitochondrial apoptosis signaling in target cells was not required for PLT-induced cell death, but increased the apoptotic response to PLT-induced Fas signaling. In vivo, PLT depletion significantly reduced apoptosis in a stroke model and an inflammation-independent model of N-methyl-d-aspartic acid-induced retinal apoptosis. Furthermore, experiments using PLT-specific PF4Cre(+) FasL(fl/fl) mice demonstrated a role of PLT-derived FasL for tissue apoptosis. Because apoptosis secondary to injury prevents inflammation, our findings describe a novel mechanism on how PLTs contribute to tissue homeostasis.

  6. Identification and characterization of novel membrane-bound PRL protein tyrosine phosphatases from Setaria cervi, a bovine filarial parasite.

    PubMed

    Singh, Neetu; Yadav, Smita; Rathaur, Sushma

    2015-11-01

    A significant amount of protein tyrosine phosphatase (PTP) activity was detected in the detergent-soluble membrane-bound fraction of Setaria cervi, a bovine filarial parasite. The membrane-bound PTP activity was significantly inhibited when the adult parasites were exposed to compounds having antifilarial activity like aspirin and SK7 as well as phenylarsine oxide, a specific PTP inhibitor suggesting that this activity is stress regulated. Further, this enzyme was purified as a single protein of apparently 21 kDa using two different chromatographic techniques. The MALDI-MS/MS analysis of its peptides showed closest match with protein tyrosine phosphatase PRL (Aedes aegypti). This purified enzyme (named as PRL) showed maximum activity at pH 5.5/37 °C and hydrolysed para nitro phenyl phosphate (pNPP) at the highest rate followed by O-P-L-tyrosine and O-P-L-threonine. It showed significant inhibition by specific inhibitors of PTP such as sodium orthovanadate, phenylarsine oxide and ammonium molybdate and was activated by dithiothreitol (DTT). The active site modification studies suggested involvement of cysteine, arginine, histidine and aspartic acid in the catalytic activity of PRL. The activity of S. cervi PRL was also found to be resistant towards the external oxidative stress. Thus, S. cervi PRL could be taken as a potential target for the management of human lymphatic filariasis. PMID:26341797

  7. 1α,25-dihydroxyvitamin D3 stimulates activin A production to fine-tune osteoblast-induced mineralization.

    PubMed

    Woeckel, V J; van der Eerden, B C J; Schreuders-Koedam, M; Eijken, M; Van Leeuwen, J P T M

    2013-11-01

    In healthy bones, mineralization has to be tightly controlled to avoid pathological phenotypes. In this study, we investigated interactions between 1α,25(OH)2 D3 (1,25D3) and activin A in the regulation of osteoblast induced mineralization. In human osteoblast cultures, we demonstrated that besides stimulation of mineralization, 1,25D3 also induced activin A, a strong inhibitor of mineralization. Simultaneously, follistatin (FST), the natural antagonist of activin A, was down-regulated by1,25D3. This resulted in an increase in activin A activity during 1,25D3 treatment. We also showed that in 1,25D3-treated osteoblasts, mineralization can be further increased when activin A activity was abrogated by adding exogenous FST. This observation implies that, besides stimulation of mineralization, 1,25D3 also controls activin A-mediated inhibition of mineralization. Besides activin A, 1,25D3 also induces osteocalcin (BGLAP), another inhibitor of mineralization. Warfarin, which has been shown to inactivate osteocalcin, increased 1,25D3-induced mineralization. Interaction between these two systems became evident from the synergistic increase in BGLAP expression upon blocking activin activity in 1,25D3-treated cultures. In conclusion, we demonstrate that 1,25D3 stimulation of mineralization by human osteoblasts is suppressed by concomitant induction of inhibitors of mineralization. Mineralization induction by 1,25D3 may actually be controlled via interplay with activin A and osteocalcin. Finally, this complex regulation of mineralization substantiates the significance of tight control of mineralization to prevent excessive mineralization and consequently reduction in bone quality and strength.

  8. Activin A, B and AB decrease progesterone production by down-regulating StAR in human granulosa cells.

    PubMed

    Chang, Hsun-Ming; Cheng, Jung-Chien; Huang, He-Feng; Shi, Feng-Tao; Leung, Peter C K

    2015-09-01

    Activins are homo- or heterodimers of inhibin β subunits that play important roles in the reproductive system. Our previous work has shown that activins A (βAβA), B (βBβB) and AB (βAβB) induce aromatase/estradiol, but suppress StAR/progesterone production in human granulosa-lutein cells. However, the underlying molecular determinants of these effects have not been examined. In this continuing study, we used immortalized human granulosa cells (SVOG) to investigate the effects of activins in regulating StAR/progesterone and the potential mechanisms of action. In SVOG cells, activins A, B and AB produced comparable down-regulation of StAR expression and progesterone production. In addition, all three activin isoforms induced equivalent phosphorylation of both SMAD2 and SMAD3. Importantly, the activin-induced down-regulation of StAR, increase in SMAD2/3 phosphorylation, and decrease in progesterone were abolished by the TGF-β type I receptor inhibitor SB431542. Interestingly, the small interfering RNA-mediated knockdown of ALK4 but not ALK5 reversed the activin-induced suppression of StAR. Furthermore, the knockdown of SMAD4 or SMAD2 but not SMAD3 abolished the inhibitory effects of all three activin isoforms on StAR expression. These results provide evidence that activins A, B and AB down-regulate StAR expression and decrease progesterone production in human granulosa cells, likely via an ALK4-mediated SMAD2/SMAD4-dependent pathway. Our findings provide important insights into the molecular mechanisms underlying the regulatory effects of activins on human granulosa cell steroidogenesis.

  9. Mg2+ is an essential activator of hydrolytic activity of membrane-bound pyrophosphatase of Rhodospirillum rubrum.

    PubMed Central

    Sosa, A; Ordaz, H; Romero, I; Celis, H

    1992-01-01

    The substrate for the hydrolytic activity of membrane-bound pyrophosphatase is the PP(i)-Mg2+ complex. The enzyme has no activity when the free Mg2+ concentration is lower than 10 microM (at 0.5 mM-PP(i)-Mg2+), and therefore free Mg2+ is an essential activator of the hydrolytic activity. The Km for the substrate changes in response to variation in free Mg2+ concentration, from 10.25 to 0.6 mM when free Mg2+ is increased from 0.03 to 1.0 mM respectively. The Km for Mg2+ depends on the substrate concentration: the Km decreases from 0.52 to 0.14 mM from 0.25 to 0.75 mM-PP(i)-Mg2+ respectively. The extrapolated Km for Mg2+ in the absence of the substrate is 0.73 mM. Imidodiphosphate-Mg2+ and free Ca2+ were used as competitive inhibitors of substrate and activator respectively. The equilibrium binding kinetics suggest an ordered mechanism for the activator and the substrate: Mg2+ ions bind the enzyme before PP(i)-Mg2+ in the formation of the catalytic complex, membrane-bound pyrophosphatase-(Mg2+)-(PP(i)-Mg2+). PMID:1315519

  10. Configuration of membrane-bound proteins by x-ray reflectivity

    NASA Astrophysics Data System (ADS)

    Chen, Chiu-Hao; Málková, Šárka; Cho, Wonhwa; Schlossman, Mark L.

    2011-11-01

    In this presentation we review our recent work using x-ray reflectivity to determine the configuration of membrane-bound proteins. The reflectivity data is analyzed in terms of the known crystallographic structure of proteins and a slab model representing the lipid layer to yield an electron density profile of the lipid/protein system. Our recent modified analysis methodology for the lipid/protein system is concisely described in this report. In addition, some results of the configuration of the membrane-bound proteins cPLA2α-C2, p40phox-PX, and PKCα-C2 are highlighted.

  11. Release of Membrane-Bound Vesicles and Inhibition of Tumor Cell Adhesion by the Peptide Neopetrosiamide A

    PubMed Central

    Austin, Pamela; Heller, Markus; Williams, David E.; McIntosh, Lawrence P.; Vogl, A. Wayne; Foster, Leonard J.; Andersen, Raymond J.; Roberge, Michel; Roskelley, Calvin D.

    2010-01-01

    Background Neopetrosiamide A (NeoA) is a 28-amino acid tricyclic peptide originally isolated from a marine sponge as a tumor cell invasion inhibitor whose mechanism of action is unknown. Methodology/Principal Findings We show that NeoA reversibly inhibits tumor cell adhesion, disassembles focal adhesions in pre-attached cells, and decreases the level of β1 integrin subunits on the cell surface. NeoA also induces the formation of dynamic, membrane-bound protrusions on the surface of treated cells and the release of membrane-bound vesicles into the culture medium. Proteomic analysis indicates that the vesicles contain EGF and transferrin receptors as well as a number of proteins involved in adhesion and migration including: β1 integrin and numerous α integrin subunits; actin and actin-binding proteins such as cofilin, moesin and myosin 1C; and membrane modulating eps15 homology domain (EHD) proteins. Surface labeling, trafficking inhibition, and real-time imaging experiments all suggest that β1 integrin-containing vesicles are released directly from NeoA-induced cell surface protrusions rather than from vesicles generated intracellularly. The biological activity of NeoA is dependent on its disulfide bond pattern and NMR spectroscopy indicates that the peptide is globular with a continuous ridge of hydrophobic groups flanked by charged amino acid residues that could facilitate a simultaneous interaction with lipids and proteins in the membrane. Conclusions/Significance NeoA is an anti-adhesive peptide that decreases cell surface integrin levels through a novel, yet to be elucidated, mechanism that involves the release of adhesion molecule-containing vesicles from the cell surface. PMID:20520768

  12. AN IMPROVED CELL FRACTIONATION PROCEDURE FOR THE PREPARATION OF RAT LIVER MEMBRANE-BOUND RIBOSOMES

    PubMed Central

    Adelman, M. R.; Blobel, Gunter; Sabatini, David D.

    1973-01-01

    A cell fractionation procedure is described which allows the preparation from rat liver of a rough microsome population containing almost 50% of the membrane-bound ribosomes of the tissue. The fraction is not contaminated with free ribosomes or smooth microsomes, and, by various other criteria, is suitable for studies of ribosome-membrane interaction. PMID:4345164

  13. Identification of two additional members of the membrane-bound dipeptidase family.

    PubMed

    Habib, Geetha M; Shi, Zheng-Zheng; Cuevas, Alan A; Lieberman, Michael W

    2003-07-01

    We have cloned two mouse cDNAs encoding previously unidentified membrane-bound dipeptidases [membrane-bound dipeptidase-2 (MBD-2) and membrane-bound dipeptidase-3 (MBD-3)] from membrane-bound dipeptidase-1 (MBD-1) deficient mice (Habib, G.M., Shi, Z-Z., Cuevas, A.A., Guo, Q., Matzuk, M.M., and Lieberman, M.W. (1998) Proc. Natl. Acad. Sci. USA 95, 4859-4863). These enzymes are closely related to MBD-1 (EC 3.4.13.19), which is known to cleave leukotriene D4 (LTD4) and cystinyl-bis-glycine. MBD-2 cDNA is 56% identical to MBD-1 with a predicted amino acid identity of 33%. The MBD-3 and MBD-1 cDNAs share a 55% nucleotide identity and a 39% predicted amino acid sequence identity. All three genes are tightly linked on the same chromosome. Expression of MBD-2 and MBD-3 in Cos cells indicated that both are membrane-bound through a glycosylphosphatidyl-inositol linkage. MBD-2 cleaves leukotriene D4 (LTD4) but not cystinyl-bis-glycine, while MBD-3 cleaves cystinyl-bis-glycine but not LTD4. MBD-1 is expressed at highest levels in kidney, lung, and heart and is absent in spleen, while MBD-2 is expressed at highest levels in lung, heart, and testis and at somewhat lower levels in spleen. Of the tissues examined, MBD-3 expression was detected only in testis. Our identification of a second enzyme capable of cleaving LTD4 raises the possibility that clearance of LTD4 during asthma and in related inflammatory conditions may be mediated by more than one enzyme.

  14. Membrane-Bound TRAIL Supplements Natural Killer Cell Cytotoxicity Against Neuroblastoma Cells

    PubMed Central

    Sheard, Michael A.; Asgharzadeh, Shahab; Liu, Yin; Lin, Tsen-Yin; Wu, Hong-Wei; Ji, Lingyun; Groshen, Susan; Lee, Dean A.; Seeger, Robert C.

    2013-01-01

    Neuroblastoma cells have been reported to be resistant to death induced by soluble, recombinant forms of TRAIL (CD253/TNFSF10) due to low or absent expression of caspase-8 and/or TRAIL-receptor 2 (TRAIL-R2/DR5/CD262/TNFRSF10b). However, their sensitivity to membrane-bound TRAIL on natural killer (NK) cells is not known. Comparing microarray gene expression and response to NK cell-mediated cytotoxicity, we observed a correlation between TRAIL-R2 expression and the sensitivity of fourteen neuroblastoma cell lines to the cytotoxicity of NK cells activated with IL-2 plus IL-15. Even though most NK cytotoxicity was dependent upon perforin, the cytotoxicity was supplemented by TRAIL in fourteen of seventeen (82%) neuroblastoma cell lines as demonstrated using an anti-TRAIL neutralizing antibody. Similarly, a recently developed NK cell expansion system employing IL-2 plus lethally irradiated K562 feeder cells constitutively expressing membrane-bound IL-21 (K562 clone 9.mbIL21) resulted in activated NK cells derived from normal healthy donors and neuroblastoma patients that also utilized TRAIL to supplement cytotoxicity. Exogenous IFNγ up-regulated expression of caspase-8 in three of four neuroblastoma cell lines and increased the contribution of TRAIL to NK cytotoxicity against two of the three lines; however, relatively little inhibition of cytotoxicity was observed when activated NK cells were treated with an anti-IFNγ neutralizing antibody. Constraining the binding of anti-TRAIL neutralizing antibody to membrane-bound TRAIL but not soluble TRAIL indicated that membrane-bound TRAIL alone was responsible for essentially all of the supplemental cytotoxicity. Together, these findings support a role for membrane-bound TRAIL in the cytotoxicity of NK cells against neuroblastoma cells. PMID:23719242

  15. Vascular Endothelial Growth Factor-A (VEGF-A) Mediates Activin A-Induced Human Trophoblast Endothelial-Like Tube Formation.

    PubMed

    Li, Yan; Zhu, Hua; Klausen, Christian; Peng, Bo; Leung, Peter C K

    2015-11-01

    Remodeling of maternal spiral arteries during pregnancy requires a subpopulation of extravillous cytotrophoblasts (EVTs) to differentiate into endovascular EVTs. Activin A, which is abundantly expressed at the maternal-fetal interface, has been shown to promote trophoblast invasion, but its role in endovascular differentiation remains unknown. Vascular endothelial growth factor-A (VEGF-A) is well recognized as a key regulator in trophoblast endovascular differentiation. Whether and how activin A might regulate VEGF-A production in human trophoblasts and its relationship to endovascular differentiation have yet to be determined. In the present study, we found that activin A increased VEGF-A production in primary and immortalized (HTR8/SVneo) human EVT cells. In addition, activin A enhanced HTR8/SVneo endothelial-like tube formation, and these effects were attenuated by pretreatment with small interfering RNA targeting VEGF-A or the VEGF receptor 1/2 inhibitor SU4312. Pretreatment with the activin/TGF-β type 1 receptor (ALK4/5/7) inhibitor SB431542 abolished the stimulatory effects of activin A on phosphorylated mothers against decapentaplegic (SMAD)-2/3 phosphorylation, VEGF-A production, and endothelial-like tube formation. Moreover, small interfering RNA-mediated down-regulation of SMAD2, SMAD3, or common SMAD4 abolished the effects of activin A on VEGF-A production and endothelial-like tube formation. In conclusion, activin A may promote human trophoblast cell endothelial-like tube formation by up-regulating VEGF-A production in an SMAD2/3-SMAD4-dependent manner. These findings provide insight into the cellular and molecular events regulated by activin A during human implantation. PMID:26327470

  16. Denitrification by plant roots? New aspects of plant plasma membrane-bound nitrate reductase.

    PubMed

    Eick, Manuela; Stöhr, Christine

    2012-10-01

    A specific form of plasma membrane-bound nitrate reductase in plants is restricted to roots. Two peptides originated from plasma membrane integral proteins isolated from Hordeum vulgare have been assigned as homologues to the subunit NarH of respiratory nitrate reductase of Escherichia coli. Corresponding sequences have been detected for predicted proteins of Populus trichocarpa with high degree of identities for the subunits NarH (75%) and NarG (65%), however, with less accordance for the subunit NarI. These findings coincide with biochemical properties, particularly in regard to the electron donors menadione and succinate. Together with the root-specific and plasma membrane-bound nitrite/NO reductase, nitric oxide is produced under hypoxic conditions in the presence of nitrate. In this context, a possible function in nitrate respiration of plant roots and an involvement of plants in denitrification processes are discussed.

  17. Development of Membrane-Bound GM-CSF and IL-18 as an Effective Tumor Vaccine

    PubMed Central

    Cheng, Ta-Chun; Chuang, Chih-Hung; Kao, Chien-Han; Hsieh, Yuan-Chin; Cheng, Kuang-Hung; Wang, Jaw-Yuan; Cheng, Chiu-Min; Chen, Chien-Shu; Cheng, Tian-Lu

    2015-01-01

    The development of effective adjuvant is the key factor to boost the immunogenicity of tumor cells as a tumor vaccine. In this study, we expressed membrane-bound granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin-18 (IL-18) as adjuvants in tumor cells to stimulate immune response. B7 transmembrane domain fused GM-CSF and IL-18 was successfully expressed in the cell membrane and stimulated mouse splenocyte proliferation. Co-expression of GM-CSF and IL-18 reduced tumorigenesis (P<0.05) and enhanced tumor protective efficacy (P<0.05) significantly in comparison with GM-CSF alone. These results indicated that the combination of GM-CSF andIL-18 will enhance the immunogenicity of a cell-based anti-tumor vaccine. This membrane-bound approach can be applied to other cytokines for the development of novel vaccine strategies. PMID:26186692

  18. Using supported bilayers to study the spatiotemporal organization of membrane bound proteins

    PubMed Central

    Field, Christine M.; Groen, Aaron C.; Mitchison, Timothy J.

    2015-01-01

    Cell division in prokaryotes and eukaryotes is commonly initiated by the well-controlled binding of proteins to the cytoplasmic side of the cell membrane. However, a precise characterization of the spatiotemporal dynamics of membrane-bound proteins is often difficult to achieve in vivo. Here, we present protocols for the use of supported lipid bilayers to rebuild the cytokinetic machineries of cells with greatly different dimensions: the bacterium Escherichia coli and eggs of the vertebrate Xenopus laevis. Combined with total internal reflection fluorescence (TIRF) microscopy, these experimental setups allow for precise quantitative analyses of membrane-bound proteins. The protocols described to obtain glass-supported membranes from bacterial and vertebrate lipids can be used as starting points for other reconstitution experiments. We believe that similar biochemical assays will be instrumental to study the biochemistry and biophysics underlying a variety of complex cellular tasks, such as signaling, vesicle trafficking and cell motility. PMID:25997350

  19. The influence of membrane bound proteins on phase separation and coarsening in cell membranes.

    PubMed

    Witkowski, Thomas; Backofen, Rainer; Voigt, Axel

    2012-11-14

    A theoretical explanation of the existence of lipid rafts in cell membranes remains a topic of lively debate. Large, micrometer sized rafts are readily observed in artificial membranes and can be explained using thermodynamic models for phase separation and coarsening. In live cells such domains are not observed and various models are proposed to describe why the systems do not coarsen. We review these attempts critically and show within a phase field approach that membrane bound proteins have the potential to explain the different behaviour observed in vitro and in vivo. Large scale simulations are performed to compute scaling laws and size distribution functions under the influence of membrane bound proteins and to observe a significant slow down of the domain coarsening at longer times and a breakdown of the self-similarity of the size-distribution function.

  20. From 'I' to 'L' and back again: the odyssey of membrane-bound M13 protein.

    PubMed

    Vos, Werner L; Nazarov, Petr V; Koehorst, Rob B M; Spruijt, Ruud B; Hemminga, Marcus A

    2009-05-01

    The major coat protein of the filamentous bacteriophage M13 is a surprising protein because it exists both as a membrane protein and as part of the M13 phage coat during its life cycle. Early studies showed that the phage-bound structure of the coat protein was a continuous I-shaped alpha-helix. However, throughout the years various structural models, both I-shaped and L-shaped, have been proposed for the membrane-bound state of the coat protein. Recently, site-directed labelling approaches have enabled the study of the coat protein under conditions that more closely mimic the in vivo membrane-bound state. Interestingly, the structure that has emerged from this work is I-shaped and similar to the structure in the phage-bound state.

  1. Biochemical and molecular characterization of mitochondrial membrane-bound arginase in Heteropneustes fossilis.

    PubMed

    Mishra, Suman; Mishra, Rajnikant

    2016-05-01

    The two predominant forms of arginase, cytosolic Arginase-I and mitochondrial Arginase-II, catalyze hydrolysis of arginine into ornithine and urea. Based on presence of arginase activity in extracts using potassium chloride (KCl), mitochondrial membrane-bound arginase has also been suggested. However, the activity of arginase in fractions obtained after KCl-treatment may be either due to leakage of mitochondrial arginase or release of adhered cytosolic arginase to cell organelles having altered net charge. Therefore, it has been intended to analyse impact of KCl on ultra-structural properties of mitochondria, and biochemical analysis of mitochondrial membrane-bound proteins and arginase of Heteropneustes fossilis. Liver of H. fossilis was used for isolating mitochondria for analysis of ultrastructural properties, preparing cytosolic, mitochondrial, and mitochondrial-membrane bound extracts after treatment of KCl. Extracts were analysed for arginase activity assay, protein profiling through SDS-PAGE and MALDI MS/MS. The KCl-mediated modulation in polypeptides and arginase were also evaluated by PANTHER, MitoProt and IPSORT servers. The effects of KCl on ultra-structural integrity of mitochondria, activity of arginase, modulation on mitochondrial proteins and enzymes including arginase were observed. The 48 kDa polypeptide of mitochondrial fraction, that showed KCl-dependent alteration matched with Myb binding protein and 30 kDa bands resembles to arginase after MALDI MS/MS analysis. Results indicate KCl-dependent ultrastructural changes in mitochondria and release of mitochondrial arginase. The proposed membrane bound mitochondrial arginase could be mitochondrial arginase-II or altered form of cytosolic arginase-I contributing to KCl-induced arginase activity in H. fossilis. PMID:26922180

  2. Structure and Dynamics of the Membrane-Bound Cytochrome P450 2C9

    PubMed Central

    Cojocaru, Vlad; Balali-Mood, Kia; Sansom, Mark S. P.; Wade, Rebecca C.

    2011-01-01

    The microsomal, membrane-bound, human cytochrome P450 (CYP) 2C9 is a liver-specific monooxygenase essential for drug metabolism. CYPs require electron transfer from the membrane-bound CYP reductase (CPR) for catalysis. The structural details and functional relevance of the CYP-membrane interaction are not understood. From multiple coarse grained molecular simulations started with arbitrary configurations of protein-membrane complexes, we found two predominant orientations of CYP2C9 in the membrane, both consistent with experiments and conserved in atomic-resolution simulations. The dynamics of membrane-bound and soluble CYP2C9 revealed correlations between opening and closing of different tunnels from the enzyme's buried active site. The membrane facilitated the opening of a tunnel leading into it by stabilizing the open state of an internal aromatic gate. Other tunnels opened selectively in the simulations of product-bound CYP2C9. We propose that the membrane promotes binding of liposoluble substrates by stabilizing protein conformations with an open access tunnel and provide evidence for selective substrate access and product release routes in mammalian CYPs. The models derived here are suitable for extension to incorporate other CYPs for oligomerization studies or the CYP reductase for studies of the electron transfer mechanism, whereas the modeling procedure is generally applicable to study proteins anchored in the bilayer by a single transmembrane helix. PMID:21852944

  3. Structure and Dynamics of the Membrane-Bound Cytochrome P450 2C9

    SciTech Connect

    Cojocaru, Vlad; Balali-Mood, Kia; Sansom, Mark S.; Wade, Rebecca C.

    2011-08-11

    The microsomal, membrane-bound, human cytochrome P450 (CYP) 2C9 is a liver-specific monooxygenase essential for drug metabolism. CYPs require electron transfer from the membrane-bound CYP reductase (CPR) for catalysis. The structural details and functional relevance of the CYP-membrane interaction are not understood. From multiple coarse grained molecular simulations started with arbitrary configurations of protein-membrane complexes, we found two predominant orientations of CYP2C9 in the membrane, both consistent with experiments and conserved in atomic-resolution simulations. The dynamics of membrane-bound and soluble CYP2C9 revealed correlations between opening and closing of different tunnels from the enzyme’s buried active site. The membrane facilitated the opening of a tunnel leading into it by stabilizing the open state of an internal aromatic gate. Other tunnels opened selectively in the simulations of product-bound CYP2C9. We propose that the membrane promotes binding of liposoluble substrates by stabilizing protein conformations with an open access tunnel and provide evidence for selective substrate access and product release routes in mammalian CYPs. The models derived here are suitable for extension to incorporate other CYPs for oligomerization studies or the CYP reductase for studies of the electron transfer mechanism, whereas the modeling procedure is generally applicable to study proteins anchored in the bilayer by a single transmembrane helix.

  4. Isolation and characterization of Xenopus follistatin and activins.

    PubMed

    Fukui, A; Nakamura, T; Sugino, K; Takio, K; Uchiyama, H; Asashima, M; Sugino, H

    1993-09-01

    Xenopus follistatin and activins were purified from a Xenopus laevis cell line (XTC-F1) by four purification steps consisting of consecutive affinity chromatography on dextran sulfate-Sepharose and Sulfate Cellulofine, fast protein liquid chromatography gel permeation, and reverse-phase high performance liquid chromatography (HPLC). Our results thus obtained indicated that almost equimolar amounts of activins A, AB, and B were found to be present as a complex with follistatin (activin-binding protein) in the conditioned medium of XTC-F1 cells. Reverse-phase HPLC of the complex gave Xenopus follistatin and activins A, AB, and B. The purified Xenopus follistatin showed four major bands in a molecular mass range from 34 to 39 kDa by SDS-polyacrylamide gel electrophoresis under nonreducing conditions. The ability of each form of the protein to specifically bind activin was determined by activin-binding assay and ligand blotting analysis. Each protein was found to have the same NH2-terminus and its sequence was very homologous to that of mammalian follistatin. Several criteria including immunoblotting analysis and various functional assays revealed the existence of three isoforms of activins A, AB, and B in Xenopus, as in mammals. Xenopus activins significantly induced both ventral and dorsal mesoderm in explants of Xenopus blastula cells that would otherwise form epidermis. In a dose-dependent manner of each isoform of activin, the induced explants were able to differentiate into blood-like cells, coelomic epithelium, mesenchyme, muscle, and notochord. The induction patterns of three Xenopus activins were essentially the same. The mesoderm induction by the purified Xenopus activins was shown to be inhibited stoichiometrically by the purified Xenopus follistatin. These results indicate that Xenopus XTC-F1 cells secrete several molecular forms of follistatin/activin-binding protein and three isoforms of activins AB and B in addition to activin A.

  5. Graded Nodal/Activin Signaling Titrates Conversion of Quantitative Phospho-Smad2 Levels into Qualitative Embryonic Stem Cell Fate Decisions

    PubMed Central

    Orlov, Yuriy Lvovich; Yit, Le Yau; Yang, Henry; Ang, Lay Teng; Poellinger, Lorenz; Lim, Bing

    2011-01-01

    Nodal and Activin are morphogens of the TGFbeta superfamily of signaling molecules that direct differential cell fate decisions in a dose- and distance-dependent manner. During early embryonic development the Nodal/Activin pathway is responsible for the specification of mesoderm, endoderm, node, and mesendoderm. In contradiction to this drive towards cellular differentiation, the pathway also plays important roles in the maintenance of self-renewal and pluripotency in embryonic and epiblast stem cells. The molecular basis behind stem cell interpretation of Nodal/Activin signaling gradients and the undertaking of disparate cell fate decisions remains poorly understood. Here, we show that any perturbation of endogenous signaling levels in mouse embryonic stem cells leads to their exit from self-renewal towards divergent differentiation programs. Increasing Nodal signals above basal levels by direct stimulation with Activin promotes differentiation towards the mesendodermal lineages while repression of signaling with the specific Nodal/Activin receptor inhibitor SB431542 induces trophectodermal differentiation. To address how quantitative Nodal/Activin signals are translated qualitatively into distinct cell fates decisions, we performed chromatin immunoprecipitation of phospho-Smad2, the primary downstream transcriptional factor of the Nodal/Activin pathway, followed by massively parallel sequencing, and show that phospho-Smad2 binds to and regulates distinct subsets of target genes in a dose-dependent manner. Crucially, Nodal/Activin signaling directly controls the Oct4 master regulator of pluripotency by graded phospho-Smad2 binding in the promoter region. Hence stem cells interpret and carry out differential Nodal/Activin signaling instructions via a corresponding gradient of Smad2 phosphorylation that selectively titrates self-renewal against alternative differentiation programs by direct regulation of distinct target gene subsets and Oct4 expression. PMID

  6. BAFF and APRIL from Activin A-Treated Dendritic Cells Upregulate the Antitumor Efficacy of Dendritic Cells In Vivo.

    PubMed

    Shurin, Michael R; Ma, Yang; Keskinov, Anton A; Zhao, Ruijing; Lokshin, Anna; Agassandian, Marianna; Shurin, Galina V

    2016-09-01

    The members of the TGFβ superfamily play a key role in regulating developmental and homeostasis programs by controlling differentiation, proliferation, polarization, and survival of different cell types. Although the role of TGFβ1 in inflammation and immunity is well evident, the contribution of other TGFβ family cytokines in the modulation of the antitumor immune response remains less documented. Here we show that activin A triggers SMAD2 and ERK1/2 pathways in dendritic cells (DC) expressing type I and II activin receptors, and upregulates production of the TNFα family cytokines BAFF (TALL-1, TNFSF13B) and APRIL (TALL-2, TNFSF13A), which is blocked by SMAD2 and ERK1/2 inhibitors, respectively. BAFF and APRIL derived from activin A-treated DCs upregulate proliferation and survival of T cells expressing the corresponding receptors, BAFF-R and TACI. In vivo, activin A-stimulated DCs demonstrate a significantly increased ability to induce tumor-specific CTLs and inhibit the growth of melanoma and lung carcinoma, which relies on DC-derived BAFF and APRIL, as knockdown of the BAFF and APRIL gene expression in activin A-treated DCs blocks augmentation of their antitumor potential. Although systemic administration of activin A, BAFF, or APRIL for the therapeutic purposes is not likely due to the pluripotent effects on malignant and nonmalignant cells, our data open a novel opportunity for improving the efficacy of DC vaccines. In fact, a significant augmentation of the antitumor activity of DC pretreated with activin A and the proven role of DC-derived BAFF and APRIL in the induction of antitumor immunity in vivo support this direction. Cancer Res; 76(17); 4959-69. ©2016 AACR. PMID:27364554

  7. The Membrane-Bound Form of IL-17A Promotes the Growth and Tumorigenicity of Colon Cancer Cells

    PubMed Central

    Van Anh, Do Thi; Park, Sang Min; Lee, Hayyoung; Kim, Young Sang

    2016-01-01

    Interleukin-17A is a member of the IL-17 family, and is known as CTLA8 in the mouse. It is produced by T lymphocytes and NK cells and has proinflammatory roles, inducing cytokine and chemokine production. However, its role in tumor biology remains controversial. We investigated the effects of locally produced IL-17A by transferring the gene encoding it into CT26 colon cancer cells, either in a secretory or a membrane-bound form. Expression of the membrane-bound form on CT26 cells dramatically enhanced their proliferation in vitro. The enhanced growth was shown to be due to an increased rate of cell cycle progression: after synchronizing cells by adding and withdrawing colcemid, the rate of cell cycle progression in the cells expressing the membrane-bound form of IL-17A was much faster than that of the control cells. Both secretory and membrane-bound IL-17A induced the expression of Sca-1 in the cancer cells. When tumor clones were grafted into syngeneic BALB/c mice, the tumor clones expressing the membrane-bound form IL-17A grew rapidly; those expressing the secretory form also grew faster than the wild type CT26 cells, but slower than the clones expressing the membrane-bound form. These results indicate that IL-17A promotes tumorigenicity by enhancing cell cycle progression. This finding should be considered in treating tumors and immune-related diseases. PMID:27378226

  8. A Multiscale Approach to Modelling Drug Metabolism by Membrane-Bound Cytochrome P450 Enzymes

    PubMed Central

    Sansom, Mark S. P.; Mulholland, Adrian J.

    2014-01-01

    Cytochrome P450 enzymes are found in all life forms. P450s play an important role in drug metabolism, and have potential uses as biocatalysts. Human P450s are membrane-bound proteins. However, the interactions between P450s and their membrane environment are not well-understood. To date, all P450 crystal structures have been obtained from engineered proteins, from which the transmembrane helix was absent. A significant number of computational studies have been performed on P450s, but the majority of these have been performed on the solubilised forms of P450s. Here we present a multiscale approach for modelling P450s, spanning from coarse-grained and atomistic molecular dynamics simulations to reaction modelling using hybrid quantum mechanics/molecular mechanics (QM/MM) methods. To our knowledge, this is the first application of such an integrated multiscale approach to modelling of a membrane-bound enzyme. We have applied this protocol to a key human P450 involved in drug metabolism: CYP3A4. A biologically realistic model of CYP3A4, complete with its transmembrane helix and a membrane, has been constructed and characterised. The dynamics of this complex have been studied, and the oxidation of the anticoagulant R-warfarin has been modelled in the active site. Calculations have also been performed on the soluble form of the enzyme in aqueous solution. Important differences are observed between the membrane and solution systems, most notably for the gating residues and channels that control access to the active site. The protocol that we describe here is applicable to other membrane-bound enzymes. PMID:25033460

  9. Ionization, partitioning, and dynamics of tryptophan octyl ester: implications for membrane-bound tryptophan residues.

    PubMed Central

    Chattopadhyay, A; Mukherjee, S; Rukmini, R; Rawat, S S; Sudha, S

    1997-01-01

    The presence of tryptophan residues as intrinsic fluorophores in most proteins makes them an obvious choice for fluorescence spectroscopic analyses of such proteins. Membrane proteins have been reported to have a significantly higher tryptophan content than soluble proteins. The role of tryptophan residues in the structure and function of membrane proteins has attracted a lot of attention. Tryptophan residues in membrane proteins and peptides are believed to be distributed asymmetrically toward the interfacial region. Tryptophan octyl ester (TOE) is an important model for membrane-bound tryptophan residues. We have characterized this molecule as a fluorescent membrane probe in terms of its ionization, partitioning, and motional characteristics in unilamellar vesicles of dioleoylphosphatidylcholine. The ionization property of this molecule in model membranes has been studied by utilizing its pH-dependent fluorescence characteristics. Analysis of pH-dependent fluorescence intensity and emission maximum shows that deprotonation of the alpha-amino group of TOE occurs with an apparent pKa of approximately 7.5 in the membrane. The fluorescence lifetime of membrane-bound TOE also shows pH dependence. The fluorescence lifetimes of TOE have been interpreted by using the rotamer model for the fluorescence decay of tryptophan. Membrane/water partition coefficients of TOE were measured in both its protonated and deprotonated forms. No appreciable difference was found in its partitioning behavior with ionization. Analysis of fluorescence polarization of TOE as a function of pH showed that there is a decrease in polarization with increasing pH, implying more rotational freedom on deprotonation. This is further supported by pH-dependent red edge excitation shift and the apparent rotational correlation time of membrane-bound TOE. TOE should prove useful in monitoring the organization and dynamics of tryptophan residues incorporated into membranes. PMID:9251800

  10. Coordination of Copper to the Membrane-Bound Form of α-Synuclein

    SciTech Connect

    Dudzik, Christopher G.; Walter, Eric D.; Abrams, Benjamin S.; Jurica, Melissa S.; Millhauser, Glenn L.

    2013-01-01

    Aggregation of the 140 amino acid protein α-synuclein (α-syn) is linked to the development of Parkinson's disease (PD). α-Syn is a copper binding protein with potential function as a regulator of metal dependent redox activity. Epidemiological studies suggest that human exposure to excess copper increases the incidence of PD. α-Syn exists in both solution and membrane bound forms. Previous work evaluated the Cu2+ uptake for α-syn in solution and identified Met1-Asp2 and His50 as primary contributors to the coordination shell, with a dissociation constant of approximately 0.1 nM. When bound to the membrane bilayer, α-syn takes on a predominantly helical conformation, which spatially separates His50 from the protein N-terminus and is therefore incompatible with the copper coordination geometry of the solution state. Here we use circular dichroism and electron paramagnetic resonance (continuous wave and pulsed) to evaluate copper coordination to the membrane bound form of α-syn. In this molecular environment, Cu2+ binds exclusively to the protein N-terminus (Met1-Asp2) with no participation from His50. Copper does not alter the membrane bound α-syn conformation, or enhance the protein's release from the bilayer. The Cu2+ affinity is similar to that identified for solution α-syn suggesting that copper coordination is retained in the membrane. Consideration of these results suggests that copper exerts its greatest conformational affect on the solution form of α-syn and this species may therefore be precursor to PD arising from environmental copper exposure.

  11. Hydrogen Production by a Hyperthermophilic Membrane-Bound Hydrogenase in Soluble Nanolipoprotein Particles

    SciTech Connect

    Baker, S E; Hopkins, R C; Blanchette, C; Walsworth, V; Sumbad, R; Fischer, N; Kuhn, E; Coleman, M; Chromy, B; Letant, S; Hoeprich, P; Adams, M W; Henderson, P T

    2008-10-22

    Hydrogenases constitute a promising class of enzymes for ex vivo hydrogen production. Implementation of such applications is currently hindered by oxygen sensitivity and, in the case of membrane-bound hydrogenases (MBH), poor water solubility. Nanolipoprotein particles (NLPs), formed from apolipoproteins and phospholipids, offer a novel means to incorporate MBH into in a well-defined water-soluble matrix that maintains the enzymatic activity and is amenable to incorporation into more complex architectures. We report the synthesis, hydrogen-evolving activity and physical characterization of the first MBH-NLP assembly. This may ultimately lead to the development of biomimetic hydrogen production devices.

  12. The prolyl hydroxylase PHD3 identifies proinflammatory macrophages and its expression is regulated by activin A.

    PubMed

    Escribese, María M; Sierra-Filardi, Elena; Nieto, Concha; Samaniego, Rafael; Sánchez-Torres, Carmen; Matsuyama, Takami; Calderon-Gómez, Elisabeth; Vega, Miguel A; Salas, Azucena; Sánchez-Mateos, Paloma; Corbí, Angel L

    2012-08-15

    Modulation of macrophage polarization underlies the onset and resolution of inflammatory processes, with polarization-specific molecules being actively sought as potential diagnostic and therapeutic tools. Based on their cytokine profile upon exposure to pathogenic stimuli, human monocyte-derived macrophages generated in the presence of GM-CSF or M-CSF are considered as proinflammatory (M1) or anti-inflammatory (M2) macrophages, respectively. We report in this study that the prolyl hydroxylase PHD3-encoding EGLN3 gene is specifically expressed by in vitro-generated proinflammatory M1(GM-CSF) human macrophages at the mRNA and protein level. Immunohistochemical analysis revealed the expression of PHD3 in CD163(+) lung macrophages under basal homeostatic conditions, whereas PHD3(+) macrophages were abundantly found in tissues undergoing inflammatory responses (e.g., Crohn's disease and ulcerative colitis) and in tumors. In the case of melanoma, PHD3 expression marked a subset of tumor-associated macrophages that exhibit a weak (e.g., CD163) or absent (e.g., FOLR2) expression of typical M2-polarization markers. EGLN3 gene expression in proinflammatory M1(GM-CSF) macrophages was found to be activin A dependent and could be prevented in the presence of an anti-activin A-blocking Ab or inhibitors of activin receptor-like kinase receptors. Moreover, EGLN3 gene expression was upregulated in response to hypoxia only in M2(M-CSF) macrophages, and the hypoxia-mediated upregulation of EGLN3 expression was significantly impaired by activin A neutralization. These results indicate that EGLN3 gene expression in macrophages is dependent on activin A both under basal and hypoxic conditions and that the expression of the EGLN3-encoded PHD3 prolyl hydroxylase identifies proinflammatory macrophages in vivo and in vitro. PMID:22778395

  13. Transferred nuclear Overhauser effect analyses of membrane-bound enkephalin analogues by sup 1 H nuclear magnetic resonance: Correlation between activities and membrane-bound conformations

    SciTech Connect

    Milon, Alain; Miyazawa, Tatsuo; Higashijima, Tsutomu )

    1990-01-09

    Leu-enkephalin, (D-Ala{sup 2})Leu-enkephalin, and (D-Ala{sup 2})Leu-enkephalinamide (agonists) and (L-Ala{sup 2})Leu-enkephalin (inactive analogue) bind to lipid bilayer consisting of phosphatidylcholine and phosphatidylserine. The conformations that these compounds assume, once bound to perdeuterated phospholipid bilayer, have been shown to be unique, as shown by the transferred nuclear Overhauser effect (TRNOE) of {sup 1}H NMR spectroscopy. In addition, their location in the bilayer was analyzed by TRNOE in the presence of spin-labeled phospholipids. These analyses showed a clear relationship between the activity and the peptide-membrane interaction. The three active peptides, when bound to membranes, adopt the same conformation, characterized by a type II{prime} {beta}-turn around Gly{sup 3}-Phe and a {gamma}-turn around Gly{sup 2} (or D-Ala{sup 2}). The inactive analogue, (L-Ala{sup 2})Leu-enkephalin, displayed a completely different TRNOE pattern corresponding to a different conformation in the membrane-bound state. The tyrosine residue of the active compounds is not inserted into the interior of membrane, but it is inserted into the bilayer for the L-Ala{sup 2} analogue. According to these results, (L-Ala{sup 2})Leu-enkephalin may be explained to be inactive because the mode of binding to the membranes is different from that of active compounds.

  14. Membrane-bound amylopullulanase is essential for starch metabolism of Sulfolobus acidocaldarius DSM639.

    PubMed

    Choi, Kyoung-Hwa; Cha, Jaeho

    2015-09-01

    Sulfolobus acidocaldarius DSM639 produced an acid-resistant membrane-bound amylopullulanase (Apu) during growth on starch as a sole carbon and energy source. The physiological role of Apu in starch metabolism was investigated by the growth and starch degradation pattern of apu disruption mutant as well as biochemical properties of recombinant Apu. The Δapu mutant lost the ability to grow in minimal medium in the presence of starch, and the amylolytic activity observed in the membrane fraction of the wild-type strain was not detected in the Δapu mutant when the cells were grown in YT medium. The purified membrane-bound Apu initially hydrolyzed starch, amylopectin, and pullulan into various sizes of maltooligosaccharides, and then produced glucose, maltose, and maltotriose in the end, indicating Apu is a typical endo-acting glycoside hydrolase family 57 (GH57) amylopullulanase. The maltose and maltotriose observed in the culture medium during the exponential and stationary phase growth indicates that Apu is the essential enzyme to initially hydrolyze the starch into small maltooligosaccharides to be transported into the cell. PMID:26104674

  15. Membrane-bound globin X protects the cell from reactive oxygen species.

    PubMed

    Koch, Jonas; Burmester, Thorsten

    2016-01-01

    Globin X (GbX) is a member of the globin family that emerged early in the evolution of Metazoa. In vertebrates, GbX is restricted to lampreys, fish, amphibians and some reptiles, and is expressed in neurons. Unlike any other metazoan globin, GbX is N-terminally acylated and anchored in the cell membrane via myristoyl and palmitoyl groups, suggesting a unique function. Here, we compared the capacity of GbX to protect a mouse neuronal cell line from hypoxia and reactive oxygen species (ROS) with that of myoglobin. To evaluate the contribution of membrane-binding, we generated a mutated version of GbX without acyl groups. All three globins enhanced cell viability under hypoxia, with myoglobin having the most pronounced effect. GbX but not myoglobin protected the cells from hydrogen peroxide (H2O2)-induced stress. Membrane-bound GbX was significantly more efficient than its mutated, soluble form. Furthermore, myoglobin and mutated GbX increased production of ROS upon H2O2-treatment, while membrane-bound GbX did not. The results indicate that myoglobin enhances O2 supply while GbX protects the cell membrane from ROS-stress. The ancient origin of GbX suggests that ROS-protection reflects the function of the early globins before they acquired a respiratory role.

  16. Membrane-bound amylopullulanase is essential for starch metabolism of Sulfolobus acidocaldarius DSM639.

    PubMed

    Choi, Kyoung-Hwa; Cha, Jaeho

    2015-09-01

    Sulfolobus acidocaldarius DSM639 produced an acid-resistant membrane-bound amylopullulanase (Apu) during growth on starch as a sole carbon and energy source. The physiological role of Apu in starch metabolism was investigated by the growth and starch degradation pattern of apu disruption mutant as well as biochemical properties of recombinant Apu. The Δapu mutant lost the ability to grow in minimal medium in the presence of starch, and the amylolytic activity observed in the membrane fraction of the wild-type strain was not detected in the Δapu mutant when the cells were grown in YT medium. The purified membrane-bound Apu initially hydrolyzed starch, amylopectin, and pullulan into various sizes of maltooligosaccharides, and then produced glucose, maltose, and maltotriose in the end, indicating Apu is a typical endo-acting glycoside hydrolase family 57 (GH57) amylopullulanase. The maltose and maltotriose observed in the culture medium during the exponential and stationary phase growth indicates that Apu is the essential enzyme to initially hydrolyze the starch into small maltooligosaccharides to be transported into the cell.

  17. Role of membrane-bound IgM in Trypanosoma cruzi evasion from immune clearance.

    PubMed

    Garcia, I E; Lima, M R; Marinho, C R; Kipnis, T L; Furtado, G C; Alvarez, J M

    1997-04-01

    We have recently described that Trypanosoma cruzi parasites of the reticulotropic Y strain increase their resistance to antibody-induced clearance during their interaction with the vertebrate host immune system. In the present study, we observed that trypomastigotes of the myotropic CL strain isolated from normal host also display an increased resistance to immune clearance when compared to parasites obtained from immunosuppressed donors. Through fluorescence-activated cell sorting analysis, we have observed that the high expression of membrane-bound IgM antibodies on Y and CL trypomastigotes correlates with their enhanced resistance to Ig-induced clearance. Trypomastigotes from normal mice were essentially refractory to the in vitro binding of immunoglobulins, showing that their membrane structures were completely covered by IgM antibodies. These findings suggest that this isotype does not efficiently mediate immune clearance. Moreover, membrane-bound IgM antibodies limited the amount of IgG attached to the parasite and, as a consequence, impaired efficient immune clearance. Through this mechanism, trypomastigotes of T. cruzi could increase their persistence in the bloodstream thus favoring parasite transmission to its hematophagous host vector in the early acute phase of the disease.

  18. Biochemical similarities between soluble and membrane-bound calcium-dependent protein kinases of barley

    SciTech Connect

    Klimczak, L.J.; Hind, G. )

    1990-04-01

    The soluble and membrane-bound forms of the calcium-dependent protein kinase from barley leaves (Hordeum vulgare L. cv. Borsoy) have been partially purified and compared. Both forms showed an active polypeptide of 37 kilodaltons on activity gels with incorporated histone as substrate. They eluted from chromatofocusing columns at an identical isoelectric point of pH 4.25 {plus minus} 0.2, and also comigrated on several other chromatographic affinity media including Matrex Gel Blue A, histone-agarose, phenyl-Sepharose, and heparin-agarose. Both activities comigrated with chicken ovalbumin during gel filtration through Sephacryl S-200, indicating a native molecular mass of 45 kilodaltons. The activities share a number of enzymatic properties including salt and pH dependence, free calcium stimulation profile, substrate specificity, and Km values. The soluble activity was shown to bind to artificial lipid vesicles. These data suggest strongly that the soluble and membrane-bound calcium-dependent protein kinases from barley are very closely related or even identical.

  19. Membrane-bound proteases of the gerbil subfornical organ and choroid plexus: an enzyme histochemical study.

    PubMed

    Mitro, A; De Bault, L E

    1994-03-01

    Using enzyme-histochemical methods, the membrane-bound peptidases, gamma-glutamyl transpeptidase (gamma-GTP), microsomal alanyl aminopeptidase (mAAP), glutamyl aminopeptidase (EAP), and dipeptidyl peptidase IV (DPP IV), were studied in microvessels of the gerbil subfornical organ (SFO), choroid plexus adjacent to the SFO, and the ependyma of brain ventricle walls in the vicinity of the SFO. Vessels and microvessels of gerbil SFO and choroid plexus were positive for gamma-GTP, mAAP, and EAP, but negative for DPP IV. Blood-brain barrier (BBB) microvessels in the surrounding brain tissue also showed positive reactions for gamma-GTP, mAAP, and EAP but a negative reaction for DPP IV. Both epithelial cells of the choroid plexus and ependymal cells of the ventricle walls were negative for all four studied enzymes. It is suggested that blood-borne peptide hormones which can be substrates for these membrane-bound proteases can be modulated by gamma-GTP, mAAP, and EAP, but not by DPP IV, when they come in contact with the plasma membrane of the endothelial cells of the vessels in gerbil SFO, choroid plexus, and surrounding brain tissue.

  20. Virtual High-Throughput Screening To Identify Novel Activin Antagonists

    PubMed Central

    Zhu, Jie; Mishra, Rama K.; Schiltz, Gary E.; Makanji, Yogeshwar; Scheidt, Karl A.; Mazar, Andrew P.; Woodruff, Teresa K.

    2015-01-01

    Activin belongs to the TGFβ superfamily, which is associated with several disease conditions, including cancer-related cachexia, preterm labor with delivery, and osteoporosis. Targeting activin and its related signaling pathways holds promise as a therapeutic approach to these diseases. A small-molecule ligand-binding groove was identified in the interface between the two activin βA subunits and was used for a virtual high-throughput in silico screening of the ZINC database to identify hits. Thirty-nine compounds without significant toxicity were tested in two well-established activin assays: FSHβ transcription and HepG2 cell apoptosis. This screening workflow resulted in two lead compounds: NUCC-474 and NUCC-555. These potential activin antagonists were then shown to inhibit activin A-mediated cell proliferation in ex vivo ovary cultures. In vivo testing showed that our most potent compound (NUCC-555) caused a dose-dependent decrease in FSH levels in ovariectomized mice. The Blitz competition binding assay confirmed target binding of NUCC-555 to the activin A:ActRII that disrupts the activin A:ActRII complex’s binding with ALK4-ECD-Fc in a dose-dependent manner. The NUCC-555 also specifically binds to activin A compared with other TGFβ superfamily member myostatin (GDF8). These data demonstrate a new in silico-based strategy for identifying small-molecule activin antagonists. Our approach is the first to identify a first-in-class small-molecule antagonist of activin binding to ALK4, which opens a completely new approach to inhibiting the activity of TGFβ receptor superfamily members. in addition, the lead compound can serve as a starting point for lead optimization toward the goal of a compound that may be effective in activin-mediated diseases. PMID:26098096

  1. Rapid effects of aldosterone in primary cultures of cardiomyocytes - do they suggest the existence of a membrane-bound receptor?

    PubMed

    Araujo, Carolina Morais; Hermidorff, Milla Marques; Amancio, Gabriela de Cassia Sousa; Lemos, Denise da Silveira; Silva, Marcelo Estáquio; de Assis, Leonardo Vinícius Monteiro; Isoldi, Mauro César

    2016-10-01

    Aldosterone acts on its target tissue through a classical mechanism or through the rapid pathway through a putative membrane-bound receptor. Our goal here was to better understand the molecular and biochemical rapid mechanisms responsible for aldosterone-induced cardiomyocyte hypertrophy. We have evaluated the hypertrophic process through the levels of ANP, which was confirmed by the analysis of the superficial area of cardiomyocytes. Aldosterone increased the levels of ANP and the cellular area of the cardiomyocytes; spironolactone reduced the aldosterone-increased ANP level and cellular area of cardiomyocytes. Aldosterone or spironolactone alone did not increase the level of cyclic 3',5'-adenosine monophosphate (cAMP), but aldosterone plus spironolactone led to increased cAMP level; the treatment with aldosterone + spironolactone + BAPTA-AM reduced the levels of cAMP. These data suggest that aldosterone-induced cAMP increase is independent of mineralocorticoid receptor (MR) and dependent on Ca(2+). Next, we have evaluated the role of A-kinase anchor proteins (AKAP) in the aldosterone-induced hypertrophic response. We have found that St-Ht31 (AKAP inhibitor) reduced the increased level of ANP which was induced by aldosterone; in addition, we have found an increase on protein kinase C (PKC) and extracellular signal-regulated kinase 5 (ERK5) activity when cells were treated with aldosterone alone, spironolactone alone and with a combination of both. Our data suggest that PKC could be responsible for ERK5 aldosterone-induced phosphorylation. Our study suggests that the aldosterone through its rapid effects promotes a hypertrophic response in cardiomyocytes that is controlled by an AKAP, being dependent on ERK5 and PKC, but not on cAMP/cAMP-dependent protein kinase signaling pathways. Lastly, we provide evidence that the targeting of AKAPs could be relevant in patients with aldosterone-induced cardiac hypertrophy and heart failure. PMID:27305962

  2. Activin signaling as an emerging target for therapeutic interventions

    PubMed Central

    Tsuchida, Kunihiro; Nakatani, Masashi; Hitachi, Keisuke; Uezumi, Akiyoshi; Sunada, Yoshihide; Ageta, Hiroshi; Inokuchi, Kaoru

    2009-01-01

    After the initial discovery of activins as important regulators of reproduction, novel and diverse roles have been unraveled for them. Activins are expressed in various tissues and have a broad range of activities including the regulation of gonadal function, hormonal homeostasis, growth and differentiation of musculoskeletal tissues, regulation of growth and metastasis of cancer cells, proliferation and differentiation of embryonic stem cells, and even higher brain functions. Activins signal through a combination of type I and II transmembrane serine/threonine kinase receptors. Activin receptors are shared by multiple transforming growth factor-β (TGF-β) ligands such as myostatin, growth and differentiation factor-11 and nodal. Thus, although the activity of each ligand is distinct, they are also redundant, both physiologically and pathologically in vivo. Activin receptors activated by ligands phosphorylate the receptor-regulated Smads for TGF-β, Smad2 and 3. The Smad proteins then undergo multimerization with the co-mediator Smad4, and translocate into the nucleus to regulate the transcription of target genes in cooperation with nuclear cofactors. Signaling through receptors and Smads is controlled by multiple mechanisms including phosphorylation and other posttranslational modifications such as sumoylation, which affect potein localization, stability and transcriptional activity. Non-Smad signaling also plays an important role in activin signaling. Extracellularly, follistatin and related proteins bind to activins and related TGF-β ligands, and control the signaling and availability of ligands. The functions of activins through activin receptors are pleiotrophic, cell type-specific and contextual, and they are involved in the etiology and pathogenesis of a variety of diseases. Accordingly, activin signaling may be a target for therapeutic interventions. In this review, we summarize the current knowledge on activin signaling and discuss the potential roles of

  3. Electrochemical insights into the mechanism of NiFe membrane-bound hydrogenases

    PubMed Central

    Flanagan, Lindsey A.; Parkin, Alison

    2016-01-01

    Hydrogenases are enzymes of great biotechnological relevance because they catalyse the interconversion of H2, water (protons) and electricity using non-precious metal catalytic active sites. Electrochemical studies into the reactivity of NiFe membrane-bound hydrogenases (MBH) have provided a particularly detailed insight into the reactivity and mechanism of this group of enzymes. Significantly, the control centre for enabling O2 tolerance has been revealed as the electron-transfer relay of FeS clusters, rather than the NiFe bimetallic active site. The present review paper will discuss how electrochemistry results have complemented those obtained from structural and spectroscopic studies, to present a complete picture of our current understanding of NiFe MBH. PMID:26862221

  4. Allosteric activation of membrane-bound glutamate receptors using coordination chemistry within living cells

    NASA Astrophysics Data System (ADS)

    Kiyonaka, Shigeki; Kubota, Ryou; Michibata, Yukiko; Sakakura, Masayoshi; Takahashi, Hideo; Numata, Tomohiro; Inoue, Ryuji; Yuzaki, Michisuke; Hamachi, Itaru

    2016-10-01

    The controlled activation of proteins in living cells is an important goal in protein-design research, but to introduce an artificial activation switch into membrane proteins through rational design is a significant challenge because of the structural and functional complexity of such proteins. Here we report the allosteric activation of two types of membrane-bound neurotransmitter receptors, the ion-channel type and the G-protein-coupled glutamate receptors, using coordination chemistry in living cells. The high programmability of coordination chemistry enabled two His mutations, which act as an artificial allosteric site, to be semirationally incorporated in the vicinity of the ligand-binding pockets. Binding of Pd(2,2‧-bipyridine) at the allosteric site enabled the active conformations of the glutamate receptors to be stabilized. Using this approach, we were able to activate selectively a mutant glutamate receptor in live neurons, which initiated a subsequent signal-transduction pathway.

  5. The purification and subunit structure of a membrane-bound ATPase from the Archaebacterium Halobacterium saccharovorum

    NASA Technical Reports Server (NTRS)

    Hochstein, Lawrence I.; Kristjansson, Hordur; Altekar, Wijaya

    1987-01-01

    The procedure for the isolation and 70-fold purification of membrane-bound cold-sensitive ATPase from Halobacterium saccharovorum is described. Upon exposure to cold, the enzyme dissociates into two major subunits, I (87 kDa) and II (60 kDa), and two minor subunits, III (29 kDa) and IV (20 kDa). The stoichiometry of the enzyme is proposed to be I2.II2.III.IV; the molecular mass of such a complex would be 343 kDa, which is in good agreement with the value of 350 kDa obtained by gel filtration. The structure of the ATPase from H. saccharovorum makes it unlike any previously described ATPase.

  6. Allosteric activation of membrane-bound glutamate receptors using coordination chemistry within living cells.

    PubMed

    Kiyonaka, Shigeki; Kubota, Ryou; Michibata, Yukiko; Sakakura, Masayoshi; Takahashi, Hideo; Numata, Tomohiro; Inoue, Ryuji; Yuzaki, Michisuke; Hamachi, Itaru

    2016-10-01

    The controlled activation of proteins in living cells is an important goal in protein-design research, but to introduce an artificial activation switch into membrane proteins through rational design is a significant challenge because of the structural and functional complexity of such proteins. Here we report the allosteric activation of two types of membrane-bound neurotransmitter receptors, the ion-channel type and the G-protein-coupled glutamate receptors, using coordination chemistry in living cells. The high programmability of coordination chemistry enabled two His mutations, which act as an artificial allosteric site, to be semirationally incorporated in the vicinity of the ligand-binding pockets. Binding of Pd(2,2'-bipyridine) at the allosteric site enabled the active conformations of the glutamate receptors to be stabilized. Using this approach, we were able to activate selectively a mutant glutamate receptor in live neurons, which initiated a subsequent signal-transduction pathway. PMID:27657873

  7. KCl-Dependent Release of Mitochondrial Membrane-Bound Arginase Appears to Be a Novel Variant of Arginase-II

    PubMed Central

    Suman, Mishra; Rajnikant, Mishra

    2016-01-01

    Arginase regulates arginine metabolism, ornithine-urea cycle, and immunological surveillance. Arginase-I is predominant in cytosol, and arginase-II is localised in the mitochondria. A mitochondrial membrane-bound arginase has also been proposed to be adsorbed with outer membrane of mitochondria which gets released by 150 mM potassium chloride (KCl). It is presumed that inclusion of 150 mM KCl in the homogenization medium would not only facilitate release of arginase bound with outer membrane of mitochondria but also affect functional anatomy of mitochondria, mitochondrial enzymes, and proteins. Therefore, it has been intended to characterize KCl-dependent release of mitochondrial membrane-bound arginase from liver of mice. Results provide advancement in the area of arginase biology and suggest that fraction of mitochondrial membrane-bound arginase contains mitochondrial arginase-II and a variant of arginase-II. PMID:27293971

  8. Genome-Based Discovery of a Novel Membrane-Bound 1,6-Dihydroxyphenazine Prenyltransferase from a Marine Actinomycete

    PubMed Central

    Zeyhle, Philipp; Bauer, Judith S.; Kalinowski, Jörn; Shin-ya, Kazuo; Gross, Harald; Heide, Lutz

    2014-01-01

    Recently, novel prenylated derivatives of 1,6-dihydroxyphenazine have been isolated from the marine sponge-associated Streptomyces sp. SpC080624SC-11. Genome sequencing of this strain now revealed a gene cluster containing all genes necessary for the synthesis of the phenazine and the isoprenoid moieties. Unexpectedly, however, the cluster did not contain a gene with similarity to previously investigated phenazine prenyltransferases, but instead a gene with modest similarity to the membrane-bound prenyltransferases of ubiquinone and menaquinone biosynthesis. Expression of this gene in E. coli and isolation of the membrane fraction proved that the encoded enzyme, Mpz10, catalyzes two successive prenylations of 1,6-dihydroxyphenazine. Mpz10 is the first example of a membrane-bound enzyme catalyzing the prenylation of a phenazine substrate, and one of few examples of membrane-bound enzymes involved in the prenylation of aromatic secondary metabolites in microorganisms. PMID:24892559

  9. Crystal structure of a membrane-bound l-amino acid deaminase from Proteus vulgaris.

    PubMed

    Ju, Yingchen; Tong, Shuilong; Gao, Yongxiang; Zhao, Wei; Liu, Qi; Gu, Qiong; Xu, Jun; Niu, Liwen; Teng, Maikun; Zhou, Huihao

    2016-09-01

    l-amino acid oxidases/deaminases (LAAOs/LAADs) are a class of oxidoreductases catalyzing the oxidative deamination of l-amino acids to α-keto acids. They are widely distributed in eukaryotic and prokaryotic organisms, and exhibit diverse substrate specificity, post-translational modifications and cellular localization. While LAAOs isolated from snake venom have been extensively characterized, the structures and functions of LAAOs from other species are largely unknown. Here, we reported crystal structure of a bacterial membrane-bound LAAD from Proteus vulgaris (pvLAAD) in complex with flavin adenine dinucleotide (FAD). We found that the overall fold of pvLAAD does not resemble typical LAAOs. Instead it, is similar to d-amino acid oxidases (DAAOs) with an additional hydrophobic insertion module on protein surface. Structural analysis and liposome-binding assays suggested that the hydrophobic module serves as an extra membrane-binding site for LAADs. Bacteria from genera Proteus and Providencia were found to encode two classes of membrane-bound LAADs. Based on our structure, the key roles of residues Q278 and L317 in substrate selectivity were proposed and biochemically analyzed. While LAADs on the membrane were proposed to transfer electrons to respiratory chain for FAD re-oxidization, we observed that the purified pvLAAD could generate a significant amount of hydrogen peroxide in vitro, suggesting it could use dioxygen to directly re-oxidize FADH2 as what typical LAAOs usually do. These findings provide a novel insights for a better understanding this class of enzymes and will help developing biocatalysts for industrial applications. PMID:27422658

  10. Hydrogen Exchange Mass Spectrometry of Functional Membrane-bound Chemotaxis Receptor Complexes

    PubMed Central

    Koshy, Seena S.; Eyles, Stephen J.; Weis, Robert M.; Thompson, Lynmarie K.

    2014-01-01

    The transmembrane signaling mechanism of bacterial chemotaxis receptors is thought to involve changes in receptor conformation and dynamics. The receptors function in ternary complexes with two other proteins, CheA and CheW, that form extended membrane-bound arrays. Previous studies have shown that attractant binding induces a small (~2 Å) piston displacement of one helix of the periplasmic and transmembrane domains towards the cytoplasm, but it is not clear how this signal propagates through the cytoplasmic domain to control the kinase activity of the CheA bound at the membrane-distal tip, nearly 200 Å away. The cytoplasmic domain has been shown to be highly dynamic, which raises the question of how a small piston motion could propagate through a dynamic domain to control CheA kinase activity. To address this, we have developed a method for measuring dynamics of the receptor cytoplasmic fragment (CF) in functional complexes with CheA and CheW. Hydrogen exchange mass spectrometry (HDX-MS) measurements of global exchange of CF demonstrate that CF exhibits significantly slower exchange in functional complexes than in solution. Since the exchange rates in functional complexes are comparable to that of other proteins of similar structure, the CF appears to be a well-structured protein within these complexes, which is compatible with its role in propagating a signal that appears to be a tiny conformational change in the periplasmic and transmembrane domains of the receptor. We also demonstrate the feasibility of this protocol for local exchange measurements, by incorporating a pepsin digest step to produce peptides with 87% sequence coverage and only 20% back exchange. This method extends HDX-MS to membrane-bound functional complexes without detergents that may perturb the stability or structure of the system. PMID:24274333

  11. A membrane-bound Fas decoy receptor expressed by human thymocytes.

    PubMed

    Jenkins, M; Keir, M; McCune, J M

    2000-03-17

    Human thymocytes at several stages of maturation express Fas, yet resist apoptosis induction through its ligation. A proximal step in apoptotic signaling through Fas is implicated in this resistance, as these cells undergo normal levels of apoptosis induction after exposure to tumor necrosis factor-alpha. We studied the Fas receptors expressed in human thymocytes to search for mechanisms of receptor-mediated inhibition of Fas signaling in these cells. We describe here a unique, membrane-bound form of Fas receptor that contained a complete extracellular domain of Fas but that lacked a death domain due to alternative splicing of exon 7. This Fas decoy receptor (FDR) was shown to have nearly wild-type ability to bind native human Fas ligand and was expressed predominantly at the plasma membrane. Unlike soluble forms of Fas receptor, FDR dominantly inhibited apoptosis induction by Fas ligand in transfected human embryonic kidney cells. Titration of FDR in Fas-expressing cells suggests that FDR may operate through the formation of mixed receptor complexes. FDR also dominantly inhibited Fas-induced apoptosis in Jurkat T cells. In mixing experiments with wild-type Fas, FDR was capable of inhibiting death signaling at molar ratios less than 0.5, and this relative level of FDR:wild type message was observed in at least some thymocytes tested. The data suggest that Fas signal pathways in primary human cells may be regulated by expression of a membrane-bound decoy receptor, analogous to the regulation of tumor necrosis factor-related apoptosis inducing ligand (TRAIL)-induced apoptosis by decoy receptors.

  12. The role of activin in mammary gland development and oncogenesis.

    PubMed

    Dunphy, Karen A; Schneyer, Alan L; Hagen, Mary J; Jerry, D Joseph

    2011-06-01

    TGFβ contributes to mammary gland development and has paradoxical roles in breast cancer because it has both tumor suppressor and tumor promoter activity. Another member of the TGFβ superfamily, activin, also has roles in the developing mammary gland, but these functions, and the role of activin in breast cancer, are not well characterized. TGFβ and activin share the same intracellular signaling pathways, but divergence in their signaling pathways are suggested. The purpose of this review is to compare the spatial and temporal expression of TGFβ and activin during mammary gland development, with consideration given to their functions during each developmental period. We also review the contributions of TGFβ and activin to breast cancer resistance and susceptibility. Finally, we consider the systemic contributions of activin in regulating obesity and diabetes; and the impact this regulation has on breast cancer. Elevated levels of activin in serum during pregnancy and its influence on pregnancy associated breast cancer are also considered. We conclude that evidence demonstrates that activin has tumor suppressing potential, without definitive indication of tumor promoting activity in the mammary gland, making it a good target for development of therapeutics.

  13. Membrane-bound guaiacol peroxidases from maize (Zea mays L.) roots are regulated by methyl jasmonate, salicylic acid, and pathogen elicitors.

    PubMed

    Mika, Angela; Boenisch, Marike Johanne; Hopff, David; Lüthje, Sabine

    2010-03-01

    Plant peroxidases are involved in numerous cellular processes in plant development and stress responses. Four plasma membrane-bound peroxidases have been identified and characterized in maize (Zea mays L.) roots. In the present study, maize seedlings were treated with different stresses and signal compounds, and a functional analysis of these membrane-bound class III peroxidases (pmPOX1, pmPOX2a, pmPOX2b, and pmPOX3) was carried out. Total guaiacol peroxidase activities from soluble and microsomal fractions of maize roots were compared and showed weak changes. By contrast, total plasma membrane and washed plasma membrane peroxidase activities, representing peripheral and integral membrane proteins, revealed strong changes after all of the stresses applied. A proteomic approach using 2D-PAGE analysis showed that pmPOX3 was the most abundant class III peroxidase at plasma membranes of control plants, followed by pmPOX2a >pmPOX2b >pmPOX1. The molecular mass (63 kDa) and the isoelectric point (9.5) of the pmPOX2a monomer were identified for the first time. The protein levels of all four enzymes changed in response to multiple stresses. While pmPOX2b was the only membrane peroxidase down-regulated by wounding, all four enzymes were differentially but strongly stimulated by methyl jasmonate, salicylic acid, and elicitors (Fusarium graminearum and Fusarium culmorum extracts, and chitosan) indicating their function in pathogen defence. Oxidative stress applied as H(2)O(2) treatment up-regulated pmPOX2b >pmPOX2a, while pmPOX3 was down-regulated. Treatment with the phosphatase inhibitor chantharidin resulted in distinct responses.

  14. The orientation of membrane bound radicals: an EPR investigation of magnetically ordered spinach chloroplasts.

    PubMed

    Dismukes, G C; Sauer, K

    1978-12-01

    The orientation of membrane-bound radicals in spinach chloroplasts is examined by electron paramagnetic resonance (EPR) spectroscopy of chloroplasts oriented by magnetic fields. Several of the membrane-bound radicals which possess g-tensor anisotropy display EPR signals with a marked dependence on the orientation of the membranes relative to the applied EPR field. The fraction of oxidized and reduced plastocyanin, P-700, iron-sulfur proteins A and B, and the X center, an early acceptor of Photosystem I, can be controlled by the light intensity during steady-state illumination and can be trapped by cooling. The X center can be photoreduced and trapped in the absence of strong reductants and high pH, conditions previously found necessary for its detection. These results confirm its role as an early electron acceptor in P-700 photo-oxidation. X is oriented with its smallest principal g-tensor axis (gx) predominantly parallel to the normal to the thylakoid membrane, the same orientation as was found for an early electron acceptor based on time-resolved electron spin polarization studies. We propose that the X center is the first example of a high potential iron-sulfur protein which functions in electron transfer in its 'superreduced' state. We present evidence which suggests that iron-sulfur proteins A and B are 4Fe-4S clusters in an 8Fe-8S protein. Center B is oriented with gy predominantly normal to the membrane plane. The spectra of center A and plastocyanin do not show significant changes with sample orientation. In the case of plastocyanin, this may indicate a lack of molecular orientation. The absence of an orientation effect for reduced center A is reconcilable with a 4Fe-4S geometry, provided that the electron obtained upon reduction can be shared between any pair of Fe atoms in the center. Orientation of the 'Rieske' iron-sulfur protein is also observed. It has axial symmetry with g parallel close to the plane of the membrane. A model is proposed for the

  15. Membrane-bound IL-21 promotes sustained ex vivo proliferation of human natural killer cells.

    PubMed

    Denman, Cecele J; Senyukov, Vladimir V; Somanchi, Srinivas S; Phatarpekar, Prasad V; Kopp, Lisa M; Johnson, Jennifer L; Singh, Harjeet; Hurton, Lenka; Maiti, Sourindra N; Huls, M Helen; Champlin, Richard E; Cooper, Laurence J N; Lee, Dean A

    2012-01-01

    NK cells have therapeutic potential for a wide variety of human malignancies. However, because NK cells expand poorly in vitro, have limited life spans in vivo, and represent a small fraction of peripheral white blood cells, obtaining sufficient cell numbers is the major obstacle for NK-cell immunotherapy. Genetically-engineered artificial antigen-presenting cells (aAPCs) expressing membrane-bound IL-15 (mbIL15) have been used to propagate clinical-grade NK cells for human trials of adoptive immunotherapy, but ex vivo proliferation has been limited by telomere shortening. We developed K562-based aAPCs with membrane-bound IL-21 (mbIL21) and assessed their ability to support human NK-cell proliferation. In contrast to mbIL15, mbIL21-expressing aAPCs promoted log-phase NK cell expansion without evidence of senescence for up to 6 weeks of culture. By day 21, parallel expansion of NK cells from 22 donors demonstrated a mean 47,967-fold expansion (median 31,747) when co-cultured with aAPCs expressing mbIL21 compared to 825-fold expansion (median 325) with mbIL15. Despite the significant increase in proliferation, mbIL21-expanded NK cells also showed a significant increase in telomere length compared to freshly obtained NK cells, suggesting a possible mechanism for their sustained proliferation. NK cells expanded with mbIL21 were similar in phenotype and cytotoxicity to those expanded with mbIL15, with retained donor KIR repertoires and high expression of NCRs, CD16, and NKG2D, but had superior cytokine secretion. The mbIL21-expanded NK cells showed increased transcription of the activating receptor CD160, but otherwise had remarkably similar mRNA expression profiles of the 96 genes assessed. mbIL21-expanded NK cells had significant cytotoxicity against all tumor cell lines tested, retained responsiveness to inhibitory KIR ligands, and demonstrated enhanced killing via antibody-dependent cell cytotoxicity. Thus, aAPCs expressing mbIL21 promote improved proliferation of

  16. Activin-A stimulates hypothalamic gonadotropin-releasing hormone release by the explanted male rat hypothalamus: interaction with inhibin and androgens.

    PubMed

    Calogero, A E; Burrello, N; Ossino, A M; Polosa, P; D'Agata, R

    1998-02-01

    The presence of activins in those hypothalamic regions containing gonadotropin-releasing hormone (GnRH)-secreting neurons suggests that these peptides may regulate the reproductive function modulating not only pituitary FSH release and biosynthesis, but also hypothalamic GnRH release. The purpose of this study was to evaluate the effects of activin-A, a homodimer of inhibin beta A subunit, on hypothalamic GnRH release in vitro and, because of their well known antithetical effects, to evaluate its interaction with inhibin. In addition, since androgens modulate the release of GnRH from male rat hypothalami, we thought it of interest to study the possible interplay between these steroids and activin on GnRH release. To accomplish this, we employed a hypothalamic organ culture system which enabled us to evaluate GnRH release from individually incubated hemi-hypothalami explanted from male rats. Activin-A stimulated GnRH release in a biphasic manner. The maximal effect was reached at a concentration of 10 ng/ml which increased GnRH output by about 75%. Inhibin abolished the stimulatory effect of a maximally effective concentration of activin-A in a dose-dependent manner, whereas alone it had no effect on GnRH output. As previously shown, testosterone (1 nmol/l) and dihydrotestosterone (DHT, 0.1 nmol/l) suppressed basal GnRH release, but only testosterone was able to inhibit the release of GnRH stimulated by activin-A. Since DHT is a non-aromatizable androgen, we evaluated whether the inhibitory effect of testosterone was due to its in vitro conversion into 17 beta-estradiol. The addition of 4-hydroxyandrostenedione, a steroidal aromatase inhibitor, did not influence the suppressive effect of testosterone on GnRH release stimulated by activin-A. In conclusion, activin-A stimulated hypothalamic GnRH release in vitro and this effect was abolished by inhibin and was blunted by testosterone. These findings suggest that activins may participate in the regulation of the

  17. Activin A balance regulates epithelial invasiveness and tumorigenesis

    PubMed Central

    Le Bras, Grégoire F.; Loomans, Holli A.; Taylor, Chase; Revetta, Frank; Andl, Claudia D.

    2015-01-01

    Activin A is a member of the TGFβ superfamily. Activin A and TGFβ have multiple common downstream targets and have been described to merge in their intracellular signaling cascades and function. We have previously demonstrated that coordinated loss of E-cadherin and TGFβ receptor II results in epithelial cell invasion. When grown in three-dimensional organotypic reconstruct cultures, esophageal keratinocytes expressing dominant-negative mutants of E-cadherin and TGFβ receptor II showed activated Smad2 in the absence of functional TGFβ receptor II. However, we could show increased levels of Activin A secretion, and Activin A was able to induce Smad2 phosphorylation. Growth factor secretion can activate autocrine and paracrine signaling, which affects crosstalk between the epithelial compartment and the surrounding microenvironment. We show that treatment with the Act A antagonist Follistatin or with a neutralizing Activin A antibody can increase cell invasion in organotypic cultures in a fibroblast- and MMP-dependent manner. Similarly, suppression of Activin A with shRNA increases cell invasion and tumorigenesis in vivo. Therefore, we conclude that maintaining a delicate balance of Activin A expression is critical for homeostasis in the esophageal microenvironment. PMID:25068654

  18. C. elegans uses Liquid-Liquid Demixing for the Assembly of Non-Membrane-Bound Compartments

    NASA Astrophysics Data System (ADS)

    Weber, Christoph A.; Juelicher, Frank; Diaz Delgadillo, Andres Felipe; Jawerth, Louise; Hyman, Anthony A.; Department Biological Physics Team; Hyman Lab Collaboration

    2015-03-01

    P granules are liquid cytoplasmic RNA/Protein condensates known to determine the germ lineage in Caenorhabditis elegans. They resemble striking similarities with liquid droplets, such as dripping, shearing and wetting. Assuming that P granules are liquid-like we consider how they form in the crowded cytoplasm. Using confocal and light-sheet microscopy, P granule formation in-vivo and in-vitro is shown to share all hallmarks with a liquid-liquid phase-separation. Specifically, demixing is determined by temperature and concentration, the droplet formation is reversible with respect to temperature quenches and there is evidence for droplet growth due to coalescence and Ostwald-ripening. Liquid-liquid demixing in-vivo breaks the paradigmatic view that a molecular machinery is necessary to build up organelles through complex biological pathways. Instead we propose that P granules form following a Flory-Huggins model. Liquid-liquid demixing could also serve as a mechanism for the assembly of non-membrane-bound compartments in other living organisms.

  19. Identification of a Membrane-bound Prepore Species Clarifies the Lytic Mechanism of Actinoporins.

    PubMed

    Morante, Koldo; Bellomio, Augusto; Gil-Cartón, David; Redondo-Morata, Lorena; Sot, Jesús; Scheuring, Simon; Valle, Mikel; González-Mañas, Juan Manuel; Tsumoto, Kouhei; Caaveiro, Jose M M

    2016-09-01

    Pore-forming toxins (PFTs) are cytolytic proteins belonging to the molecular warfare apparatus of living organisms. The assembly of the functional transmembrane pore requires several intermediate steps ranging from a water-soluble monomeric species to the multimeric ensemble inserted in the cell membrane. The non-lytic oligomeric intermediate known as prepore plays an essential role in the mechanism of insertion of the class of β-PFTs. However, in the class of α-PFTs, like the actinoporins produced by sea anemones, evidence of membrane-bound prepores is still lacking. We have employed single-particle cryo-electron microscopy (cryo-EM) and atomic force microscopy to identify, for the first time, a prepore species of the actinoporin fragaceatoxin C bound to lipid vesicles. The size of the prepore coincides with that of the functional pore, except for the transmembrane region, which is absent in the prepore. Biochemical assays indicated that, in the prepore species, the N terminus is not inserted in the bilayer but is exposed to the aqueous solution. Our study reveals the structure of the prepore in actinoporins and highlights the role of structural intermediates for the formation of cytolytic pores by an α-PFT. PMID:27445331

  20. Role of nickel in membrane-bound hydrogenase and nickel metabolism in Rhizobium japonicum

    SciTech Connect

    Stults, L.W.

    1986-01-01

    The membrane-bound hydrogenase of Rhizobium japonicum requires nickel for activity. Radioactive /sup 63/Ni co-migrates with hydrogenase activity in native gel systems and co-elutes with purified hydrogenase form an affinity matrix column. A simplified scheme for the purification of hydrogenase has been developed and constitutes the first report of the aerobic purification of this enzyme from R. japonicum. The aerobic purification utilizes the general affinity matrix. Reactive Red 120-agarose and results in higher specific activity and yield of enzyme than previously reported. The stability of aerobically purified hydrogenase to oxygen is substantially greater than that reported for anaerobically isolated enzyme. Reduction of the aerobically purified enzyme in the presence of oxygen, however, results in the rapid loss of activity. R. japonicum cells accumulate nickel during heterotrophic growth and as non-growing cells. The hydrogenase constitutive mutant SR470 accumulates substantially greater amounts of nickel under both conditions. Kinetic studies indicate that the nickel uptake system in the hydrogenase constitutive mutant SR470 is upregulated relative to SRwt cells. The uptake system is specific for nickel, although a 10-fold excess (relative to nickel) of copper or zinc inhibits nickel uptake. The nickel uptake system appears to require energy. Under nickel-free conditions hydrogenase protein is not synthesized as determined by cross-reactivity with antibodies directed against hydrogenase, indicating that nickel regulates the formation of the enzyme as well as being a constituent of the active protein.

  1. Purification and structural analysis of membrane-bound polyphenol oxidase from Fuji apple.

    PubMed

    Liu, Fang; Zhao, Jin-Hong; Wen, Xin; Ni, Yuan-Ying

    2015-09-15

    Membrane-bound polyphenol oxidase (mPPO) in Fuji apple (Malus domestica Borkh. cv. Red Fuji) was purified and analyzed with a nanoelectrospray ionization mass spectrometer. The three-dimensional model and binding site of mPPO to 4-methyl catechol were also studied using molecular docking. mPPO was purified 54.41-fold using temperature-induced phase partitioning technique and ion exchange chromatography. mPPO had a molecular weight of 67.3kDa. Even though a significant level of homology was observed between mPPO and the soluble polyphenol oxidase in the copper binding sequence, there was another region, rich in histidine residues, which differed in 13 amino acids. The three-dimensional structure of mPPO consisted of six α-helices, two short β-strands, and ten random coils. The putative substrate-binding pocket contained six polar or charged amino acids, His191, His221, Trp224, Trp228, Phe227, and Val190. Trp224 and Trp228 formed hydrogen bonds with 4-methyl-catechol.

  2. Evolutionarily divergent, Na+-regulated H+-transporting membrane-bound pyrophosphatases.

    PubMed

    Luoto, Heidi H; Nordbo, Erika; Malinen, Anssi M; Baykov, Alexander A; Lahti, Reijo

    2015-04-15

    Membrane-bound pyrophosphatase (mPPases) of various types consume pyrophosphate (PPi) to drive active H+ or Na+ transport across membranes. H+-transporting PPases are divided into phylogenetically distinct K+-independent and K+-dependent subfamilies. In the present study, we describe a group of 46 bacterial proteins and one archaeal protein that are only distantly related to known mPPases (23%-34% sequence identity). Despite this evolutionary divergence, these proteins contain the full set of 12 polar residues that interact with PPi, the nucleophilic water and five cofactor Mg2+ ions found in 'canonical' mPPases. They also contain a specific lysine residue that confers K+ independence on canonical mPPases. Two of the proteins (from Chlorobium limicola and Cellulomonas fimi) were expressed in Escherichia coli and shown to catalyse Mg2+-dependent PPi hydrolysis coupled with electrogenic H+, but not Na+ transport, in inverted membrane vesicles. Unique features of the new H+-PPases include their inhibition by Na+ and inhibition or activation, depending on PPi concentration, by K+ ions. Kinetic analyses of PPi hydrolysis over wide ranges of cofactor (Mg2+) and substrate (Mg2-PPi) concentrations indicated that the alkali cations displace Mg2+ from the enzyme, thereby arresting substrate conversion. These data define the new proteins as a novel subfamily of H+-transporting mPPases that partly retained the Na+ and K+ regulation patterns of their precursor Na+-transporting mPPases.

  3. The putative roles of nuclear and membrane-bound progesterone receptors in the female reproductive tract.

    PubMed

    Kowalik, Magdalena K; Rekawiecki, Robert; Kotwica, Jan

    2013-12-01

    Progesterone produced by the corpus luteum (CL) is a key regulator of normal cyclical reproductive functions in the females of mammalian species. The physiological effects of progesterone are mediated by the canonical genomic pathway after binding of progesterone to its specific nuclear progesterone receptor (PGR), which acts as a ligand-activated transcription factor and has two main isoforms, PGRA and PGRB. These PGR isoforms play different roles in the cell; PGRB acts as an activator of progesterone-responsive genes, while PGRA can inhibit the activity of PGRB. The ratio of these isoforms changes during the estrous cycle and pregnancy, and it corresponds to the different levels of progesterone signaling occurring in the reproductive tract. Progesterone exerts its effects on cells also by a non-genomic mechanism by the interaction with the progesterone-binding membrane proteins including the progesterone membrane component (PGRMC) 1 and 2, and the membrane progestin receptors (mPRs). These receptors rapidly activate the appropriate intracellular signal transduction pathways, and subsequently they can initiate specific cell responses or modulate genomic cell responses. The diversity of progesterone receptors and their cellular actions enhances the role of progesterone as a factor regulating the function of the reproductive system and other organs. This paper deals with the possible involvement of nuclear and membrane-bound progesterone receptors in the function of target cells within the female reproductive tract.

  4. Radiation inactivation probe of membrane-bound enzymes: gamma-glutamyltranspeptidase, aminopeptidase N, and sucrase

    SciTech Connect

    Stevens, B.R.; Kempner, E.S.; Wright, E.M.

    1986-11-01

    gamma-Glutamyltranspeptidase (GGT), aminopeptidase N (AP-N), and sucrase in purified rabbit intestinal brush border membrane vesicles were irradiated in situ at -135 degrees C using high energy electrons. Surviving activities of the enzymes were measured as a function of radiation dose, and the functional unit target sizes (corresponding to carbohydrate-free polypeptides) were determined using target analysis. The in situ functional unit sizes were GGT 59 kDa, AP-N 59 kDa, and sucrase 63 kDa. Together with biochemical data determined previously, it is concluded that the noncovalently attached large (approximately 40 kDa) and small (approximately 25 kDa) subunits of GGT are both required for catalytic activity. Furthermore, these data suggest that (i) the membrane-bound form of AP-N consists of one or more noncovalently attached subunits of 59 kDa, each of which is enzymatically active; and (ii) in situ sucrase activity is associated with a subunit of 63 kDa which is noncovalently attached within the sucrase-isomaltase complex.

  5. [Interaction of surface-active base with fraction of membrane-bound Williams's protons].

    PubMed

    Iaguzhinskiĭ, L S; Motovilov, K A; Volkov, E M; Eremeev, S A

    2013-01-01

    In the process of mitochondrial respiratory H(+)-pumps functioning, the fraction membrane-bound protons (R-protons), which have an excess of free energy is formed. According to R.J. Williams this fraction is included as energy source in the reaction of ATP synthesis. Previously, in our laboratory was found the formation of this fraction was found in the mitochondria and on the outer surface of mitoplast. On the mitoslast model we strictly shown that non-equilibrium R-proton fraction is localized on the surface of the inner mitochondrial membrane. In this paper a surface-active compound--anion of 2,4,6-trichloro-3-pentadecylphenol (TCP-C15) is described, which selectively interacts with the R-protons fraction in mitochondria. A detailed description of the specific interaction of the TCP-C15 with R-protons fraction in mitochondria is presented. Moreover, in this work it was found that phosphate transport system reacts with the R-protons fraction in mitochondria and plays the role of the endogenous volume regulation system of this fraction. The results of experiments are discussed in the terms of a local coupling model of the phosphorylation mechanism.

  6. Sex steroids regulate skin pigmentation through nonclassical membrane-bound receptors

    PubMed Central

    Natale, Christopher A; Duperret, Elizabeth K; Zhang, Junqian; Sadeghi, Rochelle; Dahal, Ankit; O'Brien, Kevin Tyler; Cookson, Rosa; Winkler, Jeffrey D; Ridky, Todd W

    2016-01-01

    The association between pregnancy and altered cutaneous pigmentation has been documented for over two millennia, suggesting that sex hormones play a role in regulating epidermal melanocyte (MC) homeostasis. Here we show that physiologic estrogen (17β-estradiol) and progesterone reciprocally regulate melanin synthesis. This is intriguing given that we also show that normal primary human MCs lack classical estrogen or progesterone receptors (ER or PR). Utilizing both genetic and pharmacologic approaches, we establish that sex steroid effects on human pigment synthesis are mediated by the membrane-bound, steroid hormone receptors G protein-coupled estrogen receptor (GPER), and progestin and adipoQ receptor 7 (PAQR7). Activity of these receptors was activated or inhibited by synthetic estrogen or progesterone analogs that do not bind to ER or PR. As safe and effective treatment options for skin pigmentation disorders are limited, these specific GPER and PAQR7 ligands may represent a novel class of therapeutics. DOI: http://dx.doi.org/10.7554/eLife.15104.001 PMID:27115344

  7. Identification of a Membrane-bound Prepore Species Clarifies the Lytic Mechanism of Actinoporins * ♦

    PubMed Central

    Bellomio, Augusto; Gil-Cartón, David; Redondo-Morata, Lorena; Sot, Jesús; Scheuring, Simon; Valle, Mikel; González-Mañas, Juan Manuel; Tsumoto, Kouhei

    2016-01-01

    Pore-forming toxins (PFTs) are cytolytic proteins belonging to the molecular warfare apparatus of living organisms. The assembly of the functional transmembrane pore requires several intermediate steps ranging from a water-soluble monomeric species to the multimeric ensemble inserted in the cell membrane. The non-lytic oligomeric intermediate known as prepore plays an essential role in the mechanism of insertion of the class of β-PFTs. However, in the class of α-PFTs, like the actinoporins produced by sea anemones, evidence of membrane-bound prepores is still lacking. We have employed single-particle cryo-electron microscopy (cryo-EM) and atomic force microscopy to identify, for the first time, a prepore species of the actinoporin fragaceatoxin C bound to lipid vesicles. The size of the prepore coincides with that of the functional pore, except for the transmembrane region, which is absent in the prepore. Biochemical assays indicated that, in the prepore species, the N terminus is not inserted in the bilayer but is exposed to the aqueous solution. Our study reveals the structure of the prepore in actinoporins and highlights the role of structural intermediates for the formation of cytolytic pores by an α-PFT. PMID:27445331

  8. Structural features of the extracellular portion of membrane-anchoring peptides on membrane-bound immunoglobulins.

    PubMed

    Major, J G; Davis, F M; Liou, R S; Chang, T W

    1996-02-01

    Membrane-bound immunoglobulins, mIgs, are displayed as transmembrane proteins on the surface of B cells, where they serve as antigen receptors. The mIgs are anchored to the membrane through a carboxy-terminal extension of the immunoglobulin heavy chain. Three distinct structural regions of these membrane-anchor peptides, of mouse and human mIgs, have been delineated: (1) a central conserved stretch of 25 hydrophobic, unchanged amino acid residues, which spans the membrane lipid bilayer; (2) a C-terminal hydrophilic region of 3-28 amino acids, which is intracytoplasmic; and (3) an N-terminal extracellular hydrophilic region of 13-67 amino acids, which is isotype-specific. Here we report predicted secondary and tertiary structures of the third structural region of the membrane anchoring peptide along with corroborating experimental evidence. The predictions of secondary and tertiary structure indicate that most of these regions can assume an chi-helical conformation. Circular dichroism spectroscopy of corresponding synthetic peptide confirms this essential feature. The choice of solvent and pH have dramatic effects on peptide helicity; solvent conditions consistent with a membrane-proximal environment promote helicity. Additional studies suggest that the two adjacent extracellular peptides may be stabilized through coiled-coil interactions similar to those described for some other transmembrane proteins.

  9. A Membrane-bound Hemoglobin from Gills of the Green Shore Crab Carcinus maenas*

    PubMed Central

    Ertas, Beyhan; Kiger, Laurent; Blank, Miriam; Marden, Michael C.; Burmester, Thorsten

    2011-01-01

    Most hemoglobins serve for the transport or storage of O2. Although hemoglobins are widespread in “entomostracan” Crustacea, malacostracans harbor the copper-containing hemocyanin in their hemolymph. Usually, only one type of respiratory protein occurs within a single species. Here, we report the identification of a hemoglobin of the shore crab Carcinus maenas (Malacostraca, Brachyura). In contrast to the dodecameric hemocyanin of this species, C. maenas hemoglobin does not reside in the hemolymph but is restricted to the gills. Immunofluorescence studies and cell fractioning showed that C. maenas hemoglobin resides in the membrane of the chief cells of the gill. To the best of our knowledge, this is the first time that a membrane-bound hemoglobin has been identified in eukaryotes. Bioinformatic evaluation suggests that C. maenas hemoglobin is anchored in the membrane by N-myristoylation. Recombinant C. maenas hemoglobin has a hexacoordinate binding scheme at the Fe2+ and an oxygen affinity of P50 = 0.5 Torr. A rapid autoxidation rate precludes a function as oxygen carrier. We rather speculate that, analogous to prokaryotic membrane-globins, C. maenas hemoglobin carries out enzymatic functions to protect the lipids in cell membrane from reactive oxygen species. Sequence comparisons and phylogenetic studies suggested that the ancestral arthropod hemoglobin was most likely an N-myristoylated protein that did not have an O2 supply function. True respiratory hemoglobins of arthropods, however, evolved independently in chironomid midges and branchiopod crustaceans. PMID:21118803

  10. Endocytic Trafficking of Membrane-Bound Cargo: A Flotillin Point of View

    PubMed Central

    Meister, Melanie; Tikkanen, Ritva

    2014-01-01

    The ubiquitous and highly conserved flotillin proteins, flotillin-1 and flotillin-2, have been shown to be involved in various cellular processes such as cell adhesion, signal transduction through receptor tyrosine kinases as well as in cellular trafficking pathways. Due to the fact that flotillins are acylated and form hetero-oligomers, they constitutively associate with cholesterol-enriched lipid microdomains. In recent years, such microdomains have been appreciated as platforms that participate in endocytosis and other cellular trafficking steps. This review summarizes the current findings on the role of flotillins in membrane-bound cargo endocytosis and endosomal trafficking events. We will discuss the proposed function of flotillins in endocytosis in the light of recent findings that point towards a role for flotillins in a step that precedes the actual endocytic uptake of cargo molecules. Recent findings have also revealed that flotillins may be important for endosomal sorting and recycling of specific cargo molecules. In addition to these aspects, the cellular trafficking pathway of flotillins themselves as potential cargo in the context of growth factor signaling will be discussed. PMID:25019426

  11. Evolutionarily divergent, Na+-regulated H+-transporting membrane-bound pyrophosphatases.

    PubMed

    Luoto, Heidi H; Nordbo, Erika; Malinen, Anssi M; Baykov, Alexander A; Lahti, Reijo

    2015-04-15

    Membrane-bound pyrophosphatase (mPPases) of various types consume pyrophosphate (PPi) to drive active H+ or Na+ transport across membranes. H+-transporting PPases are divided into phylogenetically distinct K+-independent and K+-dependent subfamilies. In the present study, we describe a group of 46 bacterial proteins and one archaeal protein that are only distantly related to known mPPases (23%-34% sequence identity). Despite this evolutionary divergence, these proteins contain the full set of 12 polar residues that interact with PPi, the nucleophilic water and five cofactor Mg2+ ions found in 'canonical' mPPases. They also contain a specific lysine residue that confers K+ independence on canonical mPPases. Two of the proteins (from Chlorobium limicola and Cellulomonas fimi) were expressed in Escherichia coli and shown to catalyse Mg2+-dependent PPi hydrolysis coupled with electrogenic H+, but not Na+ transport, in inverted membrane vesicles. Unique features of the new H+-PPases include their inhibition by Na+ and inhibition or activation, depending on PPi concentration, by K+ ions. Kinetic analyses of PPi hydrolysis over wide ranges of cofactor (Mg2+) and substrate (Mg2-PPi) concentrations indicated that the alkali cations displace Mg2+ from the enzyme, thereby arresting substrate conversion. These data define the new proteins as a novel subfamily of H+-transporting mPPases that partly retained the Na+ and K+ regulation patterns of their precursor Na+-transporting mPPases. PMID:25662511

  12. Activin Signaling in the Pathogenesis and Therapy of Neuropsychiatric Diseases

    PubMed Central

    Link, Andrea S.; Zheng, Fang; Alzheimer, Christian

    2016-01-01

    Activins are members of the transforming growth factor β (TGFβ) family and serve as multifunctional regulatory proteins in many tissues and organs. In the brain, activin A, which is formed by two disulfide-linked βA subunits, is recognized as the predominant player in activin signaling. Over the last years, considerable progress has been made in elucidating novel and unexpected functions of activin in the normal and diseased brain and in deciphering the underlying molecular mechanisms. Initially identified as a neurotrophic and protective factor during development and in several forms of acute injury, the scope of effects of activin A in the adult central nervous system (CNS) has been considerably broadened by now. Here, we will highlight recent findings that bear significance for a better understanding of the pathogenesis of various neuropsychiatric diseases and might hold promise for novel therapeutic strategies. While the basal level of activin A in the adult brain is low, significant short-term up-regulation occurs in response to increased neuronal activity. In fact, brief exposure to an enriched environment (EE) is already sufficient to considerably strengthen activin signaling. Enhancement of this pathway tunes the performance of glutamatergic and GABAergic synapses in a fashion that impacts on cognitive functions and affective behavior, counteracts death-inducing signals through extrasynaptic NMDA receptors (NMDARs), and stimulates adult neurogenesis in the hippocampus. We will discuss how impaired activin signaling is involved in anxiety disorders, depression, drug dependence, and neurodegenerative diseases such as Alzheimer’s and Parkinson’s, and how reinforcement of activin signaling might be exploited for therapeutic interventions. PMID:27242425

  13. Evidence for hydrophobic region within heavy chains of mouse B lymphocyte membrane-bound IgM

    PubMed Central

    Vassalli, Pierre; Tedghi, Rachel; Lisowska-Bernstein, Barbara; Tartakoff, Alan; Jaton, Jean-Claude

    1979-01-01

    The gel filtration behavior, in the presence of detergents, of membrane-bound IgM from normal mouse spleen B lymphocytes was compared to that of secretory IgM from mouse plasma cells. The proteins were labeled either by surface radioiodination or biosynthetically with radioactive amino acids. Cell lysates were fractionated on calibrated Sepharose 6B columns in the presence of the detergents Nonidet P-40 or deoxycholate. Eluted fractions were immunoprecipitated and the reduced or unreduced precipitates were analyzed by sodium dodecyl sulfate gel electrophoresis followed by radioautography. Surface 125I-labeled 8S IgM exhibited a gel filtration pattern in Nonidet P-40 corresponding to much higher apparent molecular weight than that of secretory 8S IgM, a difference that almost disappeared when gel filtration was performed in the presence of deoxycholate, which forms much smaller micelles than does Nonidet P-40. Biosynthetically labeled lymphocytes contain two types of IgM molecules differing in their gel filtration behavior and fate: one identical to secretory 8S IgM of plasma cells and secreted in the medium during chase periods, and the other identical to surface 125I-labeled IgM and remaining cell-associated. Because the surface-bound 8S IgM was not found to be associated with other labeled molecules, it is likely that the detergent-binding behavior of surface IgM is due to a hydrophobic segment carried by these Ig molecules. That lymphocytes synthesize two types of μ chains was also shown by the use of tunicamycin, an inhibitor of glycosylation. In its presence, two unglycosylated μ chains were observed: one identical in size to that made by tunicamycin-treated plasma cells, and the second slightly larger. Gel filtration in Nonidet P-40 of the cell lysates of tunicamycin-treated lymphocytes showed that the nonsecretory 8S IgM contains this second type of μ chains, whereas the IgM molecules of the secretory type contain plasma cell-like μ chains. It is

  14. Analysis of antibodies raised against soluble and membrane bound proteins of Nosema grylli (Microspora) spores.

    PubMed

    Sokolova, J Y; Dolgikh, V V; Weck-Heimann, A; Entzeroth, R

    2000-01-01

    Da protein connected with polar filaments as it was clearly suggested by IEM and IFA. Mab 1BD9 recognized 25, 34, 43 kDa proteins from the fraction of membrane bound proteins of spore walls, the sites of their interaction with antigens being marked with uneven fluorescence (IFA) and by gold precipitates on spore walls (IEM). Mab 1BB9 reacted with 36, 45, 65 and 75 kDa proteins, which belong mainly to the fraction of membrane-bound spore proteins, and gave a weak fluorescence associated with spores. Mab 2AB3 recognized 44 and 60 kDa proteins from the fraction of soluble spore proteins, and Mab 2AD4 acknowledged a single protein of 55 kDa from the same fraction. The obtained antibodies add to the existing microsporidian antibody bank and can be used for further work of isolation, description and sequencing the microsporidian proteins in order to understand eventually their functions.

  15. Ultrastructural localization of the membrane-bound Mg-adenosine triphosphatase activity in rat meninges.

    PubMed

    Angelov, D N; Vasilev, V A

    1989-01-01

    The distribution of the membrane-bound magnesium ions-dependent adenosine triphosphatase (Mg-ATPase) activity has been studied ultracytochemically in rat meninges by the method of Wachstein and Meisel (1957). A device specially constructed to avoid preparation artefacts has been used to obtain sections from the parietal region of the head. The meninges display an intense though irregularly distributed ATPase activity marked by depositions of electron-dense reaction product (RP) which is almost absent in the outer and middle dural layers. In the borderline zone between dura mater and the arachnoid the RP deposits are found at the outer surface of the inner dural cells and at the contact sites between these cells and the dural neurothelium. The intercellular cleft(s) between the neurothelium and the outer arachnoidal layer, occupied by an "electron-dense band", remains free of RP. The strongest accumulations of reactions granules are observed on the surface of the leptomeningeal cells of the arachnoidal space. In the contact region between the inner arachnoidal and the outer pial layers the distribution of the RP is similar to the one observed in the interface zone dura mater/arachnoid, while the pial cells themselves are definitely reaction-positive. In all meningeal vessels RP is found at the lumenal and abluminal aspects of the endothelium as well as at the cell membranes of the perivascular cells. These results emphasize the importance of the dural neurothelium for the functions of the blood-cerebrospinal fluid (CSF)-barrier between the dural blood vessels and the CSF.

  16. Membrane-Bound PenA β-Lactamase of Burkholderia pseudomallei

    PubMed Central

    Randall, Linnell B.; Dobos, Karen; Papp-Wallace, Krisztina M.; Bonomo, Robert A.

    2015-01-01

    Burkholderia pseudomallei is the etiologic agent of melioidosis, a difficult-to-treat disease with diverse clinical manifestations. β-Lactam antibiotics such as ceftazidime are crucial to the success of melioidosis therapy. Ceftazidime-resistant clinical isolates have been described, and the most common mechanism is point mutations affecting expression or critical amino acid residues of the chromosomally encoded class A PenA β-lactamase. We previously showed that PenA was exported via the twin arginine translocase system and associated with the spheroplast fraction. We now show that PenA is a membrane-bound lipoprotein. The protein and accompanying β-lactamase activity are found in the membrane fraction and can be extracted with Triton X-114. Treatment with globomycin of B. pseudomallei cells expressing PenA results in accumulation of the prolipoprotein. Mass spectrometric analysis of extracted membrane proteins reveals a protein peak whose mass is consistent with a triacylated PenA protein. Mutation of a crucial lipobox cysteine at position 23 to a serine residue results in loss of β-lactamase activity and absence of detectable PenAC23S protein. A concomitant isoleucine-to-alanine change at position 20 in the signal peptide processing site in the PenAC23S mutant results in a nonlipidated protein (PenAI20A C23S) that is processed by signal peptidase I and exhibits β-lactamase activity. The resistance profile of a B. pseudomallei strain expressing this protein is indistinguishable from the profile of the isogenic strain expressing wild-type PenA. The data show that PenA membrane association is not required for resistance and must serve another purpose. PMID:26711764

  17. Purification and properties of the membrane-bound by hydrogenase from Desulfovibrio desulfuricans.

    PubMed

    Lalla-Maharajh, W V; Hall, D O; Cammack, R; Rao, K K; Le Gall, J

    1983-02-01

    The membrane-bound hydrogenase from the anaerobic sulphate-reducing bacterium Desulfovibrio desulfuricans (Norway strain) has been purified to homogeneity, with an overall 80-fold purification and a specific activity of 70 mumol of H2 evolved/min per mg of protein. The hydrogenase had a relative molecular mass of 58 000 as determined by gel filtration and was estimated to contain six iron atoms and six acid-labile sulphur groups per molecule. The absorption spectrum of the enzyme was characteristic of an iron-sulphur protein. The E400 and E280 were 28 500 and 109 000 M-1.cm-1 respectively. The e.s.r. of the oxidized protein indicated the presence of [4Fe-4S]3+ or [3Fe-3S]3+, and another paramagnetic centre, probably Ni(III). The hydrogenase was inhibited by heavy-metal salts, carbon monoxide and high ionic strength. However, it was resistant to inhibition by thiol-blocking and metal-complexing reagents. N-Bromosuccinimide totally inhibited the enzyme activity at low concentrations. The enzyme was stable to O2 over long periods and to high temperatures. It catalyses both H2-evolution and H2-uptake with a variety of artificial electron carriers. D. desulfuricans cytochrome C3, its natural electron carrier, had a high affinity for the enzyme (Km = 2 microns). Rate enhancement was observed when cytochrome C3 was added to Methyl Viologen in the H2-evolution assay. The pH optimum for H2-evolution was 6.5. PMID:6303306

  18. Detection of oocyte perivitelline membrane-bound sperm: a tool for avian collection management

    PubMed Central

    Croyle, Kaitlin E.; Durrant, Barbara S.; Jensen, Thomas

    2015-01-01

    The success and sustainability of an avian breeding programme depend on managing productive and unproductive pairs. Given that each breeding season can be of immeasurable importance, it is critical to resolve pair fertility issues quickly. Such problems are traditionally diagnosed through behavioural observations, egg lay history and hatch rates, with a decision to re-pair generally taking one or more breeding seasons. In pairs producing incubated eggs that show little or no signs of embryonic development, determining fertility is difficult. Incorporating a technique to assess sperm presence on the oocyte could, in conjunction with behaviour and other data, facilitate a more timely re-pair decision. Detection of perivitelline membrane-bound (PVM-bound) sperm verifies successful copulation, sperm production and sperm functionality. Alternatively, a lack of detectable sperm, at least in freshly laid eggs, suggests no mating, lack of sperm production/function or sperm–oviduct incompatibility. This study demonstrated PVM-bound sperm detection by Hoechst staining in fresh to 24-day-incubated exotic eggs from 39 species representing 13 orders. However, a rapid and significant time-dependent loss of detectable PVM-bound sperm was observed following incubation of chicken eggs. The PCR detection of sperm in seven species, including two bacterially infected eggs, demonstrated that this method was not as reliable as visual detection using Hoechst staining. The absence of amplicons in visually positive PVMs was presumably due to large PVM size and low sperm count, resulting in DNA concentrations too low for standard PCR detection. In summary, this study demonstrated the feasibility and limitations of using PVM-bound sperm detection as a management tool for exotic avian species. We verified that sperm presence or absence on fluorescence microscopy can aid in the differentiation of fertile from infertile eggs to assist breeding managers in making prompt decisions for pair

  19. Chelonian perivitelline membrane-bound sperm detection: A new breeding management tool.

    PubMed

    Croyle, Kaitlin; Gibbons, Paul; Light, Christine; Goode, Eric; Durrant, Barbara; Jensen, Thomas

    2016-01-01

    Perivitelline membrane (PVM)-bound sperm detection has recently been incorporated into avian breeding programs to assess egg fertility, confirm successful copulation, and to evaluate male reproductive status and pair compatibility. Due to the similarities between avian and chelonian egg structure and development, and because fertility determination in chelonian eggs lacking embryonic growth is equally challenging, PVM-bound sperm detection may also be a promising tool for the reproductive management of turtles and tortoises. This study is the first to successfully demonstrate the use of PVM-bound sperm detection in chelonian eggs. Recovered membranes were stained with Hoechst 33342 and examined for sperm presence using fluorescence microscopy. Sperm were positively identified for up to 206 days post-oviposition, following storage, diapause, and/or incubation, in 52 opportunistically collected eggs representing 12 species. However, advanced microbial infection frequently hindered the ability to detect membrane-bound sperm. Fertile Centrochelys sulcata, Manouria emys, and Stigmochelys pardalis eggs were used to evaluate the impact of incubation and storage on the ability to detect sperm. Storage at -20°C or in formalin were found to be the best methods for egg preservation prior to sperm detection. Additionally, sperm-derived mtDNA was isolated and PCR amplified from Astrochelys radiata, C. sulcata, and S. pardalis eggs. PVM-bound sperm detection has the potential to substantially improve studies of artificial incubation and sperm storage, and could be used to evaluate the success of artificial insemination in chelonian species. Mitochondrial DNA from PVM-bound sperm has applications for parentage analysis, the study of sperm competition, and potentially species identification.

  20. Purification and properties of a membrane-bound lytic transglycosylase from Escherichia coli.

    PubMed Central

    Ursinus, A; Höltje, J V

    1994-01-01

    A membrane-bound lytic transglycosylase (Mlt) has been solubilized in the presence of 2% Triton X-100 containing 0.5 M NaCl from membranes of an Escherichia coli mutant that carries a deletion in the slt gene coding for a 70-kDa soluble lytic transglycosylase (Slt70). The enzyme was purified by a four-step procedure including anion-exchange (HiLoad SP-Sepharose and MonoS), heparin-Sepharose, and poly(U)-Sepharose 4B column chromatography. The purified protein that migrated during denaturing sodium dodecyl sulfate-polyacrylamide gel electrophoresis as a single band corresponding to an apparent molecular mass of about 38 kDa is referred to as Mlt38. Optimal activity was found in buffers with a pH between 4.0 and 4.5. The enzyme is stimulated by a factor of 2.5 in the presence of Mg2+ at a concentration of 10 mM and loses its activity rapidly at temperatures above 30 degrees C. Besides insoluble murein sacculi, the enzyme was able to degrade glycan strands isolated from murein by amidase treatment. The enzymatic reaction occurred with a maximal velocity of about 2.2 mg/liter/min with murein sacculi as a substrate. The amino acid sequences of four proteolytic peptides showed no identity with known sequences in the data bank. With Mlt38, the number of proteins in E. coli showing lytic transglycosylase activity rises to three. Images PMID:8288527

  1. Chelonian perivitelline membrane-bound sperm detection: A new breeding management tool.

    PubMed

    Croyle, Kaitlin; Gibbons, Paul; Light, Christine; Goode, Eric; Durrant, Barbara; Jensen, Thomas

    2016-01-01

    Perivitelline membrane (PVM)-bound sperm detection has recently been incorporated into avian breeding programs to assess egg fertility, confirm successful copulation, and to evaluate male reproductive status and pair compatibility. Due to the similarities between avian and chelonian egg structure and development, and because fertility determination in chelonian eggs lacking embryonic growth is equally challenging, PVM-bound sperm detection may also be a promising tool for the reproductive management of turtles and tortoises. This study is the first to successfully demonstrate the use of PVM-bound sperm detection in chelonian eggs. Recovered membranes were stained with Hoechst 33342 and examined for sperm presence using fluorescence microscopy. Sperm were positively identified for up to 206 days post-oviposition, following storage, diapause, and/or incubation, in 52 opportunistically collected eggs representing 12 species. However, advanced microbial infection frequently hindered the ability to detect membrane-bound sperm. Fertile Centrochelys sulcata, Manouria emys, and Stigmochelys pardalis eggs were used to evaluate the impact of incubation and storage on the ability to detect sperm. Storage at -20°C or in formalin were found to be the best methods for egg preservation prior to sperm detection. Additionally, sperm-derived mtDNA was isolated and PCR amplified from Astrochelys radiata, C. sulcata, and S. pardalis eggs. PVM-bound sperm detection has the potential to substantially improve studies of artificial incubation and sperm storage, and could be used to evaluate the success of artificial insemination in chelonian species. Mitochondrial DNA from PVM-bound sperm has applications for parentage analysis, the study of sperm competition, and potentially species identification. PMID:26890048

  2. Breast Cancer Risk Reduction and Membrane-Bound Catechol O-Methyltransferase Genetic Polymorphisms

    PubMed Central

    Ji, Yuan; Olson, Janet; Zhang, Jianping; Hildebrandt, Michelle; Wang, Liewei; Ingle, James; Fredericksen, Zachary; Sellers, Thomas; Miller, William R.; Dixon, J. Michael; Brauch, Hiltrud; Eichelbaum, Michel; Justenhoven, Christina; Hamann, Ute; Ko, Yon; Brüning, Thomas; Chang-Claude, Jenny; Wang-Gohrke, Shan; Schaid, Daniel; Weinshilboum, Richard

    2008-01-01

    Catechol O-methyltransferase (COMT)-catalyzed methylation of catecholestrogens has been proposed to play a protective role in estrogen-induced genotoxic carcinogenesis. We have taken a comprehensive approach to test the hypothesis that genetic variation in COMT might influence breast cancer risk. Fifteen COMT SNPs selected on the basis of in-depth resequencing of the COMT gene were genotyped in 1482 DNA samples from a Mayo Clinic breast cancer case-control study. Two common SNPs in the distal promoter for membrane-bound (MB) COMT, rs2020917 and rs737865, were associated with breast cancer risk reduction in premenopausal women in the Mayo Clinic study, with allele-specific odds ratios of 0.70 (95% CI = 0.52–0.95) and 0.68 (95% CI = 0.51–0.92), respectively. These two SNPs were then subjected to functional genomic analysis and were genotyped in an additional 3683 DNA samples from two independent case-control studies (GENICA and GESBC). Functional genomic experiments showed that these SNPs could up-regulate transcription and that they altered DNA-protein binding patterns. Furthermore, substrate kinetic and exon array analyses suggested a role for MB-COMT in catecholestrogen inactivation. The GENICA results were similar to the Mayo case-control observations, with ORs of 0.85 (95% CI = 0.72–1.00) and 0.85 (95% CI = 0.72–1.01) for the two SNPs. No significant effect was observed in the GESBC study. These studies demonstrated that two SNPs in the COMT distal promoter were associated with breast cancer risk reduction in 2 of 3 case-control studies, compatible with the results of functional genomic experiments, suggesting a role for MB-COMT in breast cancer risk. PMID:18632656

  3. Coexistence of intracytoplasmic lumens and membrane-bound vesicles in an invasive carcinoma arising in a cystosarcoma phyllodes.

    PubMed

    Gilks, B; Tavassoli, F A

    1988-01-01

    An unusual invasive breast carcinoma, arising in a cystosarcoma phyllodes and characterized by a variable cytoplasmic appearance and mucin content, was evaluated to determine the nature of the secretory material within the cells as well as the type of secretory organelle at the ultrastructural level. Histochemical studies revealed both acidic (sialic acid) and neutral mucin within the tumor cells. Ultrastructural analysis revealed secretory material within membrane-bound vesicles in some cells and within intracytoplasmic lumens in others; some cells contained both membrane-bound vesicles and intracytoplasmic lumens simultaneously. The Golgi derivation of the intracytoplasmic lumens was supported by their presence within or near hyperplastic Golgi complexes. The histochemical characteristics of the secretory material is correlated with their ultrastructural site of accumulation.

  4. Activin A is essential for neurogenesis following neurodegeneration.

    PubMed

    Abdipranoto-Cowley, Andrea; Park, Jin Sung; Croucher, David; Daniel, James; Henshall, Susan; Galbraith, Sally; Mervin, Kyle; Vissel, Bryce

    2009-06-01

    It has long been proposed that excitotoxicity contributes to nerve cell death in neurodegenerative diseases. Activin A, a member of the transforming growth factor-beta superfamily, is expressed by neurons following excitotoxicity. We show for the first time that this activin A expression is essential for neurogenesis to proceed following neurodegeneration. We found that intraventricular infusion of activin A increased the number of newborn neurons in the dentate gyrus, CA3, and CA1 layers of the normal adult hippocampus and also, following lipopolysaccharide administration, had a potent inhibitory effect on gliosis in vivo and on microglial proliferation in vivo and in vitro. Consistent with the role of activin A in regulating central nervous system inflammation and neurogenesis, intraventricular infusion of follistatin, an activin A antagonist, profoundly impaired neurogenesis and increased the number of microglia and reactive astrocytes following onset of kainic acid-induced neurodegeneration. These results show that inhibiting endogenous activin A is permissive for a potent underlying inflammatory response to neurodegeneration. We demonstrate that the anti-inflammatory actions of activin A account for its neurogenic effects following neurodegeneration because co-administration of nonsteroidal anti-inflammatory drugs reversed follistatin's inhibitory effects on neurogenesis in vivo. Our work indicates that activin A, perhaps working in conjunction with other transforming growth factor-beta superfamily molecules, is essential for neurogenesis in the adult central nervous system following excitotoxic neurodegeneration and suggests that neurons can regulate regeneration by suppressing the inflammatory response, a finding with implications for understanding and treating acute and chronic neurodegenerative diseases. PMID:19489097

  5. Membrane-bound heat shock proteins facilitate the uptake of dying cells and cross-presentation of cellular antigen.

    PubMed

    Zhu, Haiyan; Fang, Xiaoyun; Zhang, Dongmei; Wu, Weicheng; Shao, Miaomiao; Wang, Lan; Gu, Jianxin

    2016-01-01

    Heat shock proteins (HSPs) were originally identified as stress-responsive proteins and serve as molecular chaperones in different intracellular compartments. Translocation of HSPs to the cell surface and release of HSPs into the extracellular space have been observed during the apoptotic process and in response to a variety of cellular stress. Here, we report that UV irradiation and cisplatin treatment rapidly induce the expression of membrane-bound Hsp60, Hsp70, and Hsp90 upstream the phosphatidylserine exposure. Membrane-bound Hsp60, Hsp70 and Hsp90 could promote the release of IL-6 and IL-1β as well as DC maturation by the evaluation of CD80 and CD86 expression. On the other hand, Hsp60, Hsp70 and Hsp90 on cells could facilitate the uptake of dying cells by bone marrow-derived dendritic cells. Lectin-like oxidized LDL receptor-1 (LOX-1), as a common receptor for Hsp60, Hsp70, and Hsp90, is response for their recognition and mediates the uptake of dying cells. Furthermore, membrane-bound Hsp60, Hsp70 and Hsp90 could promote the cross-presentation of OVA antigen from E.G7 cells and inhibition of the uptake of dying cells by LOX-1 decreases the cross-presentation of cellular antigen. Therefore, the rapid exposure of HSPs on dying cells at the early stage allows for the recognition by and confers an activation signal to the immune system. PMID:26481477

  6. Intracellular localization of membrane-bound ATPases in the compartmentalized anammox bacterium ‘Candidatus Kuenenia stuttgartiensis’

    PubMed Central

    van Niftrik, Laura; van Helden, Mary; Kirchen, Silke; van Donselaar, Elly G; Harhangi, Harry R; Webb, Richard I; Fuerst, John A; Op den Camp, Huub J M; Jetten, Mike S M; Strous, Marc

    2010-01-01

    Anaerobic ammonium-oxidizing (anammox) bacteria are divided into three compartments by bilayer membranes (from out- to inside): paryphoplasm, riboplasm and anammoxosome. It is proposed that the anammox reaction is performed by proteins located in the anammoxosome and on its membrane giving rise to a proton-motive-force and subsequent ATP synthesis by membrane-bound ATPases. To test this hypothesis, we investigated the location of membrane-bound ATPases in the anammox bacterium ‘Candidatus Kuenenia stuttgartiensis’. Four ATPase gene clusters were identified in the K. stuttgartiensis genome: one typical F-ATPase, two atypical F-ATPases and a prokaryotic V-ATPase. K. stuttgartiensis transcriptomic and proteomic analysis and immunoblotting using antisera directed at catalytic subunits of the ATPase gene clusters indicated that only the typical F-ATPase gene cluster most likely encoded a functional ATPase under these cultivation conditions. Immunogold localization showed that the typical F-ATPase was predominantly located on both the outermost and anammoxosome membrane and to a lesser extent on the middle membrane. This is consistent with the anammox physiology model, and confirms the status of the outermost cell membrane as cytoplasmic membrane. The occurrence of ATPase in the anammoxosome membrane suggests that anammox bacteria have evolved a prokaryotic organelle; a membrane-bounded compartment with a specific cellular function: energy metabolism. PMID:20545867

  7. Effect of bacoside A on membrane-bound ATPases in the brain of rats exposed to cigarette smoke.

    PubMed

    Anbarasi, K; Vani, G; Balakrishna, K; Devi, C S Shyamala

    2005-01-01

    Membrane-bound enzymes play a vital role in neuronal function through maintenance of membrane potential and impulse propagation. We have evaluated the harmful effects of chronic cigarette smoking on membrane-bound ATPases and the protective effect of Bacoside A in rat brain. Adult male albino rats were exposed to cigarette smoke for a period of 12 weeks and simultaneously administered with Bacoside A (the active principle isolated from Bacopa monniera) at a dosage of 10 mg/kg b.w/day, p.o. The levels of lipid peroxides as marker for evaluating the extent of membrane damage, the activities of Na+/K+-ATPase, Ca2+-ATPase and Mg2+-ATPase, and associated cations sodium (Na+), potassium (K+), calcium (Ca2+), and magnesium (Mg2+) were investigated in the brain. Neuronal membrane damage was evident from the elevated levels of lipid peroxides and decreased activities of membrane-bound enzymes. Disturbances in the electrolyte balance with accumulation of Na+ and Ca2+ and depletion of K+ and Mg2+ were also observed. Administration of Bacoside A inhibited lipid peroxidation, improved the activities of ATPases, and maintained the ionic equilibrium. The results of our study indicate that Bacoside A protects the brain from cigarette smoking induced membrane damage.

  8. Mechanism of biological denitrification inhibition: procyanidins induce an allosteric transition of the membrane-bound nitrate reductase through membrane alteration.

    PubMed

    Bardon, Clément; Poly, Franck; Piola, Florence; Pancton, Muriel; Comte, Gilles; Meiffren, Guillaume; Haichar, Feth el Zahar

    2016-05-01

    Recently, it has been shown that procyanidins from Fallopia spp. inhibit bacterial denitrification, a phenomenon called biological denitrification inhibition (BDI). However, the mechanisms involved in such a process remain unknown. Here, we investigate the mechanisms of BDI involving procyanidins, using the model strain Pseudomonas brassicacearum NFM 421. The aerobic and anaerobic (denitrification) respiration, cell permeability and cell viability of P. brassicacearum were determined as a function of procyanidin concentration. The effect of procyanidins on the bacterial membrane was observed using transmission electronic microscopy. Bacterial growth, denitrification, NO3- and NO2-reductase activity, and the expression of subunits of NO3- (encoded by the gene narG) and NO2-reductase (encoded by the gene nirS) under NO3 or NO2 were measured with and without procyanidins. Procyanidins inhibited the denitrification process without affecting aerobic respiration at low concentrations. Procyanidins also disturbed cell membranes without affecting cell viability. They specifically inhibited NO3- but not NO2-reductase.Pseudomonas brassicacearum responded to procyanidins by over-expression of the membrane-bound NO3-reductase subunit (encoded by the gene narG). Our results suggest that procyanidins can specifically inhibit membrane-bound NO3-reductase inducing enzymatic conformational changes through membrane disturbance and that P. brassicacearum responds by over-expressing membrane-bound NO3-reductase. Our results lead the way to a better understanding of BDI. PMID:26906096

  9. Krypton Derivatization of an O2 -Tolerant Membrane-Bound [NiFe] Hydrogenase Reveals a Hydrophobic Tunnel Network for Gas Transport.

    PubMed

    Kalms, Jacqueline; Schmidt, Andrea; Frielingsdorf, Stefan; van der Linden, Peter; von Stetten, David; Lenz, Oliver; Carpentier, Philippe; Scheerer, Patrick

    2016-04-25

    [NiFe] hydrogenases are metalloenzymes catalyzing the reversible heterolytic cleavage of hydrogen into protons and electrons. Gas tunnels make the deeply buried active site accessible to substrates and inhibitors. Understanding the architecture and function of the tunnels is pivotal to modulating the feature of O2 tolerance in a subgroup of these [NiFe] hydrogenases, as they are interesting for developments in renewable energy technologies. Here we describe the crystal structure of the O2 -tolerant membrane-bound [NiFe] hydrogenase of Ralstonia eutropha (ReMBH), using krypton-pressurized crystals. The positions of the krypton atoms allow a comprehensive description of the tunnel network within the enzyme. A detailed overview of tunnel sizes, lengths, and routes is presented from tunnel calculations. A comparison of the ReMBH tunnel characteristics with crystal structures of other O2 -tolerant and O2 -sensitive [NiFe] hydrogenases revealed considerable differences in tunnel size and quantity between the two groups, which might be related to the striking feature of O2 tolerance.

  10. Biosynthesis of cytochrome P-450 on membrane-bound ribosomes and its subsequent incorporation into rough and smooth microsomes in rat hepatocytes

    PubMed Central

    1979-01-01

    Intracellular sites of synthesis of cytochrome P-450 and the subsequent incorporation of it into membrane structures of the endoplasmic reticulum (ER) in rat hepatocytes have been studied using an antibody monospecific for phenobarbital-inducible cytochrome P-450. The cytochrome is synthesized mainly on the "tightly bound" type of membrane-bound ribosomes whose release from the membrane requires treatment with puromycin in a high salt buffer (500 mM KCI, 5mM MgCl2, and 50 mM Tris-HCL [pH 7.5]). Subsequently the cytochrome is incorporated directly into the rough ER membranes with its major part exposed to the outer surface to the membrane and accessible to proteolytic enzymes added externally. The newly synthesized molecules, which appeared first in the rough membrane, are translocated to the smooth membrane, and are then distributed evenly between the two types of microsomeal membranes in approximately 1 h. Administration of cycloheximide, an inhibitor of protein biosynthesis, did not significantly inhibit the transfer of the enzyme from the rough to the smooth ER. It is suggested, therefore, that the translocation of the newly synthesized cythochrome P-450 between the rough and smooth microsomes is mainly due to the lateral movement of the molecules in the plane of the membranes rather than to the attachment and detachment of the ribosomes on the microsomal membranes after the ribosomal cycle for protein synthesis. PMID:457773

  11. Peptide-conformed beta2m-free class I heavy chains are intermediates in generation of soluble HLA by the membrane-bound metalloproteinase.

    PubMed

    Demaria, S; DeVito-Haynes, L D; Salter, R D; Burlingham, W J; Bushkin, Y

    1999-12-01

    Molecular mechanisms of soluble HLA-release by a membrane-bound metalloproteinase (MPase) are not defined. We have investigated the possibility that certain beta2-microglobulin (beta2m)-free heavy chains (HC) retain peptide-induced conformations before and after the cleavage by using mutant HLA-A2.242K HC with reduced affinity for beta2m. We show that dissociation of HC/beta2m complexes on the surface of C1R lymphoblastoid cells generates both conformed and non-conformed beta2m-free HC recognized by conformation-dependent antibodies. Conformed HC, having bound the HLA-A2-specific peptide HTLV-1 tax 11-19, can retain their proper conformations after dissociation of beta2m. Further, conformed and non-conformed surface beta2m-free HC are cleaved by the MPase, and some released HC preserve their conformations. Exogenous beta2m binds only to conformed HC, and protects them from cleavage as effectively as the MPase inhibitor BB-2116. We propose that soluble HLA-release requires generation of peptide-conformed beta2m-free HC intermediates on the cell surface, which are then cleaved by the MPase and in solution may reassociate with beta2m. Given the role of soluble HLA in the indirect allorecognition, the activity of this MPase may be important in transplant rejection. PMID:10626735

  12. Krypton Derivatization of an O2 -Tolerant Membrane-Bound [NiFe] Hydrogenase Reveals a Hydrophobic Tunnel Network for Gas Transport.

    PubMed

    Kalms, Jacqueline; Schmidt, Andrea; Frielingsdorf, Stefan; van der Linden, Peter; von Stetten, David; Lenz, Oliver; Carpentier, Philippe; Scheerer, Patrick

    2016-04-25

    [NiFe] hydrogenases are metalloenzymes catalyzing the reversible heterolytic cleavage of hydrogen into protons and electrons. Gas tunnels make the deeply buried active site accessible to substrates and inhibitors. Understanding the architecture and function of the tunnels is pivotal to modulating the feature of O2 tolerance in a subgroup of these [NiFe] hydrogenases, as they are interesting for developments in renewable energy technologies. Here we describe the crystal structure of the O2 -tolerant membrane-bound [NiFe] hydrogenase of Ralstonia eutropha (ReMBH), using krypton-pressurized crystals. The positions of the krypton atoms allow a comprehensive description of the tunnel network within the enzyme. A detailed overview of tunnel sizes, lengths, and routes is presented from tunnel calculations. A comparison of the ReMBH tunnel characteristics with crystal structures of other O2 -tolerant and O2 -sensitive [NiFe] hydrogenases revealed considerable differences in tunnel size and quantity between the two groups, which might be related to the striking feature of O2 tolerance. PMID:26913499

  13. Activins and inhibins: Novel regulators of thymocyte development

    SciTech Connect

    Licona-Limon, Paula; Aleman-Muench, German; Macias-Silva, Marina; Garcia-Zepeda, Eduardo A.; Fortoul, Teresa I.; Soldevila, Gloria

    2009-04-03

    Activins and inhibins are members of the transforming growth factor-{beta} superfamily that act on different cell types and regulate a broad range of cellular processes including proliferation, differentiation, and apoptosis. Here, we provide the first evidence that activins and inhibins regulate specific checkpoints during thymocyte development. We demonstrate that both activin A and inhibin A promote the DN3-DN4 transition in vitro, although they differentially control the transition to the DP stage. Whereas activin A induces the accumulation of a CD8{sup +}CD24{sup hi}TCR{beta}{sup lo} intermediate subpopulation, inhibin A promotes the differentiation of DN4 to DP. In addition, both activin A and inhibin A appear to promote CD8{sup +}SP differentiation. Moreover, inhibin {alpha} null mice have delayed in vitro T cell development, showing both a decrease in the DN-DP transition and reduced thymocyte numbers, further supporting a role for inhibins in the control of developmental signals taking place during T cell differentiation in vivo.

  14. Activins and Inhibins: novel regulators of thymocyte development

    PubMed Central

    Licona-Limón, Paula; Alemán-Muench, German; Chimal-Monroy, Jesus; Macías-Silva, Marina; García-Zepeda, Eduardo A.; Matzuk, Martin M.; Fortoul, Teresa I.; Soldevila, Gloria

    2009-01-01

    Activins and inhibins are members of the transforming growth factor β superfamily that act on different cell types and regulate a broad range of cellular processes including proliferation, differentiation, and apoptosis. Here, we provide the first evidence that activins and inhibins regulate specific checkpoints during thymocyte development. We demonstrate that both activin A and inhibin A promote the DN3-DN4 transition in vitro, although they differentially control the transition to the DP stage. Whereas activin A induces the accumulation of a CD8+CD24hiTCRβlo intermediate subpopulation, inhibin A promotes the differentiation of DN4 to DP. In addition, both activin A and inhibin A appear to promote CD8+SP differentiation Moreover, Inhibin α null mice have delayed in vitro T cell development, showing both a decrease in the DN-DP transition and reduced thymocyte numbers, further supporting a role for inhibins in the control of developmental signals taking place during T cell differentiation in vivo. PMID:19338778

  15. Structural features of membrane-bound glucocerebrosidase and α-synuclein probed by neutron reflectometry and fluorescence spectroscopy.

    PubMed

    Yap, Thai Leong; Jiang, Zhiping; Heinrich, Frank; Gruschus, James M; Pfefferkorn, Candace M; Barros, Marilia; Curtis, Joseph E; Sidransky, Ellen; Lee, Jennifer C

    2015-01-01

    Mutations in glucocerebrosidase (GCase), the enzyme deficient in Gaucher disease, are a common genetic risk factor for the development of Parkinson disease and related disorders, implicating the role of this lysosomal hydrolase in the disease etiology. A specific physical interaction exists between the Parkinson disease-related protein α-synuclein (α-syn) and GCase both in solution and on the lipid membrane, resulting in efficient enzyme inhibition. Here, neutron reflectometry was employed as a first direct structural characterization of GCase and α-syn·GCase complex on a sparsely-tethered lipid bilayer, revealing the orientation of the membrane-bound GCase. GCase binds to and partially inserts into the bilayer with its active site most likely lying just above the membrane-water interface. The interaction was further characterized by intrinsic Trp fluorescence, circular dichroism, and surface plasmon resonance spectroscopy. Both Trp fluorescence and neutron reflectometry results suggest a rearrangement of loops surrounding the catalytic site, where they extend into the hydrocarbon chain region of the outer leaflet. Taking advantage of contrasting neutron scattering length densities, the use of deuterated α-syn versus protiated GCase showed a large change in the membrane-bound structure of α-syn in the complex. We propose a model of α-syn·GCase on the membrane, providing structural insights into inhibition of GCase by α-syn. The interaction displaces GCase away from the membrane, possibly impeding substrate access and perturbing the active site. GCase greatly alters membrane-bound α-syn, moving helical residues away from the bilayer, which could impact the degradation of α-syn in the lysosome where these two proteins interact.

  16. Structural Features of Membrane-bound Glucocerebrosidase and α-Synuclein Probed by Neutron Reflectometry and Fluorescence Spectroscopy*

    PubMed Central

    Yap, Thai Leong; Jiang, Zhiping; Heinrich, Frank; Gruschus, James M.; Pfefferkorn, Candace M.; Barros, Marilia; Curtis, Joseph E.; Sidransky, Ellen; Lee, Jennifer C.

    2015-01-01

    Mutations in glucocerebrosidase (GCase), the enzyme deficient in Gaucher disease, are a common genetic risk factor for the development of Parkinson disease and related disorders, implicating the role of this lysosomal hydrolase in the disease etiology. A specific physical interaction exists between the Parkinson disease-related protein α-synuclein (α-syn) and GCase both in solution and on the lipid membrane, resulting in efficient enzyme inhibition. Here, neutron reflectometry was employed as a first direct structural characterization of GCase and α-syn·GCase complex on a sparsely-tethered lipid bilayer, revealing the orientation of the membrane-bound GCase. GCase binds to and partially inserts into the bilayer with its active site most likely lying just above the membrane-water interface. The interaction was further characterized by intrinsic Trp fluorescence, circular dichroism, and surface plasmon resonance spectroscopy. Both Trp fluorescence and neutron reflectometry results suggest a rearrangement of loops surrounding the catalytic site, where they extend into the hydrocarbon chain region of the outer leaflet. Taking advantage of contrasting neutron scattering length densities, the use of deuterated α-syn versus protiated GCase showed a large change in the membrane-bound structure of α-syn in the complex. We propose a model of α-syn·GCase on the membrane, providing structural insights into inhibition of GCase by α-syn. The interaction displaces GCase away from the membrane, possibly impeding substrate access and perturbing the active site. GCase greatly alters membrane-bound α-syn, moving helical residues away from the bilayer, which could impact the degradation of α-syn in the lysosome where these two proteins interact. PMID:25429104

  17. pH-induced conformational changes of membrane-bound influenza hemagglutinin and its effect on target lipid bilayers.

    PubMed Central

    Gray, C.; Tamm, L. K.

    1998-01-01

    Influenza virus hemagglutinin (HA) has served as a paradigm for both pH-dependent and -independent viral membrane fusion. Although large conformational changes were observed by X-ray crystallography when soluble fragments of HA were subjected to fusion-pH conditions, it is not clear whether the same changes occur in membrane-bound HA, what the spatial relationship is between the conformationally changed HA and the target and viral membranes, and in what way HA perturbs the target membrane at low pH. We have taken a spectroscopic approach using an array of recently developed FTIR techniques to address these questions. Difference attenuated total reflection FTIR spectroscopy was employed to reveal reversible and irreversible components of the pH-induced conformational change of the membrane-bound bromelain fragment of HA, BHA. Additional proteolytic fragments of BHA were produced which permitted a tentative assignment of the observed changes to the HA1 and HA2 subunits, respectively. The membrane-bound HA1 subunit undergoes a reversible conformational change, which most likely involves the loss of a small proportion of beta-sheet at low pH. BHA was found to undergo a partially reversible tilting motion relative to the target membrane upon exposure to pH 5, indicating a previously undescribed hinge near the anchoring point to the target membrane. Time-resolved amide H/D exchange experiments revealed a more dynamic (tertiary) structure of membrane-bound BHA and its HA2, but not its HA1, subunit. Finally BHA and, to a lesser degree, HA1 perturbed the lipid bilayer of the target membrane at the interface, as assessed by spectral changes of the lipid ester carbonyl groups. These results are discussed in the context of a complementary study of HA that was bound to viral membranes through its transmembrane peptide (Gray C, Tamm LK, 1997, Protein Sci 6:1993-2006). A distinctive role for the HA1 subunit in the conformational change of HA becomes apparent from these combined

  18. Membrane-bound p35 Subunit of IL-12 on Tumor Cells is Functionally Equivalent to Membrane-bound Heterodimeric Single Chain IL-12 for Induction of Anti-tumor Immunity

    PubMed Central

    Kim, Hyun-Jin; Park, Sang Min

    2016-01-01

    In this study, we compared two different tumor cell vaccines for their induction of anti-tumor immunity; one was a tumor cell clone expressing a membrane-bound form of IL-12 p35 subunit (mbIL-12 p35 tumor clone), and the other was a tumor clone expressing heterodimeric IL-12 as a single chain (mb-scIL-12 tumor clone). The stimulatory effect of mb-scIL-12 on the proliferation of ConA-activated splenocytes was higher than that of mbIL-12 p35 in vitro. However, the stimulatory effect of mbIL-12 p35 was equivalent to that of recombinant soluble IL-12 (3 ng/ml). Interestingly, both tumor clones (mbIL-12 p35 and mb-scIL-12) showed similar tumorigenicity and induction of systemic anti-tumor immunity in vivo, suggesting that tumor cell expression of the membrane-bound p35 subunit is sufficient to induce anti-tumor immunity in our tumor vaccine model. PMID:27799876

  19. Splitting placodes: effects of bone morphogenetic protein and Activin on the patterning and identity of mouse incisors.

    PubMed

    Munne, Pauliina M; Felszeghy, Szabolcs; Jussila, Maria; Suomalainen, Marika; Thesleff, Irma; Jernvall, Jukka

    2010-01-01

    The single large rodent incisor in each jaw quadrant is evolutionarily derived from a mammalian ancestor with many small incisors. The embryonic placode giving rise to the mouse incisor is considerably larger than the molar placode, and the question remains whether this large incisor placode is a developmental requisite to make a thick incisor. Here we used in vitro culture system to experiment with the molecular mechanism regulating tooth placode development and how mice have thick incisors. We found that large placodes are prone to disintegration and formation of two to three small incisor placodes. The balance between one large or multiple small placodes was altered through the regulation of bone morphogenetic protein (BMP) and Activin signaling. Exogenous Noggin, which inhibits BMP signaling, or exogenous Activin cause the development of two to three incisors. These incisors were more slender than normal incisors. Additionally, two inhibitor molecules, Sostdc1 and Follistatin, which regulate the effects of BMPs and Activin and have opposite expression patterns, are likely to be involved in the incisor placode regulation in vivo. Furthermore, inhibition of BMPs by recombinant Noggin has been previously suggested to cause a change in the tooth identity from the incisor to the molar. This evidence has been used to support a homeobox code in determining tooth identity. Our work provides an alternative interpretation, where the inhibition of BMP signaling can lead to splitting of the large incisor placode and the formation of partly separate incisors, thereby acquiring molar-like morphology without a change in tooth identity.

  20. A Soluble Activin Receptor Type IIB Does Not Improve Blood Glucose in Streptozotocin-Treated Mice

    PubMed Central

    Wang, Qian; Guo, Tingqing; Portas, Jennifer; McPherron, Alexandra C.

    2015-01-01

    Type 1 diabetes mellitus (T1DM), or insulin dependent DM, is accompanied by decreased muscle mass. The growth factor myostatin (MSTN) is a negative regulator of muscle growth, and a loss of MSTN signaling has been shown to increase muscle mass and prevent the development of obesity, insulin resistance and lipodystrophic diabetes in mice. The effects of MSTN inhibition in a T1DM model on muscle mass and blood glucose are unknown. We asked whether MSTN inhibition would increase muscle mass and decrease hyperglycemia in mice treated with streptozotocin (STZ) to destroy pancreatic beta cells. After diabetes developed, mice were treated with a soluble MSTN/activin receptor fused to Fc (ACVR2B:Fc). ACVR2B:Fc increased body weight and muscle mass compared to vehicle treated mice. Unexpectedly, ACVR2B:Fc reproducibly exacerbated hyperglycemia within approximately one week of administration. ACVR2B:Fc treatment also elevated serum levels of the glucocorticoid corticosterone. These results suggest that although MSTN/activin inhibitors increased muscle mass, they may be counterproductive in improving health in patients with T1DM. PMID:25561902

  1. Activation of signalling by the activin receptor complex.

    PubMed Central

    Attisano, L; Wrana, J L; Montalvo, E; Massagué, J

    1996-01-01

    Activin exerts its effects by simultaneously binding to two types of p rotein serine/threonine kinase receptors, each type existing in various isoforms. Using the ActR-IB and ActR-IIB receptor isoforms, we have investigated the mechanism of activin receptor activation. ActR-IIB are phosphoproteins with demonstrable affinity for each other. However, activin addition strongly promotes an interaction between these two proteins. Activin binds directly to ActR-IIB, and this complex associates with ActR-IB, which does not bind ligand on its own. In the resulting complex, ActR-IB becomes hyperphosphorylated, and this requires the kinase activity of ActR-IIB. Mutation of conserved serines and threonines in the GS domain, a region just upstream of the kinase domain in ActR-IB, abrogates both phosphorylation and signal propagation, suggesting that this domain contains phosphorylation sites required for signalling. ActR-IB activation can be mimicked by mutation of Thr-206 to aspartic acid, which yields a construct, ActR-IB(T206D), that signals in the absence of ligand. Furthermore, the signalling activity of this mutant construct is undisturbed by overexpression of a dominant negative kinase-defective ActR-IIB construct, indicating that ActR-IB(T206D) can signal independently of ActR-IIB. The evidence suggests that ActR-IIB acts as a primary activin receptor and ActR-IB acts as a downstream transducer of activin signals. PMID:8622651

  2. Contribution of liver mitochondrial membrane-bound glutathione transferase to mitochondrial permeability transition pores

    SciTech Connect

    Hossain, Quazi Sohel; Ulziikhishig, Enkhbaatar; Lee, Kang Kwang; Yamamoto, Hideyuki; Aniya, Yoko

    2009-02-15

    We recently reported that the glutathione transferase in rat liver mitochondrial membranes (mtMGST1) is activated by S-glutathionylation and the activated mtMGST1 contributes to the mitochondrial permeability transition (MPT) pore and cytochrome c release from mitochondria [Lee, K.K., Shimoji, M., Quazi, S.H., Sunakawa, H., Aniya, Y., 2008. Novel function of glutathione transferase in rat liver mitochondrial membrane: role for cytochrome c release from mitochondria. Toxcol. Appl. Pharmacol. 232, 109-118]. In the present study we investigated the effect of reactive oxygen species (ROS), generator gallic acid (GA) and GST inhibitors on mtMGST1 and the MPT. When rat liver mitochondria were incubated with GA, mtMGST1 activity was increased to about 3 fold and the increase was inhibited with antioxidant enzymes and singlet oxygen quenchers including 1,4-diazabicyclo [2,2,2] octane (DABCO). GA-mediated mtMGST1 activation was prevented by GST inhibitors such as tannic acid, hematin, and cibacron blue and also by cyclosporin A (CsA). In addition, GA induced the mitochondrial swelling which was also inhibited by GST inhibitors, but not by MPT inhibitors CsA, ADP, and bongkrekic acid. GA also released cytochrome c from the mitochondria which was inhibited completely by DABCO, moderately by GST inhibitors, and somewhat by CsA. Ca{sup 2+}-mediated mitochondrial swelling and cytochrome c release were inhibited by MPT inhibitors but not by GST inhibitors. When the outer mitochondrial membrane was isolated after treatment of mitochondria with GA, mtMGST1 activity was markedly increased and oligomer/aggregate of mtMGST1 was observed. These results indicate that mtMGST1 in the outer mitochondrial membrane is activated by GA through thiol oxidation leading to protein oligomerization/aggregation, which may contribute to the formation of ROS-mediated, CsA-insensitive MPT pore, suggesting a novel mechanism for regulation of the MPT by mtMGST1.

  3. Inhibins and activins in human ovulation, conception and pregnancy.

    PubMed

    Lockwood, G M; Muttukrishna, S; Ledger, W L

    1998-01-01

    The activins and inhibins are glycoproteins that belong to the transforming growth factor-beta superfamily and, as such, have diverse effects at many stages during growth and development. Originally identified by their effects on follicle stimulating hormone in males and females, the recent development of specific and sensitive assays for this group of polypeptides has permitted the elucidation of their role in 'fine-tuning' the hypothalamic-pituitary-gonadal axis. This review article focuses on the roles of inhibin and activin in female reproductive physiology with reference to possible future clinical applications in the investigation of infertility and abnormal pregnancy.

  4. Molecular properties of membrane-bound FAD-containing D-sorbitol dehydrogenase from thermotolerant Gluconobacter frateurii isolated from Thailand.

    PubMed

    Toyama, Hirohide; Soemphol, Wichai; Moonmangmee, Duangtip; Adachi, Osao; Matsushita, Kazunobu

    2005-06-01

    There are two types of membrane-bound D-sorbitol dehydrogenase (SLDH) reported: PQQ-SLDH, having pyrroloquinoline quinone (PQQ), and FAD-SLDH, containing FAD and heme c as the prosthetic groups. FAD-SLDH was purified and characterized from the PQQ-SLDH mutant strain of a thermotolerant Gluconobacter frateurii, having molecular mass of 61.5 kDa, 52 kDa, and 22 kDa. The enzyme properties were quite similar to those of the enzyme from mesophilic G. oxydans IFO 3254. This enzyme was shown to be inducible by D-sorbitol, but not PQQ-SLDH. The oxidation product of FAD-SLDH from D-sorbitol was identified as L-sorbose. The cloned gene of FAD-SLDH had three open reading frames (sldSLC) corresponding to the small, the large, and cytochrome c subunits of FAD-SLDH respectively. The deduced amino acid sequences showed high identity to those from G. oxydans IFO 3254: SldL showed to other FAD-enzymes, and SldC having three heme c binding motives to cytochrome c subunits of other membrane-bound dehydrogenases.

  5. Dietary Chitosan Supplementation Ameliorates Isoproterenol-Induced Aberrations in Membrane-Bound ATPases and Mineral Status of Rat Myocardium.

    PubMed

    Anandan, Rangasamy; Chatterjee, Niladri Sekhar; Sivakumar, Ramalingam; Mathew, Suseela; Asha, Kurukkan Kunnath; Ganesan, Balaraman

    2015-09-01

    Myocardial infarction is one of the major public concerns in both developed and developing countries. Recently, there is growing interest in potential healthcare applications of marine natural products in the field of cardiovascular research. In the present study, we have examined the membrane-stabilizing potential of marine mucopolysaccharide-chitosan in modulating the aberrations of thiol-dependent membrane-bound ATPases activities, mineral status, and cardiac diagnostic markers in isoproterenol-induced myocardial infarction condition in rats. Dietary intake of chitosan significantly (p < 0.05) counteracted the isoproterenol-induced lipid peroxidation and maintained the levels of thiol contents and cardiac biomarkers at concentrations analogous to that of normal controls in the rat myocardium. Chitosan administration also significantly mitigated isoproterenol-induced aberrations in the membrane-bound ATPase activities in the heart tissue and preserved the myocardial mineral status in serum and heart tissue of experimental rats at near normal value. The results of the present study have indicated that the salubrious effect of dietary chitosan supplementation in attenuating the experimentally induced myocardial infarction condition is probably ascribable to its antioxidant defense and membrane-stabilizing properties.

  6. Roles of a membrane-bound caleosin and putative peroxygenase in biotic and abiotic stress responses in Arabidopsis.

    PubMed

    Partridge, Mark; Murphy, Denis J

    2009-09-01

    We report here the localisation and properties of a new membrane-bound isoform of caleosin and its putative role as a peroxygenase involved in oxylipin metabolism during biotic and abiotic stress responses in Arabidopsis. Caleosins are a family of lipid-associated proteins that are ubiquitous in plants and true fungi. Previous research has focused on lipid-body associated, seed-specific caleosins that have peroxygenase activity. Here, we demonstrate that a separate membrane-bound constitutively expressed caleosin isoform (Clo-3) is highly upregulated following exposure to abiotic stresses, such as salt and drought, and to biotic stress such as pathogen infection. The Clo-3 protein binds one atom of calcium per molecule, is phosphorylated in response to stress, and has a similar peroxygenase activity to the seed-specific Clo-1 isoform. Clo-3 is present in microsomal and chloroplast envelope fractions and has a type I membrane orientation with about 2 kDa of the C terminal exposed to the cytosol. Analysis of Arabidopsis ABA and related mutant lines implies that Clo-3 is involved in the generation of oxidised fatty acids in stress related signalling pathways involving both ABA and salicylic acid. We propose that Clo-3 is part of an oxylipin pathway induced by multiple stresses and may also generate fatty acid derived anti-fungal compounds for plant defence. PMID:19467604

  7. X-ray structure of the membrane-bound cytochrome c quinol dehydrogenase NrfH reveals novel haem coordination

    PubMed Central

    Rodrigues, Maria Luisa; Oliveira, Tânia F; Pereira, Inês A C; Archer, Margarida

    2006-01-01

    Oxidation of membrane-bound quinol molecules is a central step in the respiratory electron transport chains used by biological cells to generate ATP by oxidative phosphorylation. A novel family of cytochrome c quinol dehydrogenases that play an important role in bacterial respiratory chains was recognised in recent years. Here, we describe the first structure of a cytochrome from this family, NrfH from Desulfovibrio vulgaris, which forms a stable complex with its electron partner, the cytochrome c nitrite reductase NrfA. One NrfH molecule interacts with one NrfA dimer in an asymmetrical manner, forming a large membrane-bound complex with an overall α4β2 quaternary arrangement. The menaquinol-interacting NrfH haem is pentacoordinated, bound by a methionine from the CXXCHXM sequence, with an aspartate residue occupying the distal position. The NrfH haem that transfers electrons to NrfA has a lysine residue from the closest NrfA molecule as distal ligand. A likely menaquinol binding site, containing several conserved and essential residues, is identified. PMID:17139260

  8. A drug delivery hydrogel system based on activin B for Parkinson's disease.

    PubMed

    Li, Juan; Darabi, Mohammadali; Gu, Jingjing; Shi, Junbin; Xue, Jinhua; Huang, Lu; Liu, Yutong; Zhang, Lei; Liu, N; Zhong, Wen; Zhang, Lin; Xing, Malcolm; Zhang, Lu

    2016-09-01

    Parkinson's disease (PD) is one of the most common neurodegenerative diseases. Activins are members of the superfamily of transforming growth factors and have many potential neuroprotective effects. Herein, at the first place, we verified activin B's neuroprotective role in a PD model, and revealed that activin B's fast release has limited function in the PD therapy. To this end, we developed a multi-functional crosslinker based thermosensitive injectable hydrogels to deliver activin B, and stereotactically injected the activin B-loaded hydrogel into the striatum of a mouse model of PD. The histological evaluation showed that activin B can be detected even 5 weeks post-surgery in PD mice implanted with activin B-loaded hydrogels, and activin B-loaded hydrogels can significantly increase the density of tyrosine hydroxylase positive (TH(+)) nerve fibers and reduce inflammatory responses. The behavioral evaluation demonstrated that activin B-loaded hydrogels significantly improved the performance of the mice in the PD model. Meanwhile, we found that hydrogels can slightly induce the activation of microglia cells and astrocytes, while cannot induce apoptosis in the striatum. Overall, our data demonstrated that the developed activin B-loaded hydrogels provide sustained release of activin B for over 5 weeks and contribute to substantial cellular protection and behavioral improvement, suggesting their potential as a therapeutic strategy for PD.

  9. Intertwining of Activin A and TGFβ Signaling: Dual Roles in Cancer Progression and Cancer Cell Invasion

    PubMed Central

    Loomans, Holli A.; Andl, Claudia D.

    2014-01-01

    In recent years, a significant amount of research has examined the controversial role of activin A in cancer. Activin A, a member of the transforming growth factor β (TGFβ) superfamily, is best characterized for its function during embryogenesis in mesoderm cell fate differentiation and reproduction. During embryogenesis, TGFβ superfamily ligands, TGFβ, bone morphogenic proteins (BMPs) and activins, act as potent morphogens. Similar to TGFβs and BMPs, activin A is a protein that is highly systemically expressed during early embryogenesis; however, post-natal expression is overall reduced and remains under strict spatiotemporal regulation. Of importance, normal post-natal expression of activin A has been implicated in the migration and invasive properties of various immune cell types, as well as endometrial cells. Aberrant activin A signaling during development results in significant morphological defects and premature mortality. Interestingly, activin A has been found to have both oncogenic and tumor suppressor roles in cancer. Investigations into the role of activin A in prostate and breast cancer has demonstrated tumor suppressive effects, while in lung and head and neck squamous cell carcinoma, it has been consistently shown that activin A expression is correlated with increased proliferation, invasion and poor patient prognosis. Activin A signaling is highly context-dependent, which is demonstrated in studies of epithelial cell tumors and the microenvironment. This review discusses normal activin A signaling in comparison to TGFβ and highlights how its dysregulation contributes to cancer progression and cell invasion. PMID:25560921

  10. XSmad2 directly activates the activin-inducible, dorsal mesoderm gene XFKH1 in Xenopus embryos.

    PubMed Central

    Howell, M; Hill, C S

    1997-01-01

    Transforming growth factor (TGF)-beta family members play a central role in mesoderm induction during early embryogenesis in Xenopus. Although a number of target genes induced as an immediate-early response to activin-like members of the family have been described, little is known about the molecular mechanisms involved. Our systematic analysis of the activin induction of the target gene XFKH1 reveals two regions that mediate activin-responsive transcription: one, in the first intron, is targeted directly by the activin-signalling pathway; the other, in the 5' flanking sequences, responds to activin indirectly, possibly being required for maintenance of gene expression. We demonstrate that a 107 bp region of the XFKH1 first intron acts as an enhancer and confers activin inducibility onto a minimal uninducible promoter in the absence of new protein synthesis. It bears little sequence similarity to other activin responsive sequences. We further demonstrate that overexpression of a constitutively active derivative of Xenopus Smad2 (XSmad2), which has been implicated as a component of the activin signalling pathway, is sufficient for direct activation of transcription via this enhancer. Moreover, we show that XSmad2 acts indirectly on the proximal promoter element induced by activin via an indirect mechanism. These results establish the XFKH1 intron enhancer as a direct nuclear target of the activin signalling pathway in Xenopus embryos, and provide strong new evidence that XSmad2 is a transducer of activin signals. PMID:9405370

  11. Structure and activation of pro-activin A

    PubMed Central

    Wang, Xuelu; Fischer, Gerhard; Hyvönen, Marko

    2016-01-01

    Activins are growth factors with multiple roles in the development and homeostasis. Like all TGF-β family of growth factors, activins are synthesized as large precursors from which mature dimeric growth factors are released proteolytically. Here we have studied the activation of activin A and determined crystal structures of the unprocessed precursor and of the cleaved pro-mature complex. Replacing the natural furin cleavage site with a HRV 3C protease site, we show how the protein gains its bioactivity after proteolysis and is as active as the isolated mature domain. The complex remains associated in conditions used for biochemical analysis with a dissociation constant of 5 nM, but the pro-domain can be actively displaced from the complex by follistatin. Our high-resolution structures of pro-activin A share features seen in the pro-TGF-β1 and pro-BMP-9 structures, but reveal a new oligomeric arrangement, with a domain-swapped, cross-armed conformation for the protomers in the dimeric protein. PMID:27373274

  12. Structure and activation of pro-activin A.

    PubMed

    Wang, Xuelu; Fischer, Gerhard; Hyvönen, Marko

    2016-01-01

    Activins are growth factors with multiple roles in the development and homeostasis. Like all TGF-β family of growth factors, activins are synthesized as large precursors from which mature dimeric growth factors are released proteolytically. Here we have studied the activation of activin A and determined crystal structures of the unprocessed precursor and of the cleaved pro-mature complex. Replacing the natural furin cleavage site with a HRV 3C protease site, we show how the protein gains its bioactivity after proteolysis and is as active as the isolated mature domain. The complex remains associated in conditions used for biochemical analysis with a dissociation constant of 5 nM, but the pro-domain can be actively displaced from the complex by follistatin. Our high-resolution structures of pro-activin A share features seen in the pro-TGF-β1 and pro-BMP-9 structures, but reveal a new oligomeric arrangement, with a domain-swapped, cross-armed conformation for the protomers in the dimeric protein. PMID:27373274

  13. Full-length membrane-bound tumor necrosis factor-α acts through tumor necrosis factor receptor 2 to modify phenotype of sensory neurons.

    PubMed

    Wu, Zetang; Wang, Shiyong; Gruber, Sandy; Mata, Marina; Fink, David J

    2013-09-01

    Neuropathic pain resulting from spinal hemisection or selective spinal nerve ligation is characterized by an increase in membrane-bound tumor necrosis factor-alpha (mTNFα) in spinal microglia without detectable release of soluble TNFα (sTNFα). In tissue culture, we showed that a full-length transmembrane cleavage-resistant TNFα (CRTNFα) construct can act through cell-cell contact to activate neighboring microglia. We undertook the current study to test the hypothesis that mTNFα expressed in microglia might also affect the phenotype of primary sensory afferents, by determining the effect of CRTNFα expressed from COS-7 cells on gene expression in primary dorsal root ganglia (DRG) neurons. Co-culture of DRG neurons with CRTNFα-expressing COS-7 cells resulted in a significant increase in the expression of voltage-gated sodium channel isoforms NaV1.7 and NaV1.8, and voltage-gated calcium channel subunit CaV3.2 at both mRNA and protein levels, and enhanced CCL2 expression and release from the DRG neurons. Exposure to sTNFα produced an increase only in CCL2 expression and release. Treatment of the cells with an siRNA against tumor necrosis factor receptor 2 (TNFR2) significantly reduced CRTNFα-induced gene expression changes in DRG neurons, whereas administration of CCR2 inhibitor had no significant effect on CRTNFα-induced increase in gene expression and CCL2 release in DRG neurons. Taken together, the results of this study suggest that mTNFα expressed in spinal microglia can facilitate pain signaling by up-regulating the expression of cation channels and CCL2 in DRG neurons in a TNFR2-dependent manner. PMID:23711481

  14. Concentration and function of membrane-bound cytochromes in cyanobacterial heterocysts

    SciTech Connect

    Houchins, J.P.; Hind, G.

    1984-10-01

    Membranes isolated from heterocysts and vegetative cells of Anabaena 7120 were assayed for content of cytochrome f, cytochrome b-563, cytochrome b-559/sub HP/, cytochrome b-559/sub LP/, and cytochrome aa/sub 3/ by use of reduced-minus-oxidized difference spectra. The level of cytochrome aa/sub 3/ in heterocyst membranes was 4 to 100 times higher than that in vegetative cells of Anabaena 7120 or other species of cyanobacteria. Heterocyst membranes lack cytochrome b-559/sub HP/ but contain cytochrome b-559/sub LP/ (E/sub m7.5/ = +77 millivolts, n = 1) at approximately the same concentration as cytochrome f. The role of cytochrome b-559/sub LP/ in the hydrogenase-dependent electron transfer pathway was investigated with the inhibitor 2-(n-heptyl)-4-hydroxyquinoline N-oxide which blocks electron flow from hydrogenase to acceptors reacting with the plastoquinone pool. Addition of inhibitor elicited no change in the reduction level of cytochrome b-559/sub LP/ indicating that this cytochrome is not directly involved in this pathway. 30 references, 6 figures, 1 table.

  15. Seminal fluid factors regulate activin A and follistatin synthesis in female cervical epithelial cells.

    PubMed

    Sharkey, David J; Schjenken, John E; Mottershead, David G; Robertson, Sarah A

    2015-12-01

    Seminal fluid induces pro-inflammatory cytokines and elicits an inflammation-like response in the cervix. Here, Affymetrix microarray and qPCR was utilised to identify activin A (INHBA) and its inhibitor follistatin (FST) amongst the cytokines induced by seminal plasma in Ect1 ectocervical epithelial cells, and a similar response was confirmed in primary ectocervical epithelial cells. TGFB is abundant in seminal plasma and all three TGFB isoforms induced INHBA in Ect1 and primary cells, and neutralisation of TGFB in seminal plasma suppressed the INHBA response. Bacterial lipopolysaccharide in seminal plasma also elicited INHBA, but potently suppressed FST production. There was moderate reciprocal inhibition between FST and INHBA, and cross-attenuating effects were seen. These data identify TGFB and potentially LPS as factors mediating seminal plasma-induced INHBA synthesis in cervical cells. INHBA and FST induced by seminal fluid in cervical tissues may thus contribute to regulation of the post-coital response in women.

  16. An epitope on membrane-bound but not secreted IgE: implications in isotype-specific regulation.

    PubMed

    Davis, F M; Gossett, L A; Chang, T W

    1991-01-01

    Immunoglobulins (Igs) on the surface of B lymphocytes are isotype-specific immunological markers of the B-cell subsets expressing them. Since these membrane-bound Igs (mIgs) are antigen receptors, their interaction with antibodies could be explored for modulating the activity of specific B-cell subsets. Targeting mIgs by antibodies in vivo, however, has not been feasible because of the presence of Igs in the circulation and the frequent association of Igs with various cell types via Fc receptors. To circumvent these problems, we proposed that the extracellular portions of the membrane-anchoring segments of the heavy chains of mIgs, referred to as "mIg isotype-specific" or "migis" peptides, may provide the antigenic sites for the isotype-specific targeting of B cells in vivo. Here we describe the exemplary development of monoclonal antibodies (mAbs) recognizing this unique epitope of mIgE.

  17. Photochemical energy conversion by membrane-bound photoredox systems. Progress report, July 1, 1989--March 1, 1992

    SciTech Connect

    Tollin, G.

    1992-03-01

    Most of our effort during the past grant period has been directed towards investigating electron transfer processes involving redox proteins at lipid bilayer/aqueous interfaces. This theme, as was noted in our previous three year renewal proposal, is consistent with our goal of developing biomimetic solar energy conversion systems which utilize the unique properties of biological electron transfer molecules. Thus, small redox proteins such as cytochrome c, plastocyanin and ferredoxin function is biological photosynthesis as mediators of electron flow between the photochemical systems localized in the membrane, and more complex soluble or membrane-bound redox proteins which are designed to carry out specific biological tasks such as transbilayer proton gradient formation, dinitrogen fixation, ATP synthesis, dihydrogen synthesis, generation of strong reductants, etc. In these studies, we have utilized two principal experimental techniques, laser flash photolysis and cyclic voltammetry, both of which permit direct measurements of electron transfer processes.

  18. Enhanced Oxygen-Tolerance of the Full Heterotrimeric Membrane-Bound [NiFe]-Hydrogenase of Ralstonia eutropha

    PubMed Central

    2014-01-01

    Hydrogenases are oxygen-sensitive enzymes that catalyze the conversion between protons and hydrogen. Water-soluble subcomplexes of membrane-bound [NiFe]-hydrogenases (MBH) have been extensively studied for applications in hydrogen–oxygen fuel cells as they are relatively tolerant to oxygen, although even these catalysts are still inactivated in oxidative conditions. Here, the full heterotrimeric MBH of Ralstonia eutropha, including the membrane-integral cytochrome b subunit, was investigated electrochemically using electrodes modified with planar tethered bilayer lipid membranes (tBLM). Cyclic voltammetry and chronoamperometry experiments show that MBH, in equilibrium with the quinone pool in the tBLM, does not anaerobically inactivate under oxidative redox conditions. In aerobic environments, the MBH is reversibly inactivated by O2, but reactivation was found to be fast even under oxidative redox conditions. This enhanced resistance to inactivation is ascribed to the oligomeric state of MBH in the lipid membrane. PMID:24866391

  19. Preliminary safety assessment of a membrane-bound delta 9 desaturase candidate protein for transgenic oilseed crops.

    PubMed

    Madduri, Krishna M; Schafer, Barry W; Hasler, James M; Lin, Gaofeng; Foster, Mendy L; Embrey, Shawna K; Sastry-Dent, Lakshmi; Song, Ping; Larrinua, Ignacio M; Gachotte, Daniel J; Herman, Rod A

    2012-10-01

    A gene encoding delta 9 desaturase (D9DS), an integral membrane protein, is being considered for incorporation into oilseed crops to reduce saturated fatty acids and thus improve human nutritional value. Typically, a safety assessment for transgenic crops involves purifying heterologously produced transgenic proteins in an active form for use in safety studies. Membrane-bound proteins have been very difficult to isolate in an active form due to their inherent physicochemical properties. Described here are methods used to derive enriched preparations of the active D9DS protein for use in early stage safety studies. Results of these studies, in combination with bioinformatic results and knowledge of the mode of action of the protein, along with a history of safe consumption of related proteins, provides a weight of evidence supporting the safety of the D9DS protein in food and feed.

  20. Seasonal changes in immunoreactivity of activin signaling component proteins in wild ground squirrel testes.

    PubMed

    Sheng, Xia; Zhang, Haolin; Zhang, Mengyuan; Zhang, Wei; Hu, Xiao; Song, Moshi; Zhou, Jiao; Xu, Meiyu; Weng, Qiang; Watanabe, Gen; Taya, Kazuyoshi

    2012-01-01

    The seasonal spermatogenesis and localization of inhibin/activin subunits (alpha, betaA, betaB) in the testes of wild ground squirrel has been previously described; however, the expression pattern of activin receptors and cytoplasmic signaling SMADs has not been detected in any seasonal breeders. The objective of this study was to investigate the abundance and cellular localization of activin signaling components in testes of the wild ground squirrel during the breeding and nonbreeding seasons. The immunolocalizations of ActRIIB (activin type II receptor B) and activin-related SMADs (phospho-SMAD2/3, SMAD4 and SMAD7) were observed by immunohistochemistry. Total proteins were extracted from testicular tissues in the breeding and nonbreeding seasons and were used for Western blotting analysis for ActRIIB and SMADs. Immunoreactivities of activin signaling components were greater in the testes of the breeding season, and then decreased to a relatively low level in the nonbreeding season. ActRIIB and related SMADs were widely spread in the active testes, while spermatogonia were the predominant cellular sites of activin signal transduction during arrested spermatogenesis. The dynamic regulation of activin type II receptor and SMADs indicated that the activin signal pathway played an important paracrine role in seasonal spermatogenesis of the wild ground squirrel. Furthermore, the distinct localizations and immunoreactivity of ActRIIB and SMADs might suggest different functions of activin in seasonal spermatogenesis.

  1. Human Renal Normal, Tumoral, and Cancer Stem Cells Express Membrane-Bound Interleukin-15 Isoforms Displaying Different Functions1

    PubMed Central

    Azzi, Sandy; Gallerne, Cindy; Romei, Cristina; Le Coz, Vincent; Gangemi, Rosaria; Khawam, Krystel; Devocelle, Aurore; Gu, Yanhong; Bruno, Stefania; Ferrini, Silvano; Chouaib, Salem; Eid, Pierre; Azzarone, Bruno; Giron-Michel, Julien

    2015-01-01

    Intrarenal interleukin-15 (IL-15) participates to renal pathophysiology, but the role of its different membrane-bound isoforms remains to be elucidated. In this study, we reassess the biology of membrane-bound IL-15 (mb-IL-15) isoforms by comparing primary cultures of human renal proximal tubular epithelial cells (RPTEC) to peritumoral (ptumTEC), tumoral (RCC), and cancer stem cells (CSC/CD105+). RPTEC express a 14 to 16 kDa mb-IL-15, whose existence has been assumed but never formally demonstrated and likely represents the isoform anchored at the cell membrane through the IL-15 receptor α (IL-15Rα) chain, because it is sensitive to acidic treatment and is not competent to deliver a reverse signal. By contrast, ptumTEC, RCC, and CSC express a novel N-hyperglycosylated, short-lived transmembrane mb-IL-15 (tmb-IL-15) isoform around 27 kDa, resistant to acidic shock, delivering a reverse signal in response to its soluble receptor (sIL-15Rα). This reverse signal triggers the down-regulation of the tumor suppressor gene E-cadherin in ptumTEC and RCC but not in CSC/CD105+, where it promotes survival. Indeed, through the AKT pathway, tmb-IL-15 protects CSC/CD105+ from non-programmed cell death induced by serum starvation. Finally, both mb-IL-15 and tmb-IL-15 are sensitive to metalloproteases, and the cleaved tmb-IL-15 (25 kDa) displays a powerful anti-apoptotic effect on human hematopoietic cells. Overall, our data indicate that both mb-IL-15 and tmb-IL-15 isoforms play a complex role in renal pathophysiology downregulating E-cadherin and favoring cell survival. Moreover, “apparently normal” ptumTEC cells, sharing different properties with RCC, could contribute to organize an enlarged peritumoral “preneoplastic” environment committed to favor tumor progression. PMID:26152359

  2. Intact Functional Fourteen-subunit Respiratory Membrane-bound [NiFe]-Hydrogenase Complex of the Hyperthermophilic Archaeon Pyrococcus furiosus*

    PubMed Central

    McTernan, Patrick M.; Chandrayan, Sanjeev K.; Wu, Chang-Hao; Vaccaro, Brian J.; Lancaster, W. Andrew; Yang, Qingyuan; Fu, Dax; Hura, Greg L.; Tainer, John A.; Adams, Michael W. W.

    2014-01-01

    The archaeon Pyrococcus furiosus grows optimally at 100 °C by converting carbohydrates to acetate, CO2, and H2, obtaining energy from a respiratory membrane-bound hydrogenase (MBH). This conserves energy by coupling H2 production to oxidation of reduced ferredoxin with generation of a sodium ion gradient. MBH is encoded by a 14-gene operon with both hydrogenase and Na+/H+ antiporter modules. Herein a His-tagged MBH was expressed in P. furiosus and the detergent-solubilized complex purified under anaerobic conditions by affinity chromatography. Purified MBH contains all 14 subunits by electrophoretic analysis (13 subunits were also identified by mass spectrometry) and had a measured iron:nickel ratio of 15:1, resembling the predicted value of 13:1. The as-purified enzyme exhibited a rhombic EPR signal characteristic of the ready nickel-boron state. The purified and membrane-bound forms of MBH both preferentially evolved H2 with the physiological donor (reduced ferredoxin) as well as with standard dyes. The O2 sensitivities of the two forms were similar (half-lives of ∼15 h in air), but the purified enzyme was more thermolabile (half-lives at 90 °C of 1 and 25 h, respectively). Structural analysis of purified MBH by small angle x-ray scattering indicated a Z-shaped structure with a mass of 310 kDa, resembling the predicted value (298 kDa). The angle x-ray scattering analyses reinforce and extend the conserved sequence relationships of group 4 enzymes and complex I (NADH quinone oxidoreductase). This is the first report on the properties of a solubilized form of an intact respiratory MBH complex that is proposed to evolve H2 and pump Na+ ions. PMID:24860091

  3. Intact functional fourteen-subunit respiratory membrane-bound [NiFe]-hydrogenase complex of the hyperthermophilic archaeon Pyrococcus furiosus.

    PubMed

    McTernan, Patrick M; Chandrayan, Sanjeev K; Wu, Chang-Hao; Vaccaro, Brian J; Lancaster, W Andrew; Yang, Qingyuan; Fu, Dax; Hura, Greg L; Tainer, John A; Adams, Michael W W

    2014-07-11

    The archaeon Pyrococcus furiosus grows optimally at 100 °C by converting carbohydrates to acetate, CO2, and H2, obtaining energy from a respiratory membrane-bound hydrogenase (MBH). This conserves energy by coupling H2 production to oxidation of reduced ferredoxin with generation of a sodium ion gradient. MBH is encoded by a 14-gene operon with both hydrogenase and Na(+)/H(+) antiporter modules. Herein a His-tagged MBH was expressed in P. furiosus and the detergent-solubilized complex purified under anaerobic conditions by affinity chromatography. Purified MBH contains all 14 subunits by electrophoretic analysis (13 subunits were also identified by mass spectrometry) and had a measured iron:nickel ratio of 15:1, resembling the predicted value of 13:1. The as-purified enzyme exhibited a rhombic EPR signal characteristic of the ready nickel-boron state. The purified and membrane-bound forms of MBH both preferentially evolved H2 with the physiological donor (reduced ferredoxin) as well as with standard dyes. The O2 sensitivities of the two forms were similar (half-lives of ∼ 15 h in air), but the purified enzyme was more thermolabile (half-lives at 90 °C of 1 and 25 h, respectively). Structural analysis of purified MBH by small angle x-ray scattering indicated a Z-shaped structure with a mass of 310 kDa, resembling the predicted value (298 kDa). The angle x-ray scattering analyses reinforce and extend the conserved sequence relationships of group 4 enzymes and complex I (NADH quinone oxidoreductase). This is the first report on the properties of a solubilized form of an intact respiratory MBH complex that is proposed to evolve H2 and pump Na(+) ions. PMID:24860091

  4. A novel adenylylation process in liver plasma membrane-bound proteins

    SciTech Connect

    San Jose, E.; Benguria, A.; Villalobo, A. )

    1990-11-25

    Rat liver plasma membrane contains five distinct polypeptides of apparent molecular mass of 130, 120, 110, 100, and 86 kDa which are labeled upon incubation with (alpha-32P)ATP as well as with (gamma-32P)ATP. Covalently bound adenosine 5'-monophosphate to some of the polypeptides was identified using nonhydrolyzable analogues of ATP. Chase experiments of alpha-32P-nucleotide-labeled polypeptides with different nonradiolabeled phosphocompounds and sensitivity to different inhibitors demonstrate that the 86-kDa polypeptide is a phosphoesterase, forming a catalytic intermediate. On the other hand, the comparative slow rate of turnover of the polypeptides of higher molecular mass (130, 120, 110, and 100 kDa) suggests that the bound AMP could play a regulatory rather than a catalytic role. Using the nonhydrolyzable ATP analogue (alpha, beta-methylene)ATP and dilution experiments with Triton X-100-solubilized membranes, it has been possible to identify the 130-kDa adenylylated polypeptide as a possible target of an adenylylating system. These polypeptides, except the 86-kDa phosphoesterase, are affected in their electrophoretic mobility in the absence of beta-mercaptoethanol. An intercatenary disulfide bond(s) appear(s) to link the polypeptide(s) of 120 kDa and/or 110 kDa in a dimeric structure of apparent molecular mass of 240 kDa. All five polypeptides labeled with (alpha-32P)ATP are glycoproteins bound to the cell plasma membrane.

  5. Transgenic models to study the roles of inhibins and activins in reproduction, oncogenesis, and development.

    PubMed

    Matzuk, M M; Kumar, T R; Shou, W; Coerver, K A; Lau, A L; Behringer, R R; Finegold, M J

    1996-01-01

    With the advent of gene targeting in pluripotent mouse embryonic stem cells, it is now possible to modify the mammalian genome to generate mutant strains of mice with precise genetic mutations. The major goal of my laboratory is to generate transgenic mice to use as physiologic models to study mammalian reproduction and development. The initial focus of our research has been to generate mice deficient in inhibins, activins, activin binding proteins (i.e., follistatin), and activin receptors (i.e., activin receptor type II) to understand their interactions and roles in the hypothalamic-pituitary-gonadal axis and mammalian development. Inhibins and activins, dimeric members of the TGF-beta superfamily, were discovered due to their role in pituitary follicle stimulating hormone homeostasis. However, these proteins have later been shown to have diverse endocrine, paracrine, and autocrine functions. Activins have been shown to mediate their signals through type I and type II serine/threonine kinase receptors. The high interspecies conservation of activins, inhibins, and activin receptors and the universal presence of activins in mammals, birds, amphibians, and fish suggest an evolutionarily conserved role of these proteins in animal development. Our initial studies have demonstrated a tumor suppressor role of inhibin in the gonads and adrenals and have also suggested a role of activins in cancer cachexia-like syndrome. To further study the gonadal tumor development and the cancer cachexia-like syndrome in these mice, we have begun to generate mice with multiple genetic alterations (e.g., mice deficient in both inhibin and Mullerian inhibiting substance). We have also generated mice deficient in other components of this complex system (e.g., activin beta A, activin receptor type II, follistatin). Analysis of these transgenic mutant models has aided our overall understanding of the critical roles these proteins play in the development of the reproductive system, in the

  6. Activin A regulation of gonadotropin-releasing hormone synthesis and release in vitro.

    PubMed

    MacConell, L A; Lawson, M A; Mellon, P L; Roberts, V J

    1999-10-01

    Activin is essential for the regulation of normal mammalian reproductive function at both the pituitary and gonadal levels. However, its central actions in the control of the hypothalamic-pituitary-gonadal axis remain largely unexplored. The present study aims to determine whether activin could regulate the reproductive axis at the level of the hypothalamus, through control of the GnRH neuroendocrine system. Using the GnRH-secreting GT1-7 neuronal cell line as a model system, we demonstrate expression of mRNAs encoding activin receptor types I, IB, and II. We examined the effects of activin A on GnRH protein secretion and mRNA levels in GT1-7 cells. Treatment with rh-activin A regulated both GnRH protein secretion and GnRH mRNA expression in the GT1-7 cells in a time-dependent fashion. Using transient transfection assays, we explored a potential transcriptional basis for these changes. Activin A increased reporter gene activity driven by minimal GnRH enhancer and promoter elements, suggesting that activin may regulate GnRH gene expression at the level of transcription. Lastly, activin A treatment of male rat hypothalami, in vitro, increased GnRH protein secretion. Collectively, molecular and physiological evidence support the presence of an activin system which might act at a hypothalamic site to regulate mammalian reproduction via activation of GnRH synthesis and release.

  7. Activin A and follistatin during the oestrous cycle and early pregnancy in ewes.

    PubMed

    O'Connell, Anne R; McNatty, Kenneth P; Hurst, Peter R; Spencer, Thomas E; Bazer, Fuller W; Reader, Karen L; Johnstone, Peter D; Davis, George H; Juengel, Jennifer L

    2016-03-01

    The activin pathway has been postulated to be involved in regulation of multiple reproductive processes important for survival of the conceptus. These processes include luteinisation of the follicular cells and thus function of the corpus luteum, early embryo development and uterine function including implantation of the conceptus. Therefore, the aim of the current study was to determine whether the concentrations of activin A and follistatin (FST), an activin-binding protein, differed between ewes with a lifetime history of enhanced or reduced embryonic survival (ES). The mRNAs encoding FST and activin A (inhibin beta A subunit; INHBA) were present in the uterus and abundant in the uterine luminal or glandular epithelia by day 18 of gestation. A peak of activin A was observed in the systemic circulation around the time of oestrus, and activin A concentrations were elevated in animals with reduced ES during the oestrous cycle and early gestation. Concentrations of activin A in uterine fluid were approximately twofold greater on day 16 of gestation in ewes with reduced ES compared to those with enhanced ES. No consistent differences in FST were observed between these groups. Treatment of luteinising ovine granulosa cells with activin A in vitro suppressed progesterone secretion providing evidence of a potential pathway whereby increased concentrations of activin A may decrease ES. PMID:26733604

  8. Novel ovarian regulatory peptides: inhibin, activin, and follistatin.

    PubMed

    Ling, N; DePaolo, L V; Bicsak, T A; Shimasaki, S

    1990-09-01

    Based on the extensive amount of research on inhibin and related polypeptides accomplished during the past 5 years, the inhibin concept put forth more than 50 years ago has not only become well established but also more complex than originally imagined. The closed-loop feedback mechanism of ovarian inhibin and pituitary FSH has been joined by possible "inhibin-like" actions of follistatin and FSH-stimulatory effects of activin. In addition, in vitro experiments suggest possible autocrine and paracrine functions for the gonadal polypeptide hormones. Figure 3 shows a simplistic diagram summarizing our current understanding of inhibin/activin and follistatin action along the hypothalamic-pituitary-gonadal axis. Hopefully, research in the coming years will allow us to remove the many question marks still remaining but will undoubtedly add others.

  9. Characterization of 19 Genes Encoding Membrane-Bound Fatty Acid Desaturases and their Expression Profiles in Gossypium raimondii Under Low Temperature.

    PubMed

    Liu, Wei; Li, Wei; He, Qiuling; Daud, Muhammad Khan; Chen, Jinhong; Zhu, Shuijin

    2015-01-01

    To produce unsaturated fatty acids, membrane-bound fatty acid desaturases (FADs) can be exploited to introduce double bonds into the acyl chains of fatty acids. In this study, 19 membrane-bound FAD genes were identified in Gossypium raimondii through database searches and were classified into four different subfamilies based on phylogenetic analysis. All 19 membrane-bound FAD proteins shared three highly conserved histidine boxes, except for GrFAD2.1, which lost the third histidine box in the C-terminal region. In the G. raimondii genome, tandem duplication might have led to the increasing size of the FAD2 cluster in the Omega Desaturase subfamily, whereas segmental duplication appeared to be the dominant mechanism for the expansion of the Sphingolipid and Front-end Desaturase subfamilies. Gene expression analysis showed that seven membrane-bound FAD genes were significantly up-regulated and that five genes were greatly suppressed in G. raimondii leaves exposed to low temperature conditions.

  10. Characterization of 19 Genes Encoding Membrane-Bound Fatty Acid Desaturases and their Expression Profiles in Gossypium raimondii Under Low Temperature

    PubMed Central

    He, Qiuling; Daud, Muhammad Khan; Chen, Jinhong; Zhu, Shuijin

    2015-01-01

    To produce unsaturated fatty acids, membrane-bound fatty acid desaturases (FADs) can be exploited to introduce double bonds into the acyl chains of fatty acids. In this study, 19 membrane-bound FAD genes were identified in Gossypium raimondii through database searches and were classified into four different subfamilies based on phylogenetic analysis. All 19 membrane-bound FAD proteins shared three highly conserved histidine boxes, except for GrFAD2.1, which lost the third histidine box in the C-terminal region. In the G. raimondii genome, tandem duplication might have led to the increasing size of the FAD2 cluster in the Omega Desaturase subfamily, whereas segmental duplication appeared to be the dominant mechanism for the expansion of the Sphingolipid and Front-end Desaturase subfamilies. Gene expression analysis showed that seven membrane-bound FAD genes were significantly up-regulated and that five genes were greatly suppressed in G. raimondii leaves exposed to low temperature conditions. PMID:25894196

  11. Molecular Dynamics Simulations to Provide Insights into Epitopes Coupled to the Soluble and Membrane-Bound MHC-II Complexes

    PubMed Central

    Bello, Martiniano; Correa-Basurto, Jose

    2013-01-01

    Epitope recognition by major histocompatibility complex II (MHC-II) is essential for the activation of immunological responses to infectious diseases. Several studies have demonstrated that this molecular event takes place in the MHC-II peptide-binding groove constituted by the α and β light chains of the heterodimer. This MHC-II peptide-binding groove has several pockets (P1-P11) involved in peptide recognition and complex stabilization that have been probed through crystallographic experiments and in silico calculations. However, most of these theoretical calculations have been performed without taking into consideration the heavy chains, which could generate misleading information about conformational mobility both in water and in the membrane environment. Therefore, in absence of structural information about the difference in the conformational changes between the peptide-free and peptide-bound states (pMHC-II) when the system is soluble in an aqueous environment or non-covalently bound to a cell membrane, as the physiological environment for MHC-II is. In this study, we explored the mechanistic basis of these MHC-II components using molecular dynamics (MD) simulations in which MHC-II was previously co-crystallized with a small epitope (P7) or coupled by docking procedures to a large (P22) epitope. These MD simulations were performed at 310 K over 100 ns for the water-soluble (MHC-IIw, MHC-II-P7w, and MHC-II-P22w) and 150 ns for the membrane-bound species (MHC-IIm, MHC-II-P7m, and MHC-II-P22m). Our results reveal that despite the different epitope sizes and MD simulation environments, both peptides are stabilized primarily by residues lining P1, P4, and P6-7, and similar noncovalent intermolecular energies were observed for the soluble and membrane-bound complexes. However, there were remarkably differences in the conformational mobility and intramolecular energies upon complex formation, causing some differences with respect to how the two peptides are

  12. Molecular dynamics simulations to provide insights into epitopes coupled to the soluble and membrane-bound MHC-II complexes.

    PubMed

    Bello, Martiniano; Correa-Basurto, Jose

    2013-01-01

    Epitope recognition by major histocompatibility complex II (MHC-II) is essential for the activation of immunological responses to infectious diseases. Several studies have demonstrated that this molecular event takes place in the MHC-II peptide-binding groove constituted by the α and β light chains of the heterodimer. This MHC-II peptide-binding groove has several pockets (P1-P11) involved in peptide recognition and complex stabilization that have been probed through crystallographic experiments and in silico calculations. However, most of these theoretical calculations have been performed without taking into consideration the heavy chains, which could generate misleading information about conformational mobility both in water and in the membrane environment. Therefore, in absence of structural information about the difference in the conformational changes between the peptide-free and peptide-bound states (pMHC-II) when the system is soluble in an aqueous environment or non-covalently bound to a cell membrane, as the physiological environment for MHC-II is. In this study, we explored the mechanistic basis of these MHC-II components using molecular dynamics (MD) simulations in which MHC-II was previously co-crystallized with a small epitope (P7) or coupled by docking procedures to a large (P22) epitope. These MD simulations were performed at 310 K over 100 ns for the water-soluble (MHC-IIw, MHC-II-P(7w), and MHC-II-P(22w)) and 150 ns for the membrane-bound species (MHC-IIm, MHC-II-P(7m), and MHC-II-P(22m)). Our results reveal that despite the different epitope sizes and MD simulation environments, both peptides are stabilized primarily by residues lining P1, P4, and P6-7, and similar noncovalent intermolecular energies were observed for the soluble and membrane-bound complexes. However, there were remarkably differences in the conformational mobility and intramolecular energies upon complex formation, causing some differences with respect to how the two peptides

  13. Identification of a unique membrane-bound molecule on a hemopoietic stem cell line and on multipotent progenitor cells.

    PubMed Central

    Han, X D; Chung, S W; Wong, P M

    1995-01-01

    Hemopoietic stem cells are a distinct population of cells that can differentiate into multilineages of hemopoietic cells and have long-term repopulation capability. A few membrane-bound molecules have been found to be preferentially, but not uniquely, present on the surface of these primitive cells. We report here the identification of a unique 105-kDa glycoprotein on the surface of hemopoietic stem cell line BL3. This molecule, recognized by the absorbed antiserum, is not present on the surface of myeloid progenitors 32D and FDC-P1 cells, EL4 T cells, and NIH 3T3 fibroblasts. This antiserum can also be used to block the proliferation of BL3 cells even in the presence of mitogen-stimulated spleen cell conditioned medium, which is known to have a stimulating activity on BL3 cells. It can also inhibit development of in vitro, fetal liver cell-derived multilineage colonies, but not other types of colonies, and of in vivo bone marrow cell-derived colony-forming unit spleen foci. These data suggest that gp105 plays an important role in hemopoietic stem cell differentiation. Images Fig. 1 Fig. 2 Fig. 3 PMID:7479927

  14. Quinone-reactive proteins devoid of haem b form widespread membrane-bound electron transport modules in bacterial respiration.

    PubMed

    Simon, Jörg; Kern, Melanie

    2008-10-01

    Many quinone-reactive enzyme complexes that are part of membrane-integral eukaryotic or prokaryotic respiratory electron transport chains contain one or more haem b molecules embedded in the membrane. In recent years, various novel proteins have emerged that are devoid of haem b but are thought to fulfil a similar function in bacterial anaerobic respiratory systems. These proteins are encoded by genes organized in various genomic arrangements and are thought to form widespread membrane-bound quinone-reactive electron transport modules that exchange electrons with redox partner proteins located at the outer side of the cytoplasmic membrane. Prototypic representatives are the multihaem c-type cytochromes NapC, NrfH and TorC (NapC/NrfH family), the putative iron-sulfur protein NapH and representatives of the NrfD/PsrC family. Members of these protein families vary in the number of their predicted transmembrane segments and, consequently, diverse quinone-binding sites are expected. Only a few of these enzymes have been isolated and characterized biochemically and high-resolution structures are limited. This mini-review briefly summarizes predicted and experimentally demonstrated properties of the proteins in question and discusses their role in electron transport and bioenergetics of anaerobic respiration.

  15. The effect of progesterone and 17-β estradiol on membrane-bound HLA-G in adipose derived stem cells.

    PubMed

    Moslehi, Akram; Hashemi-Beni, Batool; Moslehi, Azam; Akbari, Maryam Ali; Adib, Minoo

    2016-07-01

    Membrane-bound HLA-G (mHLA-G) discovery on adipose derived stem cells (ADSCs) as a tolerogenic and immunosuppressive molecule was very important. Many documents have shown that HLA-G expression can be controlled via some hormones such as progesterone (P4) and estradiol (E2). Therefore, this study was designed to evaluate progesterone and estradiol effects on mHLA-G in ADSCs at restricted and combination concentrations. Three independent cell lines were cultured in complete free phenol red DMEM and subcultured to achieve suffi cient cells. These cells were treated with P4, E2 and P4 plus E2 at physiologic and pregnancy concentrations for 3 days in cell culture conditions. The HLA-G positive ADSCs was measured via monoclonal anti HLA-G-FITC/MEMG-09 by means of flow cytometry in nine groups. Data were analyzed by one way ANOVA and Tukey's post hoc tests. There were no signifi cant values of the mean percentage of HLA-G positive cells in E2-treated and the combination of P4 plus E2-treated ADSCs compared to control cells (p value>0.05) but P4 had a signifi cant increase on mHLA-G in ADSCs (p value<0.05). High P4 concentration increased mHLA-G but E2 and the combination of P4 plus E2 could not change mHLA-G on ADSCs. PMID:27382350

  16. Application of REDOR subtraction for filtered MAS observation of labeled backbone carbons of membrane-bound fusion peptides

    NASA Astrophysics Data System (ADS)

    Yang, Jun; Parkanzky, Paul D.; Bodner, Michele L.; Duskin, Craig A.; Weliky, David P.

    2002-12-01

    Clean MAS observation of 13C-labeled carbons in membrane-bound HIV-1 and influenza fusion peptides was made by using a rotational-echo double-resonance spectroscopy (REDOR) filter of directly bonded 13C- 15N pairs. The clean filtering achieved with the REDOR approach is superior to filtering done with sample difference spectroscopy. In one labeling approach, the peptide had labels at a single 13C carbonyl and its directly bonded 15N. The resulting chemical shift distribution of the filtered signal is used to assess the distribution of local secondary structures at the labeled carbonyl. For the influenza peptide, the Leu-2 carbonyl chemical shift distribution is shown to vary markedly with lipid and detergent composition, as well as peptide:lipid ratio, suggesting that the local peptide structure also has a strong dependence on these factors. Because most carboxylic- and amino-labeled amino acids are commercially available, this REDOR approach should have broad applicability to chemically synthesized peptides as well as bacterially synthesized proteins. In a second labeling approach, the HIV-1 fusion peptide had U- 13C, 15N labeling over three sequential residues. When a 1.6 ms REDOR dephasing time is used, only backbone 13C signals are observed. The resulting spectra are used to determine spectral linewidths and to assess feasibility of assignment of uniformly labeled peptide.

  17. Crystallization and preliminary structure determination of the membrane-bound complex cytochrome c nitrite reductase from Desulfovibrio vulgaris Hildenborough

    SciTech Connect

    Rodrigues, M. L.; Oliveira, T.; Matias, P. M.; Martins, I. C.; Valente, F. M. A.; Pereira, I. A. C.; Archer, M.

    2006-06-01

    The cytochrome c nitrite reductase complex from D. vulgaris Hildenborough has been crystallized. The preliminary crystallographic structure reveals a 2:1 NrfA:NrfH complex stoichiometry. The cytochrome c nitrite reductase (cNiR) isolated from Desulfovibrio vulgaris Hildenborough is a membrane-bound complex formed of NrfA and NrfH subunits. The catalytic subunit NrfA is a soluble pentahaem cytochrome c that forms a physiological dimer of about 120 kDa. The electron-donor subunit NrfH is a membrane-anchored tetrahaem cytochrome c of about 18 kDa molecular weight and belongs to the NapC/NirT family of quinol dehydrogenases, for which no structures are known. Crystals of the native cNiR membrane complex, solubilized with dodecylmaltoside detergent (DDM), were obtained using PEG 4K as precipitant. Anomalous diffraction data were measured at the Swiss Light Source to 2.3 Å resolution. Crystals belong to the orthorhombic space group P2{sub 1}2{sub 1}2{sub 1}, with unit-cell parameters a = 79.5, b = 256.7, c = 578.2 Å. Molecular-replacement and MAD methods were combined to solve the structure. The data presented reveal that D. vulgaris cNiR contains one NrfH subunit per NrfA dimer.

  18. Comparison of membrane-bound and soluble polyphenol oxidase in Fuji apple (Malus domestica Borkh. cv. Red Fuji).

    PubMed

    Liu, Fang; Zhao, Jin-Hong; Gan, Zhi-Lin; Ni, Yuan-Ying

    2015-04-15

    This study compared membrane-bound with soluble polyphenol oxidase (mPPO and sPPO, respectively) from Fuji apple. Purified mPPO and partially purified sPPO were used. mPPO was purified by temperature-induced phase partitioning and ion exchange chromatography. The specific activity of mPPO was 34.12× higher than that of sPPO. mPPO was more stable than sPPO at pH 5.0-8.5. Although mPPO was more easily inactivated at 25-55 °C, it is still more active than sPPO in this temperature range. The optimum substrate of mPPO was 4-methyl catechol, followed by catechol. L-cysteine had the highest inhibitory effects on mPPO followed by ascorbic acid and glutathione. Surprisingly, EDTA increased mPPO activity. The results revealed that purified mPPO is a dimer with a molecular weight of approximately 67 kDa.

  19. Chelation of Membrane-Bound Cations by Extracellular DNA Activates the Type VI Secretion System in Pseudomonas aeruginosa.

    PubMed

    Wilton, Mike; Wong, Megan J Q; Tang, Le; Liang, Xiaoye; Moore, Richard; Parkins, Michael D; Lewenza, Shawn; Dong, Tao G

    2016-08-01

    Pseudomonas aeruginosa employs its type VI secretion system (T6SS) as a highly effective and tightly regulated weapon to deliver toxic molecules to target cells. T6SS-secreted proteins of P. aeruginosa can be detected in the sputum of cystic fibrosis (CF) patients, who typically present a chronic and polymicrobial lung infection. However, the mechanism of T6SS activation in the CF lung is not fully understood. Here we demonstrate that extracellular DNA (eDNA), abundant within the CF airways, stimulates the dynamics of the H1-T6SS cluster apparatus in Pseudomonas aeruginosa PAO1. Addition of Mg(2+) or DNase with eDNA abolished such activation, while treatment with EDTA mimicked the eDNA effect, suggesting that the eDNA-mediated effect is due to chelation of outer membrane-bound cations. DNA-activated H1-T6SS enables P. aeruginosa to nonselectively attack neighboring species regardless of whether or not it was provoked. Because of the importance of the T6SS in interspecies interactions and the prevalence of eDNA in the environments that P. aeruginosa inhabits, our report reveals an important adaptation strategy that likely contributes to the competitive fitness of P. aeruginosa in polymicrobial communities.

  20. Fatty acyl donor selectivity in membrane bound O-acyltransferases and communal cell fate decision-making

    PubMed Central

    Tuladhar, Rubina; Lum, Lawrence

    2015-01-01

    The post-translational modification of proteins with lipid moieties confers spatial and temporal control of protein function by restricting their subcellular distribution or movement in the extracellular milieu. Yet, little is known about the significance of lipid selectivity to the activity of proteins targeted for such modifications. Membrane bound O-acyl transferases (MBOATs) are a superfamily of multipass enzymes that transfer fatty acids on to lipid or protein substrates. Three MBOATs constitute a subfamily with secreted signalling molecules for substrates, the Wnt, Hedgehog (Hh) and Ghrelin proteins. Given their important roles in adult tissue homoeostasis, all three molecules and their respective associated acyltransferases provide a framework for interrogating the role of extracellular acylation events in cell-to-cell communication. Here, we discuss how the preference for a fatty acyl donor in the Wnt acyltransferase porcupine (Porcn) and possibly in other protein lipidation enzymes may provide a means for coupling metabolic health at the single cell level to communal cell fate decision-making in complex multicellular organisms. PMID:25849923

  1. Cloning and sequencing of the gene cluster encoding two subunits of membrane-bound alcohol dehydrogenase from Acetobacter polyoxogenes.

    PubMed

    Tamaki, T; Fukaya, M; Takemura, H; Tayama, K; Okumura, H; Kawamura, Y; Nishiyama, M; Horinouchi, S; Beppu, T

    1991-02-16

    The membrane-bound alcohol dehydrogenase (ADH) from Acetobacter polyoxogenes NBI1028 is composed of a 72 kDa subunit and a 44 kDa cytochrome c subunit. The amino acid sequences of the two regions of the 72 kDa subunit were determined to prepare oligonucleotides for the purpose of amplification of a DNA fragment corresponding to the intermediate region by the polymerase chain reaction. A 0.5 kb DNA fragment thus amplified was used as the probe to clone a 7.0 kb PstI fragment coding for the whole 72 kDa subunit. Nucleotide sequencing and immunoblot analysis revealed that the cloned fragment contained the full structural genes for the 72 kDa and the 44 kDa subunits and they were clustered with the same transcription polarity. The predicted amino acid sequence of the gene for the 72 kDa subunit showed homology with that of the 72 kDa subunit from ADH of A. aceti and those of methanol dehydrogenase from methylotrophic bacteria. The 72 and 44 kDa subunits contained one and three typical haem binding sequences, respectively.

  2. Engineering Hydrogen Gas Production from Formate in a Hyperthermophile by Heterologous Production of an 18-Subunit Membrane-bound Complex*

    PubMed Central

    Lipscomb, Gina L.; Schut, Gerrit J.; Thorgersen, Michael P.; Nixon, William J.; Kelly, Robert M.; Adams, Michael W. W.

    2014-01-01

    Biohydrogen gas has enormous potential as a source of reductant for the microbial production of biofuels, but its low solubility and poor gas mass transfer rates are limiting factors. These limitations could be circumvented by engineering biofuel production in microorganisms that are also capable of generating H2 from highly soluble chemicals such as formate, which can function as an electron donor. Herein, the model hyperthermophile, Pyrococcus furiosus, which grows optimally near 100 °C by fermenting sugars to produce H2, has been engineered to also efficiently convert formate to H2. Using a bacterial artificial chromosome vector, the 16.9-kb 18-gene cluster encoding the membrane-bound, respiratory formate hydrogen lyase complex of Thermococcus onnurineus was inserted into the P. furiosus chromosome and expressed as a functional unit. This enabled P. furiosus to utilize formate as well as sugars as an H2 source and to do so at both 80° and 95 °C, near the optimum growth temperature of the donor (T. onnurineus) and engineered host (P. furiosus), respectively. This accomplishment also demonstrates the versatility of P. furiosus for metabolic engineering applications. PMID:24318960

  3. Adropin is a brain membrane-bound protein regulating physical activity via the NB-3/Notch signaling pathway in mice.

    PubMed

    Wong, Chi-Ming; Wang, Yudong; Lee, Jimmy Tsz Hang; Huang, Zhe; Wu, Donghai; Xu, Aimin; Lam, Karen Siu Ling

    2014-09-12

    Adropin is a highly conserved polypeptide that has been suggested to act as an endocrine factor that plays important roles in metabolic regulation, insulin sensitivity, and endothelial functions. However, in this study, we provide evidence demonstrating that adropin is a plasma membrane protein expressed abundantly in the brain. Using a yeast two-hybrid screening approach, we identified NB-3/Contactin 6, a brain-specific, non-canonical, membrane-tethered Notch1 ligand, as an interaction partner of adropin. Furthermore, this interaction promotes NB3-induced activation of Notch signaling and the expression of Notch target genes. We also generated and characterized adropin knockout mice to explore the role of adropin in vivo. Adropin knockout mice exhibited decreased locomotor activity and impaired motor coordination coupled with defective synapse formation, a phenotype similar to NB-3 knockout mice. Taken together, our data suggest that adropin is a membrane-bound protein that interacts with the brain-specific Notch1 ligand NB3. It regulates physical activity and motor coordination via the NB-3/Notch signaling pathway and plays an important role in cerebellum development in mice.

  4. Bypassing tumor-associated immune suppression with recombinant adenovirus constructs expressing membrane bound or secreted GITR-L.

    PubMed

    Calmels, Bastien; Paul, Stéphane; Futin, Nicolas; Ledoux, Catherine; Stoeckel, Fabienne; Acres, Bruce

    2005-02-01

    Recent evidence has resurrected the concept of specialized populations of T lymphocytes that are able to suppress an antigen-specific immune response. T-regulatory cells (T-reg) have been characterized as CD4+ CD25+ T cells. Previous reports describing differential gene expression analysis have shown that the glucocorticoid-induced tumor necrosis family receptor family-related gene (GITR) is upregulated in these cells. Furthermore, antibodies specific for GITR have been shown to inhibit the T-suppressor function of CD4+ CD25+ T-reg. The ligands for both mouse and human GITR have been cloned recently. We have inserted the sequences for natural, membrane-bound GITR-ligand (GITR-L) and a truncated secreted form of GITR-L (GITR-Lsol) into the adenovirus-5 genome. Coculture experiments show that cells infected with Ad-GITR-L and supernatants from cells infected with Ad-GITR-Lsol can increase the proliferation of both CD4+ CD25- and CD8+ T cells in response to anti-CD3 stimulation, in the presence, as well as in the absence, of CD4+ CD25+ T cells. The virus constructs were injected into growing B16 melanoma tumors. Ad-GITR-L was shown to attract infiltration with both CD4+ and CD8+ T cells. Both constructs were shown to inhibit tumor growth. PMID:15472713

  5. The effect of progesterone and 17-β estradiol on membrane-bound HLA-G in adipose derived stem cells

    PubMed Central

    Moslehi, Akram; Hashemi-beni, Batool; Moslehi, Azam; Akbari, Maryam Ali

    2016-01-01

    Membrane-bound HLA-G (mHLA-G) discovery on adipose derived stem cells (ADSCs) as a tolerogenic and immunosuppressive molecule was very important. Many documents have shown that HLA-G expression can be controlled via some hormones such as progesterone (P4) and estradiol (E2). Therefore, this study was designed to evaluate progesterone and estradiol effects on mHLA-G in ADSCs at restricted and combination concentrations. Three independent cell lines were cultured in complete free phenol red DMEM and subcultured to achieve suffi cient cells. These cells were treated with P4, E2 and P4 plus E2 at physiologic and pregnancy concentrations for 3 days in cell culture conditions. The HLA-G positive ADSCs was measured via monoclonal anti HLA-G-FITC/MEMG-09 by means of flow cytometry in nine groups. Data were analyzed by one way ANOVA and Tukey's post hoc tests. There were no signifi cant values of the mean percentage of HLA-G positive cells in E2-treated and the combination of P4 plus E2-treated ADSCs compared to control cells (p value>0.05) but P4 had a signifi cant increase on mHLA-G in ADSCs (p value<0.05). High P4 concentration increased mHLA-G but E2 and the combination of P4 plus E2 could not change mHLA-G on ADSCs. PMID:27382350

  6. Sensing Size through Clustering in Non-Equilibrium Membranes and the Control of Membrane-Bound Enzymatic Reactions

    PubMed Central

    Vagne, Quentin; Turner, Matthew S.; Sens, Pierre

    2015-01-01

    The formation of dynamical clusters of proteins is ubiquitous in cellular membranes and is in part regulated by the recycling of membrane components. We show, using stochastic simulations and analytic modeling, that the out-of-equilibrium cluster size distribution of membrane components undergoing continuous recycling is strongly influenced by lateral confinement. This result has significant implications for the clustering of plasma membrane proteins whose mobility is hindered by cytoskeletal “corrals” and for protein clustering in cellular organelles of limited size that generically support material fluxes. We show how the confinement size can be sensed through its effect on the size distribution of clusters of membrane heterogeneities and propose that this could be regulated to control the efficiency of membrane-bound reactions. To illustrate this, we study a chain of enzymatic reactions sensitive to membrane protein clustering. The reaction efficiency is found to be a non-monotonic function of the system size, and can be optimal for sizes comparable to those of cellular organelles. PMID:26656912

  7. Reduced Levels of Membrane-Bound Alkaline Phosphatase Are Common to Lepidopteran Strains Resistant to Cry Toxins from Bacillus thuringiensis

    PubMed Central

    Jurat-Fuentes, Juan Luis; Karumbaiah, Lohitash; Jakka, Siva Rama Krishna; Ning, Changming; Liu, Chenxi; Wu, Kongming; Jackson, Jerreme; Gould, Fred; Blanco, Carlos; Portilla, Maribel; Perera, Omaththage; Adang, Michael

    2011-01-01

    Development of insect resistance is one of the main concerns with the use of transgenic crops expressing Cry toxins from the bacterium Bacillus thuringiensis. Identification of biomarkers would assist in the development of sensitive DNA-based methods to monitor evolution of resistance to Bt toxins in natural populations. We report on the proteomic and genomic detection of reduced levels of midgut membrane-bound alkaline phosphatase (mALP) as a common feature in strains of Cry-resistant Heliothis virescens, Helicoverpa armigera and Spodoptera frugiperda when compared to susceptible larvae. Reduced levels of H. virescens mALP protein (HvmALP) were detected by two dimensional differential in-gel electrophoresis (2D-DIGE) analysis in Cry-resistant compared to susceptible larvae, further supported by alkaline phosphatase activity assays and Western blotting. Through quantitative real-time polymerase chain reaction (qRT-PCR) we demonstrate that the reduction in HvmALP protein levels in resistant larvae are the result of reduced transcript amounts. Similar reductions in ALP activity and mALP transcript levels were also detected for a Cry1Ac-resistant strain of H. armigera and field-derived strains of S. frugiperda resistant to Cry1Fa. Considering the unique resistance and cross-resistance phenotypes of the insect strains used in this work, our data suggest that reduced mALP expression should be targeted for development of effective biomarkers for resistance to Cry toxins in lepidopteran pests. PMID:21390253

  8. Computational study of drug binding to the membrane-bound tetrameric M2 peptide bundle from influenza A virus.

    PubMed

    Khurana, Ekta; Devane, Russell H; Dal Peraro, Matteo; Klein, Michael L

    2011-02-01

    The M2 protein of influenza A virus performs the crucial function of transporting protons to the interior of virions enclosed in the endosome. Adamantane drugs, amantadine (AMN) and rimantidine (RMN), block the proton conduction in some strains, and have been used for the treatment and prophylaxis of influenza A infections. The structures of the transmembrane (TM) region of M2 that have been solved in micelles using NMR (residues 23-60) (Schnell and Chou, 2008) and by X-ray crystallography (residues 22-46) (Stouffer et al., 2008) suggest different drug binding sites: external and internal for RMN and AMN, respectively. We have used molecular dynamics (MD) simulations to investigate the nature of the binding site and binding mode of adamantane drugs on the membrane-bound tetrameric M2-TM peptide bundles using as initial conformations the low-pH AMN-bound crystal structure, a high-pH model derived from the drug-free crystal structure, and the high-pH NMR structure. The MD simulations indicate that under both low- and high-pH conditions, AMN is stable inside the tetrameric bundle, spanning the region between residues Val27 to Gly34. At low pH the polar group of AMN is oriented toward the His37 gate, while under high-pH conditions its orientation exhibits large fluctuations. The present MD simulations also suggest that AMN and RMN molecules do not show strong affinity to the external binding sites.

  9. The rice thylakoid membrane-bound ascorbate peroxidase OsAPX8 functions in tolerance to bacterial blight

    PubMed Central

    Jiang, Guanghuai; Yin, Dedong; Zhao, Jiying; Chen, Honglin; Guo, Lequn; Zhu, Lihuang; Zhai, Wenxue

    2016-01-01

    Thylakoid membrane-bound ascorbate peroxidase (tAPX) is a major H2O2-scavenging enzyme. To clarify its functions in tolerance to rice bacterial blight, we produced rice lines overexpressing and suppressing tAPX (OsAPX8). The overexpressing lines exhibited increased tolerance to bacterial pathogen. The RNA interference (RNAi) lines were considerably more sensitive than the control plant. Further analysis of the H2O2 content in these transgenic plants indicated that the H2O2 accumulation of OsAPX8-overexpressing plants was considerably less than that of wild-type and RNAi plants upon challenge with bacterial pathogen. Interestingly, H2O2 was the most important factor for the serious leaf dehydration and withering of rice without major resistance genes and was not the cause of hypersensitivity. It addition, wall tightening or loosening can occur according to the level of H2O2. In addition, OsAPX8 interacted with the susceptibility protein Os8N3/Xa13, and their binding repressed the reaction of OsAPX8 in tolerance to bacterial blight. PMID:27185545

  10. An investigation into membrane bound redox carriers involved in energy transduction mechanism in Brevibacterium linens DSM 20158 with unsequenced genome.

    PubMed

    Shabbiri, Khadija; Botting, Catherine H; Adnan, Ahmad; Fuszard, Matthew; Naseem, Shahid; Ahmed, Safeer; Shujaat, Shahida; Syed, Quratulain; Ahmad, Waqar

    2014-04-01

    Brevibacterium linens (B. linens) DSM 20158 with an unsequenced genome can be used as a non-pathogenic model to study features it has in common with other unsequenced pathogens of the same genus on the basis of comparative proteome analysis. The most efficient way to kill a pathogen is to target its energy transduction mechanism. In the present study, we have identified the redox protein complexes involved in the electron transport chain of B. linens DSM 20158 from their clear homology with the shot-gun genome sequenced strain BL2 of B. linens by using the SDS-Polyacrylamide gel electrophoresis coupled with nano LC-MS/MS mass spectrometry. B. linens is found to have a branched electron transport chain (Respiratory chain), in which electrons can enter the respiratory chain either at NADH (Complex I) or at Complex II level or at the cytochrome level. Moreover, we are able to isolate, purify, and characterize the membrane bound Complex II (succinate dehydrogenase), Complex III (menaquinone cytochrome c reductase cytochrome c subunit, Complex IV (cytochrome c oxidase), and Complex V (ATP synthase) of B. linens strain DSM 20158. PMID:24573306

  11. The effect of progesterone and 17-β estradiol on membrane-bound HLA-G in adipose derived stem cells.

    PubMed

    Moslehi, Akram; Hashemi-Beni, Batool; Moslehi, Azam; Akbari, Maryam Ali; Adib, Minoo

    2016-07-01

    Membrane-bound HLA-G (mHLA-G) discovery on adipose derived stem cells (ADSCs) as a tolerogenic and immunosuppressive molecule was very important. Many documents have shown that HLA-G expression can be controlled via some hormones such as progesterone (P4) and estradiol (E2). Therefore, this study was designed to evaluate progesterone and estradiol effects on mHLA-G in ADSCs at restricted and combination concentrations. Three independent cell lines were cultured in complete free phenol red DMEM and subcultured to achieve suffi cient cells. These cells were treated with P4, E2 and P4 plus E2 at physiologic and pregnancy concentrations for 3 days in cell culture conditions. The HLA-G positive ADSCs was measured via monoclonal anti HLA-G-FITC/MEMG-09 by means of flow cytometry in nine groups. Data were analyzed by one way ANOVA and Tukey's post hoc tests. There were no signifi cant values of the mean percentage of HLA-G positive cells in E2-treated and the combination of P4 plus E2-treated ADSCs compared to control cells (p value>0.05) but P4 had a signifi cant increase on mHLA-G in ADSCs (p value<0.05). High P4 concentration increased mHLA-G but E2 and the combination of P4 plus E2 could not change mHLA-G on ADSCs.

  12. The rice thylakoid membrane-bound ascorbate peroxidase OsAPX8 functions in tolerance to bacterial blight.

    PubMed

    Jiang, Guanghuai; Yin, Dedong; Zhao, Jiying; Chen, Honglin; Guo, Lequn; Zhu, Lihuang; Zhai, Wenxue

    2016-01-01

    Thylakoid membrane-bound ascorbate peroxidase (tAPX) is a major H2O2-scavenging enzyme. To clarify its functions in tolerance to rice bacterial blight, we produced rice lines overexpressing and suppressing tAPX (OsAPX8). The overexpressing lines exhibited increased tolerance to bacterial pathogen. The RNA interference (RNAi) lines were considerably more sensitive than the control plant. Further analysis of the H2O2 content in these transgenic plants indicated that the H2O2 accumulation of OsAPX8-overexpressing plants was considerably less than that of wild-type and RNAi plants upon challenge with bacterial pathogen. Interestingly, H2O2 was the most important factor for the serious leaf dehydration and withering of rice without major resistance genes and was not the cause of hypersensitivity. It addition, wall tightening or loosening can occur according to the level of H2O2. In addition, OsAPX8 interacted with the susceptibility protein Os8N3/Xa13, and their binding repressed the reaction of OsAPX8 in tolerance to bacterial blight. PMID:27185545

  13. Chelation of Membrane-Bound Cations by Extracellular DNA Activates the Type VI Secretion System in Pseudomonas aeruginosa.

    PubMed

    Wilton, Mike; Wong, Megan J Q; Tang, Le; Liang, Xiaoye; Moore, Richard; Parkins, Michael D; Lewenza, Shawn; Dong, Tao G

    2016-08-01

    Pseudomonas aeruginosa employs its type VI secretion system (T6SS) as a highly effective and tightly regulated weapon to deliver toxic molecules to target cells. T6SS-secreted proteins of P. aeruginosa can be detected in the sputum of cystic fibrosis (CF) patients, who typically present a chronic and polymicrobial lung infection. However, the mechanism of T6SS activation in the CF lung is not fully understood. Here we demonstrate that extracellular DNA (eDNA), abundant within the CF airways, stimulates the dynamics of the H1-T6SS cluster apparatus in Pseudomonas aeruginosa PAO1. Addition of Mg(2+) or DNase with eDNA abolished such activation, while treatment with EDTA mimicked the eDNA effect, suggesting that the eDNA-mediated effect is due to chelation of outer membrane-bound cations. DNA-activated H1-T6SS enables P. aeruginosa to nonselectively attack neighboring species regardless of whether or not it was provoked. Because of the importance of the T6SS in interspecies interactions and the prevalence of eDNA in the environments that P. aeruginosa inhabits, our report reveals an important adaptation strategy that likely contributes to the competitive fitness of P. aeruginosa in polymicrobial communities. PMID:27271742

  14. Functional analysis of membrane-bound complement regulatory protein on T-cell immune response in ginbuna crucian carp.

    PubMed

    Nur, Indriyani; Abdelkhalek, Nevien K; Motobe, Shiori; Nakamura, Ryota; Tsujikura, Masakazu; Somamoto, Tomonori; Nakao, Miki

    2016-02-01

    Complements have long been considered to be a pivotal component in innate immunity. Recent researches, however, highlight novel roles of complements in T-cell-mediated adaptive immunity. Membrane-bound complement regulatory protein CD46, a costimulatory protein for T cells, is a key molecule for T-cell immunomodulation. Teleost CD46-like molecule, termed Tecrem, has been newly identified in common carp and shown to function as a complement regulator. However, it remains unclear whether Tecrem is involved in T-cell immune response. We investigated Tecrem function related to T-cell responses in ginbuna crucian carp. Ginbuna Tecrem (gTecrem) proteins were detected by immunoprecipitation using anti-common carp Tecrem monoclonal antibody (mAb) and were ubiquitously expressed on blood cells including CD8α(+) and CD4(+) lymphocytes. gTecrem expression on leucocyte surface was enhanced after stimulation with the T-cell mitogen, phytohaemagglutinin (PHA). Coculture with the anti-Tecrem mAb significantly inhibited the proliferative activity of PHA-stimulated peripheral blood lymphocytes, suggesting that cross-linking of Tecrems on T-cells interferes with a signal transduction pathway for T-cell activation. These findings indicate that Tecrem may act as a T-cell moderator and imply that the complement system in teleost, as well as mammals, plays an important role for linking adaptive and innate immunity.

  15. O2-stable membrane-bound [NiFe]hydrogenase from a newly isolated Citrobacter sp. S-77.

    PubMed

    Eguchi, Shigenobu; Yoon, Ki-Seok; Ogo, Seiji

    2012-11-01

    Hydrogenases are of great interest due to their potential use in H(2)-based technology. However, most hydrogenases are highly sensitive to O(2), which have been the major bottleneck in hydrogenase studies. Here we report an O(2)-stable membrane-bound [NiFe]hydrogenase (MBH) purified from a newly isolated strain, S-77. According to the 16S rRNA gene sequence and phylogenetic analysis of the strain S-77, it belongs to the genus of Citrobacter. In vitro experiments using the cytoplasmic membrane of strain S-77 suggested that a cytochrome b acts as the physiological electron acceptor of the MBH. The purified MBH was composed of a dimer of heterodimers, consisting of two distinct subunits with the molecular weights of 58.5 and 38.5 kDa. The enzyme showed a specific activity for H(2)-oxidation of 661 U/mg, which is 35-fold greater than that for H(2)-production of 18.7 U/mg. Notably, the MBH showed a remarkable O(2)-stability, maintaining almost 95% of its original activity even after incubation for 30 h in air at 4°C. These results suggest that the O(2)-stable MBH may play an important role in the H(2)-metabolic pathway under the aerobic conditions of Citrobacter sp. S-77. This is the first report of the purification and biochemical characterization of an O(2)-stable MBH from the genus of Citrobacter.

  16. Inhibitors

    MedlinePlus

    ... Community Counts Blood Safety Inhibitors Articles & Key Findings Free Materials Videos Starting the Conversation Playing it Safe A Look at Hemophilia Joint Range of Motion My Story Links to Other Websites ...

  17. Survival, mobility, and membrane-bound enzyme activities of freshwater planarian, Dugesia japonica, exposed to synthetic and natural surfactants.

    PubMed

    Li, Mei-Hui

    2012-04-01

    Surfactants are a major class of emerging pollutants widely used in large quantities in everyday life and commonly found in surface waters worldwide. Freshwater planarian was selected to examine the effects of different surfactants by measuring mortality, mobility, and membrane-bound enzyme activities. Among the 10 surfactants tested, the acute toxicities of betaine and polyethylene glycol (PEG-200) to planarians were relatively low, with a median lethal concentration (LC50) greater than 10,000 mg/L. The toxicity to planarians of the other eight surfactants based on 48-h LC50 could be arranged in the descending order of cetylpyridinum chloride (CPC) > 4-tert-octylphenol (4-tert-OP) > ammonium lauryl sulfate > benzalkonium chloride > saponin > sodium lauroylsarcosinate > dioctyl sulfosuccinate > dodecyl trimethyl ammonium bromide (DTAB). Both CPC and 4-tert-OP were very toxic to planarians, with 48-h LC50 values <1 mg/L. The median effective concentrations (EC50s) of planarian mobility were in the 0.1 to 50 mg/L range and were in the same range as the 24-h LC50 of planarians exposed to different surfactants, except for DTAB. In addition, significant inhibition of cholinesterase activity activities was found in planarians exposed to 4-tert-OP at 2.5 and 5 mg/L and to saponin at 10 mg/L after 2-h treatments. This result suggests that planarian mobility responses can be used as an alternative indicator for acute toxicity of surfactants after a very short exposure period.

  18. A heteromeric membrane-bound prenyltransferase complex from hop catalyzes three sequential aromatic prenylations in the bitter acid pathway.

    PubMed

    Li, Haoxun; Ban, Zhaonan; Qin, Hao; Ma, Liya; King, Andrew J; Wang, Guodong

    2015-03-01

    Bitter acids (α and β types) account for more than 30% of the fresh weight of hop (Humulus lupulus) glandular trichomes and are well known for their contribution to the bitter taste of beer. These multiprenylated chemicals also show diverse biological activities, some of which have potential benefits to human health. The bitter acid biosynthetic pathway has been investigated extensively, and the genes for the early steps of bitter acid synthesis have been cloned and functionally characterized. However, little is known about the enzyme(s) that catalyze three sequential prenylation steps in the β-bitter acid pathway. Here, we employed a yeast (Saccharomyces cerevisiae) system for the functional identification of aromatic prenyltransferase (PT) genes. Two PT genes (HlPT1L and HlPT2) obtained from a hop trichome-specific complementary DNA library were functionally characterized using this yeast system. Coexpression of codon-optimized PT1L and PT2 in yeast, together with upstream genes, led to the production of bitter acids, but no bitter acids were detected when either of the PT genes was expressed by itself. Stepwise mutation of the aspartate-rich motifs in PT1L and PT2 further revealed the prenylation sequence of these two enzymes in β-bitter acid biosynthesis: PT1L catalyzed only the first prenylation step, and PT2 catalyzed the two subsequent prenylation steps. A metabolon formed through interactions between PT1L and PT2 was demonstrated using a yeast two-hybrid system, reciprocal coimmunoprecipitation, and in vitro biochemical assays. These results provide direct evidence of the involvement of a functional metabolon of membrane-bound prenyltransferases in bitter acid biosynthesis in hop. PMID:25564559

  19. Rubredoxin-related Maturation Factor Guarantees Metal Cofactor Integrity during Aerobic Biosynthesis of Membrane-bound [NiFe] Hydrogenase*

    PubMed Central

    Fritsch, Johannes; Siebert, Elisabeth; Priebe, Jacqueline; Zebger, Ingo; Lendzian, Friedhelm; Teutloff, Christian; Friedrich, Bärbel; Lenz, Oliver

    2014-01-01

    The membrane-bound [NiFe] hydrogenase (MBH) supports growth of Ralstonia eutropha H16 with H2 as the sole energy source. The enzyme undergoes a complex biosynthesis process that proceeds during cell growth even at ambient O2 levels and involves 14 specific maturation proteins. One of these is a rubredoxin-like protein, which is essential for biosynthesis of active MBH at high oxygen concentrations but dispensable under microaerobic growth conditions. To obtain insights into the function of HoxR, we investigated the MBH protein purified from the cytoplasmic membrane of hoxR mutant cells. Compared with wild-type MBH, the mutant enzyme displayed severely decreased hydrogenase activity. Electron paramagnetic resonance and infrared spectroscopic analyses revealed features resembling those of O2-sensitive [NiFe] hydrogenases and/or oxidatively damaged protein. The catalytic center resided partially in an inactive Niu-A-like state, and the electron transfer chain consisting of three different Fe-S clusters showed marked alterations compared with wild-type enzyme. Purification of HoxR protein from its original host, R. eutropha, revealed only low protein amounts. Therefore, recombinant HoxR protein was isolated from Escherichia coli. Unlike common rubredoxins, the HoxR protein was colorless, rather unstable, and essentially metal-free. Conversion of the atypical iron-binding motif into a canonical one through genetic engineering led to a stable reddish rubredoxin. Remarkably, the modified HoxR protein did not support MBH-dependent growth at high O2. Analysis of MBH-associated protein complexes points toward a specific interaction of HoxR with the Fe-S cluster-bearing small subunit. This supports the previously made notion that HoxR avoids oxidative damage of the metal centers of the MBH, in particular the unprecedented Cys6[4Fe-3S] cluster. PMID:24448806

  20. Bacillus thuringiensis subsp. israelensis Cyt1Aa synergizes Cry11Aa toxin by functioning as a membrane-bound receptor.

    PubMed

    Pérez, Claudia; Fernandez, Luisa E; Sun, Jianguang; Folch, Jorge Luis; Gill, Sarjeet S; Soberón, Mario; Bravo, Alejandra

    2005-12-20

    Bacillus thuringiensis subsp. israelensis produces crystal proteins, Cry (4Aa, 4Ba, 10Aa, and 11Aa) and Cyt (1Aa and 2Ba) proteins, toxic to mosquito vectors of human diseases. Cyt1Aa overcomes insect resistance to Cry11Aa and Cry4 toxins and synergizes the toxicity of these toxins. However, the molecular mechanism of synergism remains unsolved. Here, we provide evidence that Cyt1Aa functions as a receptor of Cry11Aa. Sequential-binding analysis of Cyt1Aa and Cry11Aa revealed that Cyt1Aa binding to Aedes aegypti brush border membrane vesicles enhanced the binding of biotinylated-Cry11Aa. The Cyt1Aa- and Cry11Aa-binding epitopes were mapped by means of the yeast two-hybrid system, peptide arrays, and heterologous competition assays with synthetic peptides. Two exposed regions in Cyt1Aa, loop beta6-alphaE and part of beta7, bind Cry11Aa. On the other side, Cry11Aa binds Cyt1Aa proteins by means of domain II-loop alpha8 and beta-4, which are also involved in midgut receptor interaction. Characterization of single-point mutations in Cry11Aa and Cyt1Aa revealed key Cry11Aa (S259 and E266) and Cyt1Aa (K198, E204 and K225) residues involved in the interaction of both proteins and in synergism. Additionally, a Cyt1Aa loop beta6-alphaE mutant (K198A) with enhanced synergism to Cry11Aa was isolated. Data provided here strongly indicates that Cyt1Aa synergizes or suppresses resistance to Cry11Aa toxin by functioning as a membrane-bound receptor. Bacillus thuringiensis subsp. israelensis is a highly effective pathogenic bacterium because it produces a toxin and also its functional receptor, promoting toxin binding to the target membrane and causing toxicity. PMID:16339907

  1. Cellular chaperones and folding enzymes are vital contributors to membrane bound replication and movement complexes during plant RNA virus infection

    PubMed Central

    Verchot, Jeanmarie

    2012-01-01

    Cellular chaperones and folding enzymes play central roles in the formation of positive-strand and negative-strand RNA virus infection. This article examines the key cellular chaperones and discusses evidence that these factors are diverted from their cellular functions to play alternative roles in virus infection. For most chaperones discussed, their primary role in the cell is to ensure protein quality control. They are system components that drive substrate protein folding, complex assembly or disaggregation. Their activities often depend upon co-chaperones and ATP hydrolysis. During plant virus infection, Hsp70 and Hsp90 proteins play central roles in the formation of membrane-bound replication complexes for certain members of the tombusvirus, tobamovirus, potyvirus, dianthovirus, potexvirus, and carmovirus genus. There are several co-chaperones, including Yjd1, RME-8, and Hsp40 that associate with the bromovirus replication complex, pomovirus TGB2, and tospovirus Nsm movement proteins. There are also examples of plant viruses that rely on chaperone systems in the endoplasmic reticulum (ER) to support cell-to-cell movement. TMV relies on calreticulin to promote virus intercellular transport. Calreticulin also resides in the plasmodesmata and plays a role in calcium sequestration as well as glycoprotein folding. The pomovirus TGB2 interacts with RME-8 in the endosome. The potexvirus TGB3 protein stimulates expression of ER resident chaperones via the bZIP60 transcription factor. Up-regulating factors involved in protein folding may be essential to handling the load of viral proteins translated along the ER. In addition, TGB3 stimulates SKP1 which is a co-factor in proteasomal degradation of cellular proteins. Such chaperones and co-factors are potential targets for antiviral defense. PMID:23230447

  2. Thylakoid membrane-bound ascorbate peroxidase is a limiting factor of antioxidative systems under photo-oxidative stress.

    PubMed

    Yabuta, Yukinori; Motoki, Takashi; Yoshimura, Kazuya; Takeda, Toru; Ishikawa, Takahiro; Shigeoka, Shigeru

    2002-12-01

    To evaluate the physiological importance of thylakoid membrane-bound ascorbate peroxidase (tAPX) in the active oxygen species-scavenging system of chloroplasts, the level of tAPX in tobacco plants was altered by expression of the tAPX cDNA in both sense and antisense orientation. The tobacco plants transformed with constructs of antisense tAPXs from spinach and tobacco could not be obtained, suggesting that the suppression of tAPX in higher plants had a severe effect on the growth even under normal conditions. In contrast, the transgenic tobacco plants (TpTAP-12) overexpressing tAPX, which had approximately 37-fold higher activity than that of the wild-type plants, were generated. The TpTAP-12 plants showed increased tolerance to oxidative stress caused by application of methylviologen (MV, 50 microm) under light intensity (300 and 1600 microE m(-2) sec(-1)) and by chilling stress with high light intensity (4 degrees C, 1000 microE m(-2) sec(-1)). At 24 h after the MV treatment under illumination at 300 microE m-2 sec-1, destruction of chlorophyll was observed in the wild-type plants, but not in the TpTAP-12 plants. The activities of thiol-modulated enzymes in the Calvin cycle, the level and redox status of ascorbate (AsA), and the activity of tAPX in the wild-type plants significantly decreased, while those in the TpTAP-12 plants were hardly changed. These observations suggest that tAPX is a limiting factor of antioxidative systems under photo-oxidative stress in chloroplasts, and that the enhanced activity of tAPX functions to maintain the AsA content and the redox status of AsA under stress conditions.

  3. Structure of membrane-bound α-synuclein from site-directed spin labeling and computational refinement

    PubMed Central

    Jao, Christine C.; Hegde, Balachandra G.; Chen, Jeannie; Haworth, Ian S.; Langen, Ralf

    2008-01-01

    α-Synuclein is known to play a causative role in Parkinson disease. Although its physiological functions are not fully understood, α-synuclein has been shown to interact with synaptic vesicles and modulate neurotransmitter release. However, the structure of its physiologically relevant membrane-bound state remains unknown. Here we developed a site-directed spin labeling and EPR-based approach for determining the structure of α-synuclein bound to a lipid bilayer. Continuous-wave EPR was used to assign local secondary structure and to determine the membrane immersion depth of lipid-exposed residues, whereas pulsed EPR was used to map long-range distances. The structure of α-synuclein was built and refined by using simulated annealing molecular dynamics restrained by the immersion depths and distances. We found that α-synuclein forms an extended, curved α-helical structure that is over 90 aa in length. The monomeric helix has a superhelical twist similar to that of right-handed coiled-coils which, like α-synuclein, contain 11-aa repeats, but which are soluble, oligomeric proteins (rmsd = 0.82 Å). The α-synuclein helix extends parallel to the curved membrane in a manner that allows conserved Lys and Glu residues to interact with the zwitterionic headgroups, while uncharged residues penetrate into the acyl chain region. This structural arrangement is significantly different from that of α-synuclein in the presence of the commonly used membrane-mimetic detergent SDS, which induces the formation of two antiparallel helices. Our structural analysis emphasizes the importance of studying membrane protein structure in a bilayer environment. PMID:19066219

  4. The structure of Serratia marcescens Lip, a membrane-bound component of the type VI secretion system

    SciTech Connect

    Rao, Vincenzo A.; Shepherd, Sharon M.; English, Grant; Coulthurst, Sarah J.; Hunter, William N.

    2011-12-01

    The high-resolution crystal structure of S. marcescens Lip reveals a new member of the transthyretin family of proteins. Lip, a core component of the type VI secretion apparatus, is localized to the outer membrane and is positioned to interact with other proteins forming this complex system. Lip is a membrane-bound lipoprotein and a core component of the type VI secretion system found in Gram-negative bacteria. The structure of a Lip construct (residues 29–176) from Serratia marcescens (SmLip) has been determined at 1.92 Å resolution. Experimental phases were derived using a single-wavelength anomalous dispersion approach on a sample cocrystallized with iodide. The membrane localization of the native protein was confirmed. The structure is that of the globular domain lacking only the lipoprotein signal peptide and the lipidated N-terminus of the mature protein. The protein fold is dominated by an eight-stranded β-sandwich and identifies SmLip as a new member of the transthyretin family of proteins. Transthyretin and the only other member of the family fold, 5-hydroxyisourate hydrolase, form homotetramers important for their function. The asymmetric unit of SmLip is a tetramer with 222 symmetry, but the assembly is distinct from that previously noted for the transthyretin protein family. However, structural comparisons and bacterial two-hybrid data suggest that the SmLip tetramer is not relevant to its role as a core component of the type VI secretion system, but rather reflects a propensity for SmLip to participate in protein–protein interactions. A relatively low level of sequence conservation amongst Lip homologues is noted and is restricted to parts of the structure that might be involved in interactions with physiological partners.

  5. Overproduction of the membrane-bound [NiFe]-hydrogenase in Thermococcus kodakarensis and its effect on hydrogen production

    PubMed Central

    Kanai, Tamotsu; Simons, Jan-Robert; Tsukamoto, Ryohei; Nakajima, Akihito; Omori, Yoshiyuki; Matsuoka, Ryoji; Beppu, Haruki; Imanaka, Tadayuki; Atomi, Haruyuki

    2015-01-01

    The hyperthermophilic archaeon Thermococcus kodakarensis can utilize sugars or pyruvate for growth. In the absence of elemental sulfur, the electrons via oxidation of these substrates are accepted by protons, generating molecular hydrogen (H2). The hydrogenase responsible for this reaction is a membrane-bound [NiFe]-hydrogenase (Mbh). In this study, we have examined several possibilities to increase the protein levels of Mbh in T. kodakarensis by genetic engineering. Highest levels of intracellular Mbh levels were achieved when the promoter of the entire mbh operon (TK2080-TK2093) was exchanged to a strong constitutive promoter from the glutamate dehydrogenase gene (TK1431) (strain MHG1). When MHG1 was cultivated under continuous culture conditions using pyruvate-based medium, a nearly 25% higher specific hydrogen production rate (SHPR) of 35.3 mmol H2 g-dcw−1 h−1 was observed at a dilution rate of 0.31 h−1. We also combined mbh overexpression using an even stronger constitutive promoter from the cell surface glycoprotein gene (TK0895) with disruption of the genes encoding the cytosolic hydrogenase (Hyh) and an alanine aminotransferase (AlaAT), both of which are involved in hydrogen consumption (strain MAH1). At a dilution rate of 0.30 h−1, the SHPR was 36.2 mmol H2 g-dcw−1 h−1, corresponding to a 28% increase compared to that of the host T. kodakarensis strain. Increasing the dilution rate to 0.83 h−1 or 1.07 h−1 resulted in a SHPR of 120 mmol H2 g-dcw−1 h−1, which is one of the highest production rates observed in microbial fermentation. PMID:26379632

  6. Formation of 4-keto-D-aldopentoses and 4-pentulosonates (4-keto-D-pentonates) with unidentified membrane-bound enzymes from acetic acid bacteria.

    PubMed

    Adachi, Osao; Hours, Roque A; Shinagawa, Emiko; Akakabe, Yoshihiko; Yakushi, Toshiharu; Matsushita, Kazunobu

    2011-01-01

    In our previous study, a new microbial reaction yielding 4-keto-D-arabonate from 2,5-diketo-D-gluconate was identified with Gluconacetobacter liquefaciens RCTMR 10. It appeared that decarboxylation and dehydrogenation took place together in the reaction. To analyze the nature of the reaction, investigations were done with the membrane fraction of the organism, and 4-keto-D-arabinose was confirmed as the direct precursor of 4-keto-D-arabonate. Two novel membrane-bound enzymes, 2,5-diketo-D-gluconate decarboxylase and 4-keto-D-aldopentose 1-dehydrogenase, were involved in the reaction. Alternatively, D-arabonate was oxidized to 4-keto-D-arabonate by another membrane-bound enzyme, D-arabonate 4-dehydrogenase. More directly, D-arabinose oxidation was examined with growing cells and with the membrane fraction of G. suboxydans IFO 12528. 4-Keto-D-arabinose, the same intermediate as that from 2,5-diketo-D-gluconate, was detected, and it was oxidized to 4-keto-D-arabonate. Likewise, D-ribose was oxidized to 4-keto-D-ribose and then it was oxidized to 4-keto-D-ribonate. In addition to 4-keto-D-aldopentose 1-dehydrogenase, the presence of a novel membrane-bound enzyme, D-aldopentose 4-dehydrogenase, was confirmed in the membrane fraction. The formation of 4-keto-D-aldopentoses and 4-keto-D-pentonates (4-pentulosonates) was finally confirmed as reaction products of four different novel membrane-bound enzymes.

  7. Formation of 4-keto-D-aldopentoses and 4-pentulosonates (4-keto-D-pentonates) with unidentified membrane-bound enzymes from acetic acid bacteria.

    PubMed

    Adachi, Osao; Hours, Roque A; Shinagawa, Emiko; Akakabe, Yoshihiko; Yakushi, Toshiharu; Matsushita, Kazunobu

    2011-01-01

    In our previous study, a new microbial reaction yielding 4-keto-D-arabonate from 2,5-diketo-D-gluconate was identified with Gluconacetobacter liquefaciens RCTMR 10. It appeared that decarboxylation and dehydrogenation took place together in the reaction. To analyze the nature of the reaction, investigations were done with the membrane fraction of the organism, and 4-keto-D-arabinose was confirmed as the direct precursor of 4-keto-D-arabonate. Two novel membrane-bound enzymes, 2,5-diketo-D-gluconate decarboxylase and 4-keto-D-aldopentose 1-dehydrogenase, were involved in the reaction. Alternatively, D-arabonate was oxidized to 4-keto-D-arabonate by another membrane-bound enzyme, D-arabonate 4-dehydrogenase. More directly, D-arabinose oxidation was examined with growing cells and with the membrane fraction of G. suboxydans IFO 12528. 4-Keto-D-arabinose, the same intermediate as that from 2,5-diketo-D-gluconate, was detected, and it was oxidized to 4-keto-D-arabonate. Likewise, D-ribose was oxidized to 4-keto-D-ribose and then it was oxidized to 4-keto-D-ribonate. In addition to 4-keto-D-aldopentose 1-dehydrogenase, the presence of a novel membrane-bound enzyme, D-aldopentose 4-dehydrogenase, was confirmed in the membrane fraction. The formation of 4-keto-D-aldopentoses and 4-keto-D-pentonates (4-pentulosonates) was finally confirmed as reaction products of four different novel membrane-bound enzymes. PMID:21897028

  8. Polymorphisms in the Tumor Necrosis Factor Receptor Genes Affect the Expression Levels of Membrane-Bound Type I and Type II Receptors

    PubMed Central

    Sennikov, Sergey V.; Vasilyev, Filipp F.; Lopatnikova, Julia A.; Shkaruba, Nadezhda S.; Silkov, Alexander N.

    2014-01-01

    The level of TNF receptors on various cells of immune system and its association with the gene polymorphism were investigated. Determining the levels of membrane-bound TNFα receptors on peripheral blood mononuclear cells (PBMCs) was performed by flow cytometry using BD QuantiBRITE calibration particles. Soluble TNFα receptor (sTNFRs) levels were determined by ELISA and genotyping was determined by PCR-RFLP. Homozygous TT individuals at SNP −609G/T TNFRI (rs4149570) showed lower levels of sTNFRI compared to GG genotype carriers. Homozygous carriers of CC genotype at SNP −1207G/C TNFRI (rs4149569) had lower expression densities of membrane-bound TNFRI on intact CD14+ monocytes compared to individuals with the GC genotype. The frequency differences in the CD3+ and CD19+ cells expressing TNFRII in relation to SNP −1709A/T TNFRII (rs652625) in healthy individuals were also determined. The genotype CC in SNP −3609C/T TNFRII (rs590368) was associated with a lower percentage of CD14+ cells expressing TNFRII compared to individuals with the CT genotype. Patients with rheumatoid arthritis had no significant changes in the frequencies of genotypes. Reduced frequency was identified for the combination TNFRI −609GT + TNFRII −3609CC only. The polymorphisms in genes represent one of cell type-specific mechanisms affecting the expression levels of membrane-bound TNFα receptors and TNFα-mediated signaling. PMID:24782596

  9. Activin A Predicts Left Ventricular Remodeling and Mortality in Patients with ST-Elevation Myocardial Infarction

    PubMed Central

    Lin, Jeng-Feng; Hsu, Shun-Yi; Teng, Ming-Sheng; Wu, Semon; Hsieh, Chien-An; Jang, Shih-Jung; Liu, Chih-Jen; Huang, Hsuan-Li; Ko, Yu-Lin

    2016-01-01

    Background Activin A levels increase in a variety of heart diseases including ST-elevation myocardial infarction (STEMI). The aim of this study is to investigate whether the level of activin A can be beneficial in predicting left ventricular remodeling, heart failure, and death in patients with ST-elevation myocardial infarction (STEMI). Methods We enrolled 278 patients with STEMI who had their activin A levels measured on day 2 of hospitalization. Echocardiographic studies were performed at baseline and were repeated 6 months later. Thereafter, the clinical events of these patients were followed for a maximum of 3 years, including all-cause death and readmission for heart failure. Results During hospitalization, higher activin A level was associated with higher triglyceride level, lower left ventricular ejection fraction (LVEF), and lower left ventricular end diastolic ventricular volume index (LVEDVI) in multivariable linear regression model. During follow-up, patients with activin A levels > 129 pg/ml had significantly lower LVEF, and higher LVEDVI at 6 months. Kaplan-Meier survival curves showed that activin A level > 129 pg/ml was a predictor of all-cause death (p = 0.022), but not a predictor of heart failure (p = 0.767). Conclusions Activin A level > 129 pg/ml predicts worse left ventricular remodeling and all-cause death in STEMI. PMID:27471355

  10. Constitutively active FOXO1 diminishes activin induction of Fshb transcription in immortalized gonadotropes.

    PubMed

    Park, Chung Hyun; Skarra, Danalea V; Rivera, Alissa J; Arriola, David J; Thackray, Varykina G

    2014-01-01

    In the present study, we investigate whether the FOXO1 transcription factor modulates activin signaling in pituitary gonadotropes. Our studies show that overexpression of constitutively active FOXO1 decreases activin induction of murine Fshb gene expression in immortalized LβT2 cells. We demonstrate that FOXO1 suppression of activin induction maps to the -304/-95 region of the Fshb promoter containing multiple activin response elements and that the suppression requires the FOXO1 DNA-binding domain (DBD). FOXO1 binds weakly to the -125/-91 region of the Fshb promoter in a gel-shift assay. Since this region of the promoter contains a composite SMAD/FOXL2 binding element necessary for activin induction of Fshb transcription, it is possible that FOXO1 DNA binding interferes with SMAD and/or FOXL2 function. In addition, our studies demonstrate that FOXO1 directly interacts with SMAD3/4 but not SMAD2 in a FOXO1 DBD-dependent manner. Moreover, we show that SMAD3/4 induction of Fshb-luc and activin induction of a multimerized SMAD-binding element-luc are suppressed by FOXO1 in a DBD-dependent manner. These results suggest that FOXO1 binding to the proximal Fshb promoter as well as FOXO1 interaction with SMAD3/4 proteins may result in decreased activin induction of Fshb in gonadotropes.

  11. Upregulation of activin A gene by butyrate in human colon cancer cell lines.

    PubMed

    Sonoyama, Kei; Pholnukulkit, Pimara; Toyoda, Masahiko; Rutatip, Suriya; Kasai, Takanori

    2003-06-01

    Activin A has been reported to play a role in the progression of colorectal cancer. Because dietary fiber protects against colorectal cancer, we hypothesized that butyrate, a fermentation product of dietary fiber, may affect the expression of activin A in colon cancer cells. Semiquantitative RT-PCR demonstrated that the activin A gene was upregulated by sodium butyrate in the human colon cancer cell lines HT-29 and Caco-2 in a concentration- and time-dependent manner. However, the activin A gene did not respond to sodium butyrate in the human normal colonic cell line FHC, rat normal intestinal epithelial cell (IEC) line IEC-6, and the explant of rat colon. Flow cytometry and agarose gel electrophoresis of genomic DNA revealed that cell cycle arrest and apoptosis were induced by sodium butyrate but not exogenous activin A in HT-29 cells, indicating that activin A could not act as an autocrine factor in colon cancer cells. By assuming that activin A promotes colorectal cancer spread as a paracrine factor, our findings suggest that butyrate could act as a tumor promoter in some circumstances.

  12. A membrane-bound form of glutamate dehydrogenase possesses an ATP-dependent high-affinity microtubule-binding activity.

    PubMed Central

    Rajas, F; Rousset, B

    1993-01-01

    We previously identified a 50 kDa membrane protein which bound to in vitro assembled microtubules [Mithieux and Rousset (1989) J. Biol. Chem. 264, 4664-4668]. This protein exhibited the expected properties for mediating the ATP-dependent association of vesicles with microtubules [Mithieux, Audebet and Rousset (1988) Biochim. Biophys. Acta 969, 121-130]. The 50 kDa membrane protein (MP50), initially extracted in very low amount from isolated pig thyroid lysosomes/endosomes, has now been purified from membrane preparations of crude vesicle fractions from pig liver and brain. MP50 was isolated from detergent-solubilized membrane protein by affinity chromatography on immobilized ATP; 3-5 mg of MP50 was obtained from 100 g of liver tissue. Phase partitioning in Triton X-114 indicated that MP50 is a peripheral membrane protein. Radioiodinated liver MP50 bound to microtubules assembled in vitro. The binding was inhibited by ATP (Ki = 0.76 mM) and displaced by unlabelled liver or brain MP50. Equilibrium binding studies yielded KD values of 1.8 x 10(-7) M. By N-terminal amino acid sequence analysis, MP50 was identified as glutamate dehydrogenase (GDH), by comparison of V8 protease peptide maps of MP50 with purified liver GDH. Liver MP50 exhibited a low GDH activity; 4-5 units/mg compared with 18 and 34 units/mg for purified bovine and rat liver GDH respectively. Bovine and rat liver GDH yielded six spots from pI 5.7 to 7.2 when analysed by two-dimensional electrophoresis; in contrast, MP50 gave one main spot (corresponding to spot 2 of liver GDH) with a pI of approx. 6.5. Soluble liver GDH from commercial sources exhibited a very low or no microtubule-binding activity. In conclusion, we have found a membrane-bound form of GDH capable of specific and nucleotide-sensitive interaction with microtubules. Our data suggest that GDH isoproteins, the number of which has been undervalued up to now, could have cellular functions other than that of an enzyme. Images Figure 1 Figure 3

  13. Effects of Activin A on the phenotypic properties of human periodontal ligament cells.

    PubMed

    Sugii, Hideki; Maeda, Hidefumi; Tomokiyo, Atsushi; Yamamoto, Naohide; Wada, Naohisa; Koori, Katsuaki; Hasegawa, Daigaku; Hamano, Sayuri; Yuda, Asuka; Monnouchi, Satoshi; Akamine, Akifumi

    2014-09-01

    Periodontal ligament (PDL) tissue plays an important role in tooth preservation by structurally maintaining the connection between the tooth root and the bone. The mechanisms involved in the healing and regeneration of damaged PDL tissue, caused by bacterial infection, caries and trauma, have been explored. Accumulating evidence suggests that Activin A, a member of the transforming growth factor-β (TGF-β) superfamily and a dimer of inhibinβa, contributes to tissue healing through cell proliferation, migration, and differentiation of various target cells. In bone, Activin A has been shown to exert an inhibitory effect on osteoblast maturation and mineralization. However, there have been no reports examining the expression and function of Activin A in human PDL cells (HPDLCs). Thus, we aimed to investigate the biological effects of Activin A on HPDLCs. Activin A was observed to be localized in HPDLCs and rat PDL tissue. When PDL tissue was surgically damaged, Activin A and IL-1β expression increased and the two proteins were shown to be co-localized around the lesion. HPDLCs treated with IL-1β or TNF-α also up-regulated the expression of the gene encoding inhibinβa. Activin A promoted chemotaxis, migration and proliferation of HPDLCs, and caused an increase in fibroblastic differentiation of these cells while down-regulating their osteoblastic differentiation. These osteoblastic inhibitory effects of Activin A, however, were only noted during the early phase of HPDLC osteoblastic differentiation, with later exposures having no effect on differentiation. Collectively, our results suggest that Activin A could be used as a therapeutic agent for healing and regenerating PDL tissue in response to disease, trauma or surgical reconstruction. PMID:24928494

  14. Effects of Activin A on the phenotypic properties of human periodontal ligament cells.

    PubMed

    Sugii, Hideki; Maeda, Hidefumi; Tomokiyo, Atsushi; Yamamoto, Naohide; Wada, Naohisa; Koori, Katsuaki; Hasegawa, Daigaku; Hamano, Sayuri; Yuda, Asuka; Monnouchi, Satoshi; Akamine, Akifumi

    2014-09-01

    Periodontal ligament (PDL) tissue plays an important role in tooth preservation by structurally maintaining the connection between the tooth root and the bone. The mechanisms involved in the healing and regeneration of damaged PDL tissue, caused by bacterial infection, caries and trauma, have been explored. Accumulating evidence suggests that Activin A, a member of the transforming growth factor-β (TGF-β) superfamily and a dimer of inhibinβa, contributes to tissue healing through cell proliferation, migration, and differentiation of various target cells. In bone, Activin A has been shown to exert an inhibitory effect on osteoblast maturation and mineralization. However, there have been no reports examining the expression and function of Activin A in human PDL cells (HPDLCs). Thus, we aimed to investigate the biological effects of Activin A on HPDLCs. Activin A was observed to be localized in HPDLCs and rat PDL tissue. When PDL tissue was surgically damaged, Activin A and IL-1β expression increased and the two proteins were shown to be co-localized around the lesion. HPDLCs treated with IL-1β or TNF-α also up-regulated the expression of the gene encoding inhibinβa. Activin A promoted chemotaxis, migration and proliferation of HPDLCs, and caused an increase in fibroblastic differentiation of these cells while down-regulating their osteoblastic differentiation. These osteoblastic inhibitory effects of Activin A, however, were only noted during the early phase of HPDLC osteoblastic differentiation, with later exposures having no effect on differentiation. Collectively, our results suggest that Activin A could be used as a therapeutic agent for healing and regenerating PDL tissue in response to disease, trauma or surgical reconstruction.

  15. The potential role of activin and follistatin in lung transplant dysfunction.

    PubMed

    Snell, James N; Westall, Glen P; Snell, Gregory I

    2015-01-01

    Activin A, a member of the transforming growth factor β super-family, is a key regulator of multiple biological pathways including the physiological processes of organ development and homeostasis; as well as the pathological processes of inflammation, remodelling and fibrosis. Dysregulation of activin A and its naturally occurring antagonist follistatin, contribute to the development of disease in multiple organ systems. In this review, we summarize the regulation of activin A, its dysregulated expression in a number of respiratory diseases and postulate its potential role in contributing to allograft dysfunction following lung transplantation.

  16. Mutual effects of melatonin and activin on induction of aldosterone production by human adrenocortical cells.

    PubMed

    Hara, Takayuki; Otsuka, Fumio; Tsukamoto-Yamauchi, Naoko; Inagaki, Kenichi; Hosoya, Takeshi; Nakamura, Eri; Terasaka, Tomohiro; Komatsubara, Motoshi; Makino, Hirofumi

    2015-08-01

    Melatonin has been reported to suppress adrenocorticotropin (ACTH) secretion in the anterior pituitary and cortisol production in the adrenal by different mechanisms. However, the effect of melatonin on aldosterone production has remained unknown. In this study, we investigated the role of melatonin in the regulation of aldosterone production using human adrenocortical H295R cells by focusing on the activin system expressed in the adrenal. Melatonin receptor MT1 mRNA and protein were expressed in H295R cells and the expression levels of MT1 were increased by activin treatment. Activin increased ACTH-induced, but not angiotensin II (Ang II)-induced, aldosterone production. Melatonin alone did not affect basal synthesis of either aldosterone or cortisol. However, melatonin effectively enhanced aldosterone production induced by co-treatment with ACTH and activin, although melatonin had no effect on aldosterone production induced by Ang II in combination with activin. These changes in steroidogenesis became apparent when the steroid production was evaluated by the ratio of aldosterone/cortisol. Melatonin also enhanced dibutyryl-AMP-induced aldosterone/cortisol levels in the presence of activin, suggesting a functional link to the cAMP-PKA pathway for induction of aldosterone production by melatonin and activin. In accordance with the data for steroids, ACTH-induced, but not Ang II-induced, cAMP synthesis was also amplified by co-treatment with melatonin and activin. Furthermore, the ratio of ACTH-induced mRNA level of CYP11B2 compared with that of CYP17 was amplified in the condition of treatment with both melatonin and activin. In addition, melatonin increased expression of the activin type-I receptor ALK-4 but suppressed expression of inhibitory Smads6/7, leading to the enhancement of Smad2 phosphorylation. Collectively, the results showed that melatonin facilitated aldosterone production induced by ACTH and activin via the cAMP-PKA pathway. The results also

  17. An Activin Receptor IA/Activin-Like Kinase-2 (R206H) Mutation in Fibrodysplasia Ossificans Progressiva.

    PubMed

    Herrera-Esparza, Rafael; Pacheco-Tovar, Deyanira; Bollain-Y-Goytia, Juan José; Torres Del Muro, Felipe; Ramírez-Sandoval, Roxana; Pacheco-Tovar, María Guadalupe; Castañeda-Ureña, María; Avalos-Díaz, Esperanza

    2013-01-01

    Fibrodysplasia ossificans progressiva (FOP) is an exceptionally rare genetic disease that is characterised by congenital malformations of the great toes and progressive heterotopic ossification (HO) in specific anatomical areas. This disease is caused by a mutation in activin receptor IA/activin-like kinase-2 (ACVR1/ALK2). A Mexican family with one member affected by FOP was studied. The patient is a 19-year-old female who first presented with symptoms of FOP at 8 years old; she developed spontaneous and painful swelling of the right scapular area accompanied by functional limitation of movement. Mutation analysis was performed in which genomic DNA as PCR amplified using primers flanking exons 4 and 6, and PCR products were digested with Cac8I and HphI restriction enzymes. The most informative results were obtained with the exon 4 flanking primers and the Cac8I restriction enzyme, which generated a 253 bp product that carries the ACVR1 617G>A mutation, which causes an amino acid substitution of histidine for arginine at position 206 of the glycine-serine (GS) domain, and its mutation results in the dysregulation of bone morphogenetic protein (BMP) signalling that causes FOP. PMID:23653868

  18. TAK-1/p38/nNFκB signaling inhibits myoblast differentiation by increasing levels of Activin A

    PubMed Central

    2012-01-01

    Background Skeletal-muscle differentiation is required for the regeneration of myofibers after injury. The differentiation capacity of satellite cells is impaired in settings of old age, which is at least one factor in the onset of sarcopenia, the age-related loss of skeletal-muscle mass and major cause of frailty. One important cause of impaired regeneration is increased levels of transforming growth factor (TGF)-β accompanied by reduced Notch signaling. Pro-inflammatory cytokines are also upregulated in aging, which led us hypothesize that they might potentially contribute to impaired regeneration in sarcopenia. Thus, in this study, we further analyzed the muscle differentiation-inhibition pathway mediated by pro-inflammatory cytokines in human skeletal muscle cells (HuSKMCs). Methods We studied the modulation of HuSKMC differentiation by the pro-inflammatory cytokines interleukin (IL)-1α and tumor necrosis factor (TNF)-α The grade of differentiation was determined by either imaging (fusion index) or creatine kinase (CK) activity, a marker of muscle differentiation. Secretion of TGF-β proteins during differentiation was assessed by using a TGF-β-responsive reporter-gene assay and further identified by means of pharmacological and genetic inhibitors. In addition, signaling events were monitored by western blotting and reverse transcription PCR, both in HuSKMC cultures and in samples from a rat sarcopenia study. Results The pro-inflammatory cytokines IL-1α and TNF-α block differentiation of human myoblasts into myotubes. This anti-differentiation effect requires activation of TGF-β-activated kinase (TAK)-1. Using pharmacological and genetic inhibitors, the TAK-1 pathway could be traced to p38 and NFκB. Surprisingly, the anti-differentiation effect of the cytokines required the transcriptional upregulation of Activin A, which in turn acted through its established signaling pathway: ActRII/ALK/SMAD. Inhibition of Activin A signaling was able to rescue human

  19. Targeting tumour vasculature by inhibiting activin receptor-like kinase (ALK)1 function.

    PubMed

    de Vinuesa, Amaya García; Bocci, Matteo; Pietras, Kristian; Ten Dijke, Peter

    2016-08-15

    Angiogenesis is a hallmark of cancer and is now a validated therapeutic target in the clinical setting. Despite the initial success, anti-angiogenic compounds impinging on the vascular endothelial growth factor (VEGF) pathway display limited survival benefits in patients and resistance often develops due to activation of alternative pathways. Thus, finding and validating new targets is highly warranted. Activin receptor-like kinase (ALK)1 is a transforming growth factor beta (TGF-β) type I receptor predominantly expressed in actively proliferating endothelial cells (ECs). ALK1 has been shown to play a pivotal role in regulating angiogenesis by binding to bone morphogenetic protein (BMP)9 and 10. Two main pharmacological inhibitors, an ALK1-Fc fusion protein (Dalantercept/ACE-041) and a fully human antibody against the extracellular domain of ALK1 (PF-03446962) are currently under clinical development. Herein, we briefly recapitulate the role of ALK1 in blood vessel formation and the current status of the preclinical and clinical studies on inhibition of ALK1 signalling as an anti-angiogenic strategy. Future directions in terms of new combination regimens will also be presented. PMID:27528762

  20. ACTIVIN IIB RECEPTOR BLOCKADE ATTENUATES DYSTROPHIC PATHOLOGY IN A MOUSE MODEL OF DUCHENNE MUSCULAR DYSTROPHY

    PubMed Central

    Morine, Kevin J.; Bish, Lawrence T.; Selsby, Joshua T.; Gazzara, Jeffery A.; Pendrak, Klara; Sleeper, Meg M.; Barton, Elisabeth R.; Lee, Se-Jin; Sweeney, H. Lee

    2015-01-01

    Modulation of transforming growth factor-β (TGF-β) signaling to promote muscle growth holds tremendous promise for the muscular dystrophies and other disorders involving the loss of functional muscle mass. Previous studies have focused on the TGF-β family member myostatin and demonstrated that inhibition of myostatin leads to muscle growth in normal and dystrophic mice. We describe a unique method of systemic inhibition of activin IIB receptor signaling via adeno-associated virus (AAV)-mediated gene transfer of a soluble form of the extracellular domain of the activin IIB receptor to the liver. Treatment of mdx mice with activin IIB receptor blockade led to increased skeletal muscle mass, increased force production in the extensor digitorum longus (EDL), and reduced serum creatine kinase. No effect on heart mass or function was observed. Our results indicate that activin IIB receptor blockade represents a novel and effective therapeutic strategy for the muscular dystrophies. PMID:20730876

  1. Abnormal concentration of maternal serum activin-A in gestational diseases.

    PubMed

    Petraglia, F; De Vita, D; Gallinelli, A; Aguzzoli, L; Genazzani, A R; Romero, R; Woodruff, T K

    1995-02-01

    Serum plasma activin-A is measurable in the maternal circulation of healthy pregnant women, increases in specimens collected during the third trimester of gestation, and is highest at parturition. Hormone abnormalities are known to be associated with preterm labor or diabetes in pregnancy. Therefore, in the present study serum activin-A levels in normal controls were compared to those in pregnant women with preterm labor or gestational diabetes. In some cases, values were obtained before and after insulin therapy. In other controls and patients with preterm labor, the activin-A concentration in cord serum was also studied. A newly developed two-site immunotest was used to determine activin-A levels. Subjects included normal controls (n = 7), who were sampled throughout gestation every 5 weeks; pregnant women at term (38-40 weeks) not in labor (n = 22); pregnant women at term in spontaneous labor (< 3.0 cm dilated; n = 42); women in preterm labor (25-35 weeks; n = 38); and women with gestational diabetes (20-39 weeks; n = 9). In control women, serum activin-A levels increased from 4.8 +/- 5.5 micrograms/L (mean +/- SD) at 20 weeks to 25.4 +/- 27.8 micrograms/L at 40 weeks (P < 0.01), and values correlated with gestational age. Pregnant women in preterm labor had serum activin-A concentrations (89.04 +/- 173.31 micrograms/L) higher than those in normal controls (P < 0.01), and no significant correlation to gestational age was found in this group of pregnant women. Healthy women in labor showed serum activin-A concentrations higher than those in women at term but not in labor (P < 0.01). Diabetic patients had serum activin-A concentrations (52.39 +/- 23.32 micrograms/L) significantly higher than those in normal controls. In these patients, maternal serum activin-A concentrations significantly decreased to the range in healthy controls at the same gestational age after insulin therapy (9.48 +/- 3.82 micrograms/L). The present study shows that preterm labor is

  2. Arabidopsis Type II Phosphatidylinositol 4-Kinase PI4Kγ5 Regulates Auxin Biosynthesis and Leaf Margin Development through Interacting with Membrane-Bound Transcription Factor ANAC078.

    PubMed

    Tang, Yong; Zhao, Chun-Yan; Tan, Shu-Tang; Xue, Hong-Wei

    2016-08-01

    Normal leaf margin development is important for leaf morphogenesis and contributes to diverse leaf shapes in higher plants. We here show the crucial roles of an atypical type II phosphatidylinositol 4-kinase, PI4Kγ5, in Arabidopsis leaf margin development. PI4Kγ5 presents a dynamics expression pattern along with leaf development and a T-DNA mutant lacking PI4Kγ5, pi4kγ5-1, presents serrated leaves, which is resulted from the accelerated cell division and increased auxin concentration at serration tips. Studies revealed that PI4Kγ5 interacts with and phosphorylates a membrane-bound NAC transcription factor, ANAC078. Previous studies demonstrated that membrane-bound transcription factors regulate gene transcription by undergoing proteolytic process to translocate into nucleus, and ANAC078 undergoes proteolysis by cleaving off the transmembrane region and carboxyl terminal. Western blot analysis indeed showed that ANAC078 deleting of carboxyl terminal is significantly reduced in pi4kγ5-1, indicating that PI4Kγ5 is important for the cleavage of ANAC078. This is consistent with the subcellular localization observation showing that fluorescence by GFP-ANAC078 is detected at plasma membrane but not nucleus in pi4kγ5-1 mutant and that expression of ANAC078 deleting of carboxyl terminal, driven by PI4Kγ5 promoter, could rescue the leaf serration defects of pi4kγ5-1. Further analysis showed that ANAC078 suppresses the auxin synthesis by directly binding and regulating the expression of auxin synthesis-related genes. These results indicate that PI4Kγ5 interacts with ANAC078 to negatively regulate auxin synthesis and hence influences cell proliferation and leaf development, providing informative clues for the regulation of in situ auxin synthesis and cell division, as well as the cleavage and functional mechanism of membrane-bound transcription factors. PMID:27529511

  3. Arabidopsis Type II Phosphatidylinositol 4-Kinase PI4Kγ5 Regulates Auxin Biosynthesis and Leaf Margin Development through Interacting with Membrane-Bound Transcription Factor ANAC078

    PubMed Central

    Tan, Shu-Tang; Xue, Hong-Wei

    2016-01-01

    Normal leaf margin development is important for leaf morphogenesis and contributes to diverse leaf shapes in higher plants. We here show the crucial roles of an atypical type II phosphatidylinositol 4-kinase, PI4Kγ5, in Arabidopsis leaf margin development. PI4Kγ5 presents a dynamics expression pattern along with leaf development and a T-DNA mutant lacking PI4Kγ5, pi4kγ5–1, presents serrated leaves, which is resulted from the accelerated cell division and increased auxin concentration at serration tips. Studies revealed that PI4Kγ5 interacts with and phosphorylates a membrane-bound NAC transcription factor, ANAC078. Previous studies demonstrated that membrane-bound transcription factors regulate gene transcription by undergoing proteolytic process to translocate into nucleus, and ANAC078 undergoes proteolysis by cleaving off the transmembrane region and carboxyl terminal. Western blot analysis indeed showed that ANAC078 deleting of carboxyl terminal is significantly reduced in pi4kγ5–1, indicating that PI4Kγ5 is important for the cleavage of ANAC078. This is consistent with the subcellular localization observation showing that fluorescence by GFP-ANAC078 is detected at plasma membrane but not nucleus in pi4kγ5–1 mutant and that expression of ANAC078 deleting of carboxyl terminal, driven by PI4Kγ5 promoter, could rescue the leaf serration defects of pi4kγ5–1. Further analysis showed that ANAC078 suppresses the auxin synthesis by directly binding and regulating the expression of auxin synthesis-related genes. These results indicate that PI4Kγ5 interacts with ANAC078 to negatively regulate auxin synthesis and hence influences cell proliferation and leaf development, providing informative clues for the regulation of in situ auxin synthesis and cell division, as well as the cleavage and functional mechanism of membrane-bound transcription factors. PMID:27529511

  4. Two specific membrane-bound aminopeptidase N isoforms from Aedes aegypti larvae serve as functional receptors for the Bacillus thuringiensis Cry4Ba toxin implicating counterpart specificity.

    PubMed

    Aroonkesorn, Aratee; Pootanakit, Kusol; Katzenmeier, Gerd; Angsuthanasombat, Chanan

    2015-05-29

    The interaction between Bacillus thuringiensis Cry toxins and their receptors on midgut cells of susceptible insect larvae is the critical determinant in toxin specificity. Besides GPI-linked alkaline phosphatase in Aedes aegypti mosquito-larval midguts, membrane-bound aminopeptidase N (AaeAPN) is widely thought to serve as a Cry4Ba receptor. Here, two full-length AaeAPN isoforms, AaeAPN2778 and AaeAPN2783, predicted to be GPI-linked were cloned and successfully expressed in Spodoptera frugiperda (Sf9) cells as 112- and 107-kDa membrane-bound proteins, respectively. In the cytotoxicity assay, Sf9 cells expressing each of the two AaeAPN isoforms showed increased sensitivity to the Cry4Ba mosquito-active toxin. Double immunolocalization revealed specific binding of Cry4Ba to each individual AaeAPN expressed on the cell membrane surface. Sequence analysis and homology-based modeling placed these two AaeAPNs to the M1 aminopeptidase family as they showed similar four-domain structures, with the most conserved domain II being the catalytic component. Additionally, the most variable domain IV containing negatively charged surface patches observed only in dipteran APNs could be involved in insect specificity. Overall results demonstrated that these two membrane-bound APN isoforms were responsible for mediating Cry4Ba toxicity against AaeAPN-expressed Sf9 cells, suggesting their important role as functional receptors for the toxin counterpart in A. aegypti mosquito larvae. PMID:25871797

  5. Change in the Gastro-Intestinal Tract by Overexpressed Activin Beta A

    PubMed Central

    Kim, Mi-Nyeu; Kim, Young Il; Cho, Chunghee; Mayo, Kelly E.; Cho, Byung-Nam

    2015-01-01

    Originally, activins were identified as stimulators of FSH release in reproduction. Other activities, including secondary axis formation in development, have since been revealed. Here, we investigated the influence of activin βA on the body, including the gastro-intestinal (GI) tract. Initially, the activin βA protein was detected in the serum proportional to the amount of pCMV-rAct plasmid injected. The induced level of activin βA in muscle was higher in female than male mice. Subsequent results revealed that stomach and intestine were severely damaged in pCMV-rAct-injected mice. At the cellular level, loss of parietal cells was observed, resulting in increased pH within the stomach. This phenomenon was more severe in male than female mice. Consistent with damage of the stomach and intestine, activin βA often led to necrosis in the tip of the tail or foot, and loss of body weight was observed in pCMV-rAct-injected male but not female mice. Finally, in pCMV-rAct-injected mice, circulating activin βA led to death at supraphysiological doses, and this was dependent on the strain of mice used. Taken together, these results indicate that activin βA has an important role outside of reproduction and development, specifically in digestion. These data also indicate that activin βA must be controlled within a narrow range because of latent lethal activity. In addition, our approach can be used effectively for functional analysis of secreted proteins. PMID:26608361

  6. Activin A is associated with asthma in underweight and overweight patients.

    PubMed

    Zhang, L L; Liu, C T

    2015-01-01

    There are limited data regarding the effects of activin A on underweight, normal weight, and overweight patients with asthma. We determined serum levels of activin A in asthmatic patients in relation to body mass index. The study protocol included questionnaires, measurement of exhaled nitric oxide, blood sampling for inflammatory biomarkers, and high-resolution computed tomography of the lungs to identify bronchial wall thickening. Serum and sputum activin A levels were measured using an enzyme-linked immunosorbent assay in 94 asthmatic patients. Mean serum levels of activin A were significantly (P = 0.001) higher in underweight (1781 ± 327.3 pg/mL) than in normal weight and overweight asthmatic patients, regardless of gender. After stratification by gender, significantly higher mean values of activin A were observed in females compared to males in the normal and underweight groups (P = 0.003 and P = 0.0002, respectively). Significant differences between groups were found in airway wall area (%) (P < 0.0001). We also observed a much higher percentage of sputum lymphocytes in the underweight group compared to the other groups (P < 0.0001). Correlations between bronchial wall thickness and activin A were found in the underweight (r = 0.67, P = 0.48) and normal weight groups (r = 0.51, P = 0.042). Correlations between fractional of exhaled nitric oxide, forced expiratory volume in 1 s, delayed treatment years, and activin A in different groups were also observed. Increased serum level of activin A indicates its role in the pathogenesis of asthma, particularly in underweight and overweight patients.

  7. Activin a signaling regulates cell invasion and proliferation in esophageal adenocarcinoma

    PubMed Central

    Le Bras, Gregoire F.; Koumangoye, Rainelli B.; Romero-Morales, Alejandra I.; Quast, Laura L.; Zaika, Alexander I.; El-Rifai, Wael; Andl, Thomas; Andl, Claudia D.

    2015-01-01

    TGFβ signaling has been implicated in the metaplasia from squamous epithelia to Barrett's esophagus and, ultimately, esophageal adenocarcinoma. The role of the family member Activin A in Barrett's tumorigenesis is less well established. As tumorigenesis is influenced by factors in the tumor microenvironment, such as fibroblasts and the extracellular matrix, we aimed to determine if epithelial cell-derived Activin affects initiation and progression differently than Activin signaling stimulation from a mimicked stromal source. Using Barrett's esophagus cells, CPB, and the esophageal adenocarcinoma cell lines OE33 and FLO-1, we showed that Activin reduces colony formation only in CPB cells. Epithelial cell overexpression of Activin increased cell migration and invasion in Boyden chamber assays in CPB and FLO-1 cells, which exhibited mesenchymal features such as the expression of the CD44 standard form, vimentin, and MT1-MMP. When grown in organotypic reconstructs, OE33 cells expressed E-cadherin and Keratin 8. As mesenchymal characteristics have been associated with the acquisition of stem cell-like features, we analyzed the expression and localization of SOX9, showing nuclear localization of SOX9 in esophageal CPB and FLO-1 cells. In conclusion, we show a role for autocrine Activin signaling in the regulation of colony formation, cell migration and invasion in Barrett's tumorigenesis. PMID:26447543

  8. The Biology Of Activin: Recent Advances In Structure, Regulation And Function

    PubMed Central

    Xia, Yin; Schneyer, Alan L.

    2009-01-01

    Activin was discovered in the 1980’s as a gonadal protein that stimulated FSH release from pituitary gonadotropes and was thought of as a reproductive hormone. In the ensuing decades many additional activities of activin were described and it was found to be produced in a wide variety of cell types at nearly all stages of development. Its signaling and actions are regulated intracellularly as well as by extracellular antagonists. Over the past 5 years a number of important advances have been made that clarify our understanding of the structural basis for signaling and regulation, as well as the biological roles of activin in stem cells, embryonic development, and in adults. These include the crystallization of activin in complex with the activin type II receptor ActRIIB, or with the binding proteins follistatin and follistatin-like 3 (FSTL3), and identification of the activin roles in gonadal sex development, follicle development and luteolysis, in β-cell proliferation and function in the islet, in stem cell self-renewal and differentiation into different cell types, and in immune cells. These advances are reviewed to provide perspective for future studies. PMID:19273500

  9. Immunohistochemical localization of inhibin/activin subunits in the wild ground squirrel (Citellus dauricus Brandt) ovary.

    PubMed

    Sheng, Xia; Weng, Jiaju; Zhang, Haolin; Li, Xiaonan; Zhang, Mengyuan; Xu, Meiyu; Weng, Qiang; Watanabe, Gen; Taya, Kazuyoshi

    2012-01-01

    The intraovarian function of gonadally produced inhibin and activin has been extensively studied in experimental models for decades, yet their presence and function have been rarely reported in wild rodents. With our seasonal breeding model, the wild ground squirrel, we aimed to investigate the possible roles of these peptides in the seasonal folliculogenesis. Immunohistochemical staining and Western blotting have been used to detect the cellular localization and expression patterns of inhibin/activin subunits (α, β(A) and β(B)). In the breeding season ovary, all three subunits were present in granulosa cells, theca cells of antral follicles and interstitial cells, with the strongest immunostaining in granulosa cells. Following ovulation, the corpora lutea become a major site of inhibin/activin synthesis. In the nonbreeding season ovary, inhibin/activin α and β(A) subunits were weakly immunopositive in granulosa cells of early stage follicles, while β(B) subunit was undetectable. The expression level of inhibin/activin subunit proteins were generally higher in the ovaries of the breeding season, and then decreased to a relatively low level during the nonbreeding season. The dynamic expression of inhibin/activin subunits indicated that they might play important paracrine and/or autocrine roles during the seasonal folliculogenesis of the wild ground squirrel.

  10. Activin B promotes initiation and development of hair follicles in mice.

    PubMed

    Jia, Qin; Zhang, Min; Kong, Yanan; Chen, Shixuan; Chen, Yinghua; Wang, Xueer; Zhang, Lei; Lang, Weiya; Zhang, Lu; Zhang, Lin

    2013-01-01

    Activin B has been reported to promote the regeneration of hair follicles during wound healing. However, its role in the development and life cycle of hair follicles has not been elucidated. In our study, the effect of activin B on mouse hair follicles of cultured and neonatal mouse skin was investigated. In these models, PBS or activin B (5, 10 or 50 ng/ml) was applied, and hair follicle development was monitored. Hair follicle initiation and development was examined using hematoxylin and eosin staining, alkaline phosphatase activity staining, Oil Red O+ staining, and the detection of TdT-mediated dUTP-biotin nick end-labeling cell apoptosis. Activin B was found to efficiently induce the initiation of hair follicles in the skin of both cultured and neonatal mice and to promote the development of hair follicles in neonatal mouse skin. Moreover, activin-B-treated hair follicles were observed to enter the anagen stage from the telogen stage and to remain in the anagen stage. These results demonstrate that activin B promotes the initiation and development of hair follicles in mice.

  11. Lysophosphatidylethanolamine acyltransferase 1/membrane-bound O-acyltransferase 1 regulates morphology and function of P19C6 cell-derived neurons.

    PubMed

    Tabe, Shirou; Hikiji, Hisako; Ariyoshi, Wataru; Hashidate-Yoshida, Tomomi; Shindou, Hideo; Okinaga, Toshinori; Shimizu, Takao; Tominaga, Kazuhiro; Nishihara, Tatsuji

    2016-07-01

    Glycerophospholipids, which are components of biomembranes, are formed de novo by the Kennedy pathway and subsequently mature through the Lands cycle. Lysophospholipid acyltransferases (LPLATs) are key enzymes in both pathways and influence the fatty acid composition of biomembranes. Neuronal differentiation is characterized by neurite outgrowth, which requires biomembrane biosynthesis. However, the role of LPLATs in neuronal differentiation remains unknown. In this study, we examined whether LPLATs are involved in neuronal differentiation using all-trans-retinoic acid (ATRA)-treated P19C6 cells. In these cells, mRNA levels of lysophosphatidylethanolamine acyltransferase (LPEAT)-1/membrane-bound O-acyltransferase (MBOAT)-1 were higher than those in undifferentiated cells. LPEAT enzymatic activity increased with 16:0- and 18:1-CoA as acyl donors. When LPEAT1/MBOAT1 was knocked down with small interfering RNA (siRNA), outgrowth of neurites and expression of neuronal markers decreased in ATRA-treated P19C6 cells. Voltage-dependent calcium channel activity was also suppressed in these cells transfected with LPEAT1/MBOAT1 siRNA. These results suggest that LPEAT1/MBOAT1 plays an important role in neurite outgrowth and function.-Tabe, S., Hikiji, H., Ariyoshi, W., Hashidate-Yoshida, T., Shindou, H., Okinaga, T., Shimizu, T., Tominaga, K., Nishihara, T. Lysophosphatidylethanolamine acyltransferase 1/membrane-bound O-acyltransferase 1 regulates morphology and function of P19C6 cell-derived neurons. PMID:27048541

  12. An organelle-free assay for pea chloroplast Mg-chelatase: Resolution of the activity into soluble and membrane bound fractions

    SciTech Connect

    Walker, C.J.; Weinstein, J.D. )

    1991-05-01

    Mg-chelatase, which catalyzes the insertion of magnesium into protoporphyrin, lies at the branchpoint of heme and chlorophyll biosynthesis in chloroplasts. Since magnesium chelation is the first step unique to chlorophyll synthesis, one would expect this step to be highly regulated. However, to date little is known about the enzymology or regulation of Mg-chelatase due mostly to an inability to assay it's activity outside of the intact plastid. Here the authors report the first truly in vitro i.e. organelle-free, assay for Mg-chelatase. Mg-chelatase activity in intact pea chloroplasts which is 3 to 4 fold higher than in cucumber chloroplasts, survived chloroplast lysis and could be fractionated, by centrifugation, into supernatant and pellet components. Both of these fractions were required to reconstitute Mg-chelatase activity and both were inactivated by boiling; indicating that the enzyme is composed of soluble and membrane bound protein(s). The specific activity of the reconstituted system was typically 1 nmol Mg-Deuteroporphyrin/h/mg protein and activity was linear for at least 60 min under our assay conditions. ATP and magnesium were required for Mg-chelatase activity. The soluble component could be fractionated with ammonium sulfate. The product of the reaction was confirmed fluorometrically as the magnesium chelate of the porphyrin substrate. Crude separation of chloroplast membranes into thylakoids and envelopes, suggested that the membrane-bound component of Mg-chelatase is probably located in the envelope.

  13. Characterization and inactivation of the membrane-bound polyol dehydrogenase in Gluconobacter oxydans DSM 7145 reveals a role in meso-erythritol oxidation.

    PubMed

    Voss, Jörn; Ehrenreich, Armin; Liebl, Wolfgang

    2010-06-01

    The growth of Gluconobacter oxydans DSM 7145 on meso-erythritol is characterized by two stages: in the first stage, meso-erythritol is oxidized almost stoichiometrically to L-erythrulose according to the Bertrand-Hudson rule. The second phase is distinguished from the first phase by a global metabolic change from membrane-bound meso-erythritol oxidation to L-erythrulose assimilation with concomitant accumulation of acetic acid. The membrane-associated erythritol-oxidizing enzyme was found to be encoded by a gene homologous to sldA known from other species of acetic acid bacteria. Disruption of this gene in the genome of G. oxydans DSM 7145 revealed that the membrane-bound polyol dehydrogenase not only oxidizes meso-erythritol but also has a broader substrate spectrum which includes C3-C6 polyols and D-gluconate and supports growth on these substrates. Cultivation of G. oxydans DSM 7145 on different substrates indicated that expression of the polyol dehydrogenase was not regulated, implying that the production of biomass of G. oxydans to be used as whole-cell biocatalysts in the biotechnological conversion of meso-erythritol to L-erythrulose, which is used as a tanning agent in the cosmetics industry, can be conveniently carried out with glucose as the growth substrate.

  14. The Autocrine Mitogenic Loop of the Ciliate Euplotes raikovi: The Pheromone Membrane-bound Forms Are the Cell Binding Sites and Potential Signaling Receptors of Soluble Pheromones

    PubMed Central

    Ortenzi, Claudio; Alimenti, Claudio; Vallesi, Adriana; Di Pretoro, Barbara; Terza, Antonietta La; Luporini, Pierangelo

    2000-01-01

    Homologous proteins, denoted pheromones, promote cell mitotic proliferation and mating pair formation in the ciliate Euplotes raikovi, according to whether they bind to cells in an autocrine- or paracrine-like manner. The primary transcripts of the genes encoding these proteins undergo alternate splicing, which generates at least two distinct mRNAs. One is specific for the soluble pheromone, the other for a pheromone isoform that remains anchored to the cell surface as a type II protein, whose extracellular C-terminal region is structurally equivalent to the secreted form. The 15-kDa membrane-bound isoform of pheromone Er-1, denoted Er-1mem and synthesized by the same E. raikovi cells that secrete Er-1, has been purified from cell membranes by affinity chromatography prepared with matrix-bound Er-1, and its extracellular and cytoplasmic regions have been expressed as recombinant proteins. Using the purified material and these recombinant proteins, it has been shown that Er-1mem has the property of binding pheromones competitively through its extracellular pheromone-like domain and associating reversibly and specifically with a guanine nucleotide-binding protein through its intracellular domain. It has been concluded that the membrane-bound pheromone isoforms of E. raikovi represent the cell effective pheromone binding sites and are functionally equipped for transducing the signal generated by this binding. PMID:10749941

  15. The role of myostatin and activin receptor IIB in the regulation of unloading-induced myofiber type-specific skeletal muscle atrophy.

    PubMed

    Babcock, Lyle W; Knoblauch, Mark; Clarke, Mark S F

    2015-09-15

    Chronic unloading induces decrements in muscle size and strength. This adaptation is governed by a number of molecular factors including myostatin, a potent negative regulator of muscle mass. Myostatin must first be secreted into the circulation and then bind to the membrane-bound activin receptor IIB (actRIIB) to exert its atrophic action. Therefore, we hypothesized that myofiber type-specific atrophy observed after hindlimb suspension (HLS) would be related to myofiber type-specific expression of myostatin and/or actRIIB. Wistar rats underwent HLS for 10 days, after which the tibialis anterior was harvested for frozen cross sectioning. Simultaneous multichannel immunofluorescent staining combined with differential interference contrast imaging was employed to analyze myofiber type-specific expression of myostatin and actRIIB and myofiber type cross-sectional area (CSA) across fiber types, myonuclei, and satellite cells. Hindlimb suspension (HLS) induced significant myofiber type-specific atrophy in myosin heavy chain (MHC) IIx (P < 0.05) and MHC IIb myofibers (P < 0.05). Myostatin staining associated with myonuclei was less in HLS rats compared with controls, while satellite cell staining for myostatin remained unchanged. In contrast, the total number myonuclei and satellite cells per myofiber was reduced in HLS compared with ambulatory control rats (P < 0.01). Sarcoplasmic actRIIB staining differed between myofiber types (I < IIa < IIx < IIb) independent of loading conditions. Myofiber types exhibiting the greatest cytoplasmic staining of actRIIB corresponded to those exhibiting the greatest degree of atrophy following HLS. Our data suggest that differential expression of actRIIB may be responsible for myostatin-induced myofiber type-selective atrophy observed during chronic unloading. PMID:26205544

  16. Serum activin A and B, and follistatin in critically ill patients with influenza A(H1N1) infection

    PubMed Central

    2014-01-01

    Background Activin A and its binding protein follistatin (FS) are increased in inflammatory disorders and sepsis. Overexpression of activin A in the lung causes similar histopathological changes as acute respiratory distress syndrome (ARDS). ARDS and severe respiratory failure are complications of influenza A(H1N1) infection. Interleukin 6 (IL-6), which in experimental studies increases after activin A release, is known to be related to the severity of H1N1 infection. Our aim was to evaluate the levels of activin A, activin B, FS, IL-6 and IL-10 and their association with the severity of respiratory failure in critically ill H1N1 patients. Methods A substudy of a prospective, observational cohort of H1N1 patients in Finnish intensive care units (ICU). Clinical information was recorded during ICU treatment, and serum activin A, activin B, FS, IL-6 and IL-10 were measured at admission to ICU and on days 2 and 7. Results Blood samples from 29 patients were analysed. At the time of admission to intensive care unit, elevated serum levels above the normal range for respective age group and sex were observed in 44% for activin A, 57% for activin B, and 39% for FS. In 13 of the 29 patients, serial samples at all time points were available and in these the highest activin A, activin B and FS were above the normal range in 85%, 100% and 46% of the patients, respectively. No difference in baseline or highest activin A or activin B was found in patients with or without acute lung injury (ALI) or ARDS (P > 0.05 for all). Peak levels of IL-6 were significantly elevated in ALI/ARDS patients. Peak activin A and activin A/FS were associated with ventilatory support free-days, severity of acute illness and length of ICU stay (P < 0.05 for all). Conclusions Higher than normal values of these proteins were common in patients with H1N1 infection but we found no association with the severity of their respiratory failure. PMID:24885241

  17. Activin Modulates the Transcriptional Response of LβT2 Cells to Gonadotropin-Releasing Hormone and Alters Cellular Proliferation

    PubMed Central

    Zhang, Hao; Bailey, Janice S.; Coss, Djurdjica; Lin, Bo; Tsutsumi, Rie; Lawson, Mark A.; Mellon, Pamela L.; Webster, Nicholas J. G.

    2009-01-01

    Both GnRH and activin are crucial for the correct function of pituitary gonadotrope cells. GnRH regulates LH and FSH synthesis and secretion and gonadotrope proliferation, whereas activin is essential for expression of FSH. Little is known, however, about the interplay of signaling downstream of these two hormones. In this study, we undertook expression profiling to determine how activin pre-treatment alters the transcriptional response of LβT2 gonadotrope cells to GnRH stimulation. Activin treatment alone altered the transcriptional profile of 303 genes including inducing that of the 17β-hydroxysteroid dehydrogenase B1 gene that converts estrone to 17β-estradiol, altering the sensitivity of the cells to estrone. Furthermore, activin had a dramatic effect on the response of LβT2 cells to GnRH. Hierarchical clustering of 2453 GnRH-responsive genes identified groups of genes the response of which to GnRH was either enhanced or blunted after activin treatment. Mapping of these genes to gene ontology classifications or signaling pathways highlighted significant differences in the classes of altered genes. In the presence of activin, GnRH regulates genes in pathways controlling cell energetics, cytoskeletal rearrangements, organelle organization, and mitosis in the absence of activin, but genes controlling protein processing, cell differentiation, and secretion. Therefore, we demonstrated that activin enhanced GnRH induction of p38MAPK activity, caused GnRH-dependent phosphorylation of p53, and reduced the ability of GnRH to cause G1 arrest. Thus, although activin alone changes a modest number of transcripts, activin pretreatment dramatically alters the response to GnRH from an antiproliferative response to a more differentiated, synthetic response appropriate for a secretory cell. PMID:16772531

  18. Activin Signaling Targeted by Insulin/dFOXO Regulates Aging and Muscle Proteostasis in Drosophila

    PubMed Central

    Bai, Hua; Kang, Ping; Hernandez, Ana Maria; Tatar, Marc

    2013-01-01

    Reduced insulin/IGF signaling increases lifespan in many animals. To understand how insulin/IGF mediates lifespan in Drosophila, we performed chromatin immunoprecipitation-sequencing analysis with the insulin/IGF regulated transcription factor dFOXO in long-lived insulin/IGF signaling genotypes. Dawdle, an Activin ligand, is bound and repressed by dFOXO when reduced insulin/IGF extends lifespan. Reduced Activin signaling improves performance and protein homeostasis in muscles of aged flies. Activin signaling through the Smad binding element inhibits the transcription of Autophagy-specific gene 8a (Atg8a) within muscle, a factor controlling the rate of autophagy. Expression of Atg8a within muscle is sufficient to increase lifespan. These data reveal how insulin signaling can regulate aging through control of Activin signaling that in turn controls autophagy, representing a potentially conserved molecular basis for longevity assurance. While reduced Activin within muscle autonomously retards functional aging of this tissue, these effects in muscle also reduce secretion of insulin-like peptides at a distance from the brain. Reduced insulin secretion from the brain may subsequently reinforce longevity assurance through decreased systemic insulin/IGF signaling. PMID:24244197

  19. The immunoregulatory and fibrotic roles of activin A in allergic asthma

    PubMed Central

    Hardy, C L; Rolland, J M; O'Hehir, R E

    2015-01-01

    Activin A, a member of the TGF-β superfamily of cytokines, was originally identified as an inducer of follicle stimulating hormone release, but has since been ascribed roles in normal physiological processes, as an immunoregulatory cytokine and as a driver of fibrosis. In the last 10–15 years, it has also become abundantly clear that activin A plays an important role in the regulation of asthmatic inflammation and airway remodelling. This review provides a brief introduction to the activin A/TGF-β superfamily, focussing on the regulation of receptors and signalling pathways. We examine the contradictory evidence for generalized pro- vs. anti-inflammatory effects of activin A in inflammation, before appraising its role in asthmatic inflammation and airway remodelling specifically by evaluating data from both murine models and clinical studies. We identify key issues to be addressed, paving the way for safe exploitation of modulation of activin A function for treatment of allergic asthma and other inflammatory lung diseases. PMID:25962695

  20. Xenopus axis formation: induction of goosecoid by injected Xwnt-8 and activin mRNAs.

    PubMed

    Steinbeisser, H; De Robertis, E M; Ku, M; Kessler, D S; Melton, D A

    1993-06-01

    In this study, we compare the effects of three mRNAs-goosecoid, activin and Xwnt-8- that are able to induce partial or complete secondary axes when injected into Xenopus embryos. Xwnt-8 injection produces complete secondary axes including head structures whereas activin and goosecoid injection produce partial secondary axes at high frequency that lack head structures anterior to the auditory vesicle and often lack notochord. Xwnt-8 can activate goosecoid only in the deep marginal zone, i.e., in the region in which this organizer-specific homeobox gene is normally expressed on the dorsal side. Activin B mRNA, however, can turn on goosecoid in all regions of the embryo. We also tested the capacity of these gene products to restore axis formation in embryos in which the cortical rotation was blocked by UV irradiation. Whereas Xwnt-8 gives complete rescue of anterior structures, both goosecoid and activin give partial rescue. Rescued axes including hindbrain structures up to level of the auditory vesicle can be obtained at high frequency even in the absence of notochord structures. The possible functions of Wnt-like and activin-like signals and of the goosecoid homeobox gene, and their order of action in the formation of Spemann's organizer are discussed. PMID:7900991

  1. Activin-like factor from a Xenopus laevis cell line responsible for mesoderm induction.

    PubMed

    van den Eijnden-Van Raaij, A J; van Zoelent, E J; van Nimmen, K; Koster, C H; Snoek, G T; Durston, A J; Huylebroeck, D

    1990-06-21

    Induction of mesoderm during early amphibian embryogenesis can be mimicked in vitro by adding growth factors, including heparin-binding and type-beta transforming growth factors (TGF-beta), to isolated ectoderm explants from Xenopus laevis embryos. Although the mesoderm-inducing factor (MIF) from X. laevis XTC cells (XTC-MIF) has properties similar to TGF-beta, this factor is still unidentified. Recently, we obtained a number of homogeneous cell lines from the heterogeneous XTC population, which differ in their MIF production. Only one, XTC-GTX-11, produced MIF, although it was similar to the rest of the clones in its production of known growth factors, including TGF-beta activity. This observation, together with the identification of activin A as a potent MIF led us to study the parallel activities of MIF and activin. Here we report an analysis of activin-like activity from XTC cells and some of the XTC clones, including XTC-GTX-11. There is a clear consistent correlation between MIF activity and presence of activin activity, indicating that XTC-MIF is the Xenopus homologue of mammalian activin.

  2. Intermolecular communication on a liposomal membrane: enzymatic amplification of a photonic signal with a gemini peptide lipid as a membrane-bound artificial receptor.

    PubMed

    Mukai, Masaru; Maruo, Kohei; Sasaki, Yoshihiro; Kikuchi, Jun-ichi

    2012-03-12

    A supramolecular system that can activate an enzyme through photo-isomerization was constructed by using a liposomal membrane scaffold. The design of the system was inspired by natural signal transduction systems, in which enzymes amplify external signals to control signal transduction pathways. The liposomal membrane, which provided a scaffold for the system, was prepared by self-assembly of a photoresponsive receptor and a cationic synthetic lipid. NADH-dependent L-lactate dehydrogenase, the signal amplifier, was immobilized on the liposomal surface by electrostatic interactions. Recognition of photonic signals by the membrane-bound receptor induced photo-isomerization, which significantly altered the receptor's metal-binding affinity. The response to the photonic signal was transmitted to the enzyme by Cu(2+) ions. The enzyme amplified the chemical information through a catalytic reaction to generate the intended output signal.

  3. Resolution of Distinct Membrane-Bound Enzymes from Enterobacter cloacae SLD1a-1 That Are Responsible for Selective Reduction of Nitrate and Selenate Oxyanions

    PubMed Central

    Ridley, Helen; Watts, Carys A.; Richardson, David J.; Butler, Clive S.

    2006-01-01

    Enterobacter cloacae SLD1a-1 is capable of reductive detoxification of selenate to elemental selenium under aerobic growth conditions. The initial reductive step is the two-electron reduction of selenate to selenite and is catalyzed by a molybdenum-dependent enzyme demonstrated previously to be located in the cytoplasmic membrane, with its active site facing the periplasmic compartment (C. A. Watts, H. Ridley, K. L. Condie, J. T. Leaver, D. J. Richardson, and C. S. Butler, FEMS Microbiol. Lett. 228:273-279, 2003). This study describes the purification of two distinct membrane-bound enzymes that reduce either nitrate or selenate oxyanions. The nitrate reductase is typical of the NAR-type family, with α and β subunits of 140 kDa and 58 kDa, respectively. It is expressed predominantly under anaerobic conditions in the presence of nitrate, and while it readily reduces chlorate, it displays no selenate reductase activity in vitro. The selenate reductase is expressed under aerobic conditions and expressed poorly during anaerobic growth on nitrate. The enzyme is a heterotrimeric (αβγ) complex with an apparent molecular mass of ∼600 kDa. The individual subunit sizes are ∼100 kDa (α), ∼55 kDa (β), and ∼36 kDa (γ), with a predicted overall subunit composition of α3β3γ3. The selenate reductase contains molybdenum, heme, and nonheme iron as prosthetic constituents. Electronic absorption spectroscopy reveals the presence of a b-type cytochrome in the active complex. The apparent Km for selenate was determined to be ∼2 mM, with an observed Vmax of 500 nmol SeO42− min−1 mg−1 (kcat, ∼5.0 s−1). The enzyme also displays activity towards chlorate and bromate but has no nitrate reductase activity. These studies report the first purification and characterization of a membrane-bound selenate reductase. PMID:16885262

  4. Effect of feeding lipids recovered from fish processing waste by lactic acid fermentation and enzymatic hydrolysis on antioxidant and membrane bound enzymes in rats.

    PubMed

    Rai, Amit Kumar; Bhaskar, N; Baskaran, V

    2015-06-01

    Fish oil recovered from fresh water fish visceral waste (FVW-FO) through lactic acid fermentation (FO-LAF) and enzymatic hydrolysis (FO-EH) were fed to rats to study their influence on lipid peroxidation and activities of antioxidant and membrane bound enzyme in liver, heart and brain. Feeding of FO-LAF and FO-EH resulted in increase (P < 0.05) in lipid peroxides level in serum, liver, brain and heart tissues compared to ground nut oil (control). Activity of catalase (40-235 %) and superoxide dismutase (17-143 %) also increased (P < 0.05) with incremental level of EPA + DHA in diet. The increase was similar to cod liver oil fed rats at same concentration of EPA + DHA. FO-LAF and FO-EH increased (P < 0.05) the Na(+)K(+) ATPase activity in liver and brain microsomes, Ca(+)Mg(+) ATPase in heart microsome and acetylcholine esterase in brain microsomes when fed with 5 % EPA + DHA. There was also significant change in fatty acid composition and cholesterol/phospholipid ratio in microsomes of rat fed with FVW-FO. Feeding FVW-FO recovered by biotechnological approaches enhanced the activity of antioxidant enzymes in tissues, modulates the activities of membrane bound enzymes and improved the fatty acid composition in microsomes of tissues similar to CLO. Utilization of these processing wastes for the production of valuable biofunctional products can reduce the mounting economic values of fish oil and minimize the environmental pollution problems.

  5. ATPaseTb2, a Unique Membrane-bound FoF1-ATPase Component, Is Essential in Bloodstream and Dyskinetoplastic Trypanosomes

    PubMed Central

    Šubrtová, Karolína; Panicucci, Brian; Zíková, Alena

    2015-01-01

    In the infectious stage of Trypanosoma brucei, an important parasite of humans and livestock, the mitochondrial (mt) membrane potential (Δψm) is uniquely maintained by the ATP hydrolytic activity and subsequent proton pumping of the essential FoF1-ATPase. Intriguingly, this multiprotein complex contains several trypanosome-specific subunits of unknown function. Here, we demonstrate that one of the largest novel subunits, ATPaseTb2, is membrane-bound and localizes with monomeric and multimeric assemblies of the FoF1-ATPase. Moreover, RNAi silencing of ATPaseTb2 quickly leads to a significant decrease of the Δψm that manifests as a decreased growth phenotype, indicating that the FoF1-ATPase is impaired. To further explore the function of this protein, we employed a trypanosoma strain that lacks mtDNA (dyskinetoplastic, Dk) and thus subunit a, an essential component of the proton pore in the membrane Fo-moiety. These Dk cells generate the Δψm by combining the hydrolytic activity of the matrix-facing F1-ATPase and the electrogenic exchange of ATP4- for ADP3- by the ATP/ADP carrier (AAC). Surprisingly, in addition to the expected presence of F1-ATPase, the monomeric and multimeric FoF1-ATPase complexes were identified. In fact, the immunoprecipitation of a F1-ATPase subunit demonstrated that ATPaseTb2 was a component of these complexes. Furthermore, RNAi studies established that the membrane-bound ATPaseTb2 subunit is essential for maintaining normal growth and the Δψm of Dk cells. Thus, even in the absence of subunit a, a portion of the FoF1-ATPase is assembled in Dk cells. PMID:25714685

  6. Conversion of membrane-bound Fas(CD95) ligand to its soluble form is associated with downregulation of its proapoptotic activity and loss of liver toxicity.

    PubMed

    Schneider, P; Holler, N; Bodmer, J L; Hahne, M; Frei, K; Fontana, A; Tschopp, J

    1998-04-20

    Human Fas ligand (L) (CD95L) and tumor necrosis factor (TNF)-alpha undergo metalloproteinase-mediated proteolytic processing in their extracellular domains resulting in the release of soluble trimeric ligands (soluble [s]FasL, sTNF-alpha) which, in the case of sFasL, is thought to be implicated in diseases such as hepatitis and AIDS. Here we show that the processing of sFasL occurs between Ser126 and Leu127. The apoptotic-inducing capacity of naturally processed sFasL was reduced by >1,000-fold compared with membrane-bound FasL, and injection of high doses of recombinant sFasL in mice did not induce liver failure. However, soluble FasL retained its capacity to interact with Fas, and restoration of its cytotoxic activity was achieved both in vitro and in vivo with the addition of cross-linking antibodies. Similarly, the marginal apoptotic activity of recombinant soluble TNF-related apoptosis-inducing ligand (sTRAIL), another member of the TNF ligand family, was greatly increased upon cross-linking. These results indicate that the mere trimerization of the Fas and TRAIL receptors may not be sufficient to trigger death signals. Thus, the observation that sFasL is less cytotoxic than membrane-bound FasL may explain why in certain types of cancer, systemic tissue damage is not detected, even though the levels of circulating sFasL are high. PMID:9547332

  7. Tryptophan orientations in membrane-bound gramicidin and melittin-a comparative linear dichroism study on transmembrane and surface-bound peptides.

    PubMed

    Svensson, Frida R; Lincoln, Per; Nordén, Bengt; Esbjörner, Elin K

    2011-01-01

    In the search for methods to study structure and function of membrane-associated proteins and peptides flow linear dichroism, LD, spectroscopy has emerged as a promising technique. Using shear-aligned lipid vesicles, conformations and binding geometries of membrane-bound bio-macromolecules can be assessed. Here we investigate anchoring properties and specific orientations of tryptophan relative to the peptide backbone and to the membrane normal for the model peptides gramicidin and melittin. We have monitored the conformational change associated with the refolding of non-channel gramicidin into its channel form, and quantitatively determined the average orientations of its tryptophan transition moments, suggesting that these residues adopt a well-defined orientation at the membrane interface. An important conclusion regards the structural variation of gramicidin between these two distinct transmembrane forms. Whilst circular dichroism (CD) spectra, as has been reported before, vary strongly between the two forms suggesting their structures might be quite different, the LD results clearly evidence both the peptide backbone orientation and tryptophan side-chain positioning to be very similar. The latter are oriented in accord with what is expected from their role to anchor peptide termini to the membrane surface. The variations in CD could be due to, the in LD observed, minor shifts in mutual orientation and distance between neighbouring tryptophans sensitively determining their exciton interactions. Our data dispute that the non-channel form of membrane-bound gramicidin would be any of the intertwined forms often observed in crystal as the positioning of tryptophans along the peptide axis would not be compatible with the strong interfacial positioning observed here. The general role of tryptophans as interfacial anchors is further assessed for melittin whose conformation shows considerable angular spread, consistent with a carpet model of its mechanism for induced

  8. Both soluble and membrane-bound forms of Flt3 ligand enhance tumor immunity following "suicide" gene therapy in a murine colon carcinoma model.

    PubMed

    Alsheikhly, Abdul-Razzak; Zweiri, Jehad; Walmesley, Alice J; Watson, Alastair J M; Christmas, Stephen E

    2004-11-01

    In prodrug-activated ("suicide") gene therapy, tumor cells are transfected with the gene for an enzyme that converts an inactive prodrug, such as ganciclovir (GCV), to a toxic compound. Transfected cells are killed on administration of GCV, as also are untransfected "bystander" cells. The ability of the dendritic cell stimulatory cytokine Flt3 ligand (Flt3-L) to modulate prodrug-activated gene therapy has been investigated. Transfectants of the murine colon carcinoma MC26 were generated expressing soluble (FLS) and membrane-bound forms of Flt3-L. They were inoculated together with wild-type MC26 cells and cells expressing herpes simplex virus-1 (HSV1) thymidine kinase into BALB/c mice, which were then administered GCV. Expression of Flt3-L or FLS prevented regrowth of tumor in most mice, which was comparable to the effect of granulocyte-macrophage colony-stimulating factor (GM-CSF), while tumors recurred in all mice receiving "suicide" gene therapy alone. Recurring tumor cells were resistant to direct killing by GCV but sensitive to "bystander" killing in vitro. Mice without tumor recurrence were rechallenged with unmodified MC26 cells. Of those mice given transfectants expressing GM-CSF, Flt3-L, or FLS, approximately 50% were immune to rechallenge. These mice also showed cytotoxic and proliferative responses to MC26 cells. These experiments show that both soluble and membrane-bound forms of Flt3-L were able to induce a protective immune response to colon carcinoma cells in a fashion similar to GM-CSF.

  9. Activins and inhibins in mammalian testis development: new models, new insights.

    PubMed

    Barakat, B; Itman, C; Mendis, S H; Loveland, K L

    2012-08-15

    The discovery of activin and inhibins as modulators of the hypothalamic-pituitary-gonadal axis has set the foundation for understanding their central importance to many facets of development and disease. This review contains an overview of the processes and cell types that are central to testis development and spermatogenesis and then provides an update focussed on information gathered over the past five years to address new concepts about how these proteins function to control testis development in fetal and juvenile life. Current knowledge about the interactive nature of the transforming growth factor-β (TGFβ) superfamily signalling network is applied to recent findings about activins and inhibins in the testis. Information about the regulated synthesis of signalling components and signalling regulators in the testis is integrated with new concepts that demonstrate their functional significance. The importance of activin bioactivity levels or dosage in controlling balanced growth of spermatogonial cells and their niche at different stages of testis development is highlighted.

  10. Activin/Nodal signalling before implantation: setting the stage for embryo patterning

    PubMed Central

    Papanayotou, Costis; Collignon, Jérôme

    2014-01-01

    Activins and Nodal are members of the transforming growth factor beta (TGF-β) family of growth factors. Their Smad2/3-dependent signalling pathway is well known for its implication in the patterning of the embryo after implantation. Although this pathway is active early on at preimplantation stages, embryonic phenotypes for loss-of-function mutations of prominent components of the pathway are not detected before implantation. It is only fairly recently that an understanding of the role of the Activin/Nodal signalling pathway at these stages has started to emerge, notably from studies detailing how it controls the expression of target genes in embryonic stem cells. We review here what is currently known of the TGF-β-related ligands that determine the activity of Activin/Nodal signalling at preimplantation stages, and recent advances in the elucidation of the Smad2/3-dependent mechanisms underlying developmental progression. PMID:25349448

  11. Activin/Nodal signalling before implantation: setting the stage for embryo patterning.

    PubMed

    Papanayotou, Costis; Collignon, Jérôme

    2014-12-01

    Activins and Nodal are members of the transforming growth factor beta (TGF-β) family of growth factors. Their Smad2/3-dependent signalling pathway is well known for its implication in the patterning of the embryo after implantation. Although this pathway is active early on at preimplantation stages, embryonic phenotypes for loss-of-function mutations of prominent components of the pathway are not detected before implantation. It is only fairly recently that an understanding of the role of the Activin/Nodal signalling pathway at these stages has started to emerge, notably from studies detailing how it controls the expression of target genes in embryonic stem cells. We review here what is currently known of the TGF-β-related ligands that determine the activity of Activin/Nodal signalling at preimplantation stages, and recent advances in the elucidation of the Smad2/3-dependent mechanisms underlying developmental progression.

  12. Low miR-143/miR-145 Cluster Levels Induce Activin A Overexpression in Oral Squamous Cell Carcinomas, Which Contributes to Poor Prognosis

    PubMed Central

    Bufalino, Andreia; Cervigne, Nilva K.; de Oliveira, Carine Ervolino; Fonseca, Felipe Paiva; Rodrigues, Priscila Campioni; Macedo, Carolina Carneiro Soares; Sobral, Lays Martin; Miguel, Marcia Costa; Lopes, Marcio Ajudarte; Leme, Adriana Franco Paes; Lambert, Daniel W.; Salo, Tuula A.; Kowalski, Luiz Paulo; Graner, Edgard; Coletta, Ricardo D.

    2015-01-01

    Deregulated expression of activin A is reported in several tumors, but its biological functions in oral squamous cell carcinoma (OSCC) are unknown. Here, we investigate whether activin A can play a causal role in OSCCs. Activin A expression was assessed by qPCR and immunohistochemistry in OSCC tissues. Low activin A-expressing cells were treated with recombinant activin A and assessed for apoptosis, proliferation, adhesion, migration, invasion and epithelial-mesenchymal transition (EMT). Those phenotypes were also evaluated in high activin A-expressing cells treated with follistatin (an activin A antagonist) or stably expressing shRNA targeting activin A. Transfections of microRNA mimics were performed to determine whether the overexpression of activin A is regulated by miR-143/miR-145 cluster. Activin A was overexpressed in OSCCs in comparison with normal oral mucosa, and high activin A levels were significantly associated with lymph node metastasis, tumor differentiation and poor survival. High activin A levels promoted multiple properties associated with malignant transformation, including decreased apoptosis and increased proliferation, migration, invasion and EMT. Both miR-143 and miR-145 were markedly downregulated in OSCC cell lines and in clinical specimens, and inversely correlated to activin A levels. Forced expression of miR-143 and miR-145 in OSCC cells significantly decreased the expression of activin A. Overexpression of activin A in OSCCs, which is controlled by downregulation of miR-143/miR-145 cluster, regulates apoptosis, proliferation and invasiveness, and it is clinically correlated with lymph node metastasis and poor survival. PMID:26317418

  13. Ramipril attenuates left ventricular remodeling by regulating the expression of activin A-follistatin in a rat model of heart failure

    PubMed Central

    Wei, Qun; Liu, Haiyan; Liu, Miao; Yang, Chunyan; Yang, Jie; Liu, Zhonghui; Yang, Ping

    2016-01-01

    Prior studies have shown that overexpression of ACT A can lead to ventricular remodeling in rat models of heart failure. Furthermore, recently work studying demonstrated that stimulation of activin An expression in rat aortic smooth muscle (RASM) cells by angiotensin II (Ang II). Ramipril is a recently developed angiotensin converting enzyme (ACE) inhibitor. To investigate the effects of Ramipril on expression of ACT A-FS, we established the rat model of heart failure after myocardial infarction (MI), and divided into either a sham operation (SO), MI, or MI-Ramipril group. We found that Ramipril significantly attenuates collagen-I and III deposition (col-I and III). Notably, we determined that expression of ACT A and II activin receptor (ActRII) were significantly down-regulated in the non-infarcted area of the left ventricle in the Ramipril group, whereas the mRNA and protein levels of FS were markedly up-regulated. Our data suggested that Ramipril benefited left ventricular remodeling by reducing fibrosis and collagen accumulation in the left ventricle of rats after myocardial infarction. This observation was also associated with down-regulation of ACT A expression. This study elucidated a new protective mechanism of Ramipril and suggests a novel strategy for treatment of post-infarct remodeling and subsequent heart failure. PMID:27642098

  14. Ramipril attenuates left ventricular remodeling by regulating the expression of activin A-follistatin in a rat model of heart failure.

    PubMed

    Wei, Qun; Liu, Haiyan; Liu, Miao; Yang, Chunyan; Yang, Jie; Liu, Zhonghui; Yang, Ping

    2016-01-01

    Prior studies have shown that overexpression of ACT A can lead to ventricular remodeling in rat models of heart failure. Furthermore, recently work studying demonstrated that stimulation of activin An expression in rat aortic smooth muscle (RASM) cells by angiotensin II (Ang II). Ramipril is a recently developed angiotensin converting enzyme (ACE) inhibitor. To investigate the effects of Ramipril on expression of ACT A-FS, we established the rat model of heart failure after myocardial infarction (MI), and divided into either a sham operation (SO), MI, or MI-Ramipril group. We found that Ramipril significantly attenuates collagen-I and III deposition (col-I and III). Notably, we determined that expression of ACT A and II activin receptor (ActRII) were significantly down-regulated in the non-infarcted area of the left ventricle in the Ramipril group, whereas the mRNA and protein levels of FS were markedly up-regulated. Our data suggested that Ramipril benefited left ventricular remodeling by reducing fibrosis and collagen accumulation in the left ventricle of rats after myocardial infarction. This observation was also associated with down-regulation of ACT A expression. This study elucidated a new protective mechanism of Ramipril and suggests a novel strategy for treatment of post-infarct remodeling and subsequent heart failure. PMID:27642098

  15. Diagnostic accuracy of serum activin A in detection of ectopic pregnancy

    PubMed Central

    Roghaei, Mohammad Ali; Sabet, Fahimeh; Mohamadi, Keivan

    2012-01-01

    Background: Ectopic pregnancy (EP) still remains a main cause of maternal mortalities. This study is designed to evaluate the accuracy of serum Activin A in detection of ectopic pregnancy. Methods: This prospective observational study was conducted from 2009 to 2010 at two main referral university hospitals, Isfahan University of Medical Sciences, Isfahan, Iran. Two hundred subjects who were under 10 week's pregnancy with clinical presentations of abdominal pain and vaginal bleeding were enrolled. After sampling serum Activin A, patients underwent ultrasonography, titer of B-HCG and surgery (if indicated) and were divided into two groups: EP (n = 100) and intrauterine pregnancy (IUP) (n = 100). The mean of Activin A was compared between groups and by ROC curve, the optimal cut off with sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) were determined. Results: The mean age of women with IUP was 25.4 ± 4.3 years (15-40 years) compared with 25.9 ± 4.1 years in women in EP group (P = 0.448). Statistical difference was not found between EP versus IUP groups in gestational age (6.32 ± 1.03 vs. 6.85 ± 1.82 weeks, P = 0.124). The mean of serum Activin A in EP group was 0.264 ± 0.0703 ng/ml versus 0.949 ± 0.5283 ng/ml in IUP group (P < 0.05). According to ROC curve (area under the curve = 0.981, P < 0.05, confidence interval: 0.961-1.000), the optimal cut off was estimated as 0.504 ng/ml with sensitivity of 97% and specificity of 93.5%. Conclusion: This study indicated that the mean of serum Activin A is lower in EP compared with IUP. The serum Activin A has a fair accuracy in detecting EP. PMID:23267401

  16. Activin-receptor signaling regulates cocaine-primed behavioral and morphological plasticity

    PubMed Central

    Gancarz, Amy M.; Wang, Zi-Jun; Schroeder, Gabrielle L.; Damez-Werno, Diane; Braunscheidel, Kevin; Mueller, Lauren E.; Humby, Monica S.; Caccamise, Aaron; Martin, Jennifer A.; Dietz, Karen C.; Neve, Rachael L.; Dietz, David M.

    2015-01-01

    Cocaine addiction is a life-long relapsing disorder that results from long-term adaptations within the brain. We find that Activin-receptor signaling, including the transcription factor Smad3, is upregulated in the rat nucleus accumbens shell following withdrawal from cocaine. Direct genetic and pharmacological manipulations of this pathway bidirectionally alter cocaine seeking, while governing morphological plasticity in nucleus accumbens neurons. These findings reveal that Activin/Smad3 signaling is induced following withdrawal from cocaine, and such regulation may be a key molecular mechanism underlying behavioral and cellular plasticity in the brain following cocaine self-administration. PMID:26030849

  17. Regulation of the activin-inhibin-follistatin system by bone morphogenetic proteins in the zebrafish ovary.

    PubMed

    Li, Cheuk Wun; Ge, Wei

    2013-09-01

    In the zebrafish, the dynamic expression of the activin-inhibin-follistatin system during folliculogenesis and its exclusive localization (except follistatin) in follicle cells suggests that the system plays important roles in follicle development and that its expression is subject to tight controls, probably by external factors including those derived from the oocyte. We have previously identified zebrafish bone morphogenetic proteins (BMPs) as oocyte factors that may act on follicle cells; however, the targets of BMPs in the follicle cells remain unknown. Considering their spatiotemporal expression in the follicle, we hypothesized that members of the activin-inhibin-follistatin system in follicle cells could be potential target genes of BMPs. In the present study, we developed a novel coculture system to co-incubate zebrafish bone morphogenetic protein 2b or 4 (zfBMP2b/4)-producing Chinese hamster ovary (CHO) cells with zebrafish follicle cells. During incubation, the zfBMPs secreted from the CHO cells would act directly on the follicle cells in a paracrine manner. Our results showed that all activin beta subunits (inhbaa, inhbab, and inhbb) were down-regulated by both zfBMP2b and zfBMP4, while follistatin (fst, an activin-binding protein) and inhibin alpha (inha, an activin antagonist) were significantly up-regulated. The specificity of bone morphogenetic protein (BMP) actions was confirmed by short interfering RNA knockdown of zfBMP4 expression in the CHO cells. The robust response of inha to zfBMPs, together with our previous observation that inha expression surged at the full-grown stage prior to oocyte maturation, led us to hypothesize that the full-grown oocyte may signal upper levels of the hypothalamic-pituitary-gonadal axis its readiness to mature by releasing BMPs, which in turn stimulate inhibin production. As an ovarian hormone and activin antagonist, inhibin may suppress the action of activin in the pituitary to reduce follicle-stimulating hormone

  18. ACVR1B (ALK4, activin receptor type 1B) gene mutations in pancreatic carcinoma

    PubMed Central

    Su, Gloria H.; Bansal, Ravi; Murphy, Kathleen M.; Montgomery, Elizabeth; Yeo, Charles J.; Hruban, Ralph H.; Kern, Scott E.

    2001-01-01

    DPC4 is known to mediate signals initiated by type β transforming growth factor (TGFβ) as well as by other TGFβ superfamily ligands such as activin and BMP (bone morphogenic proteins), but mutational surveys of such non-TGFβ receptors have been negative to date. Here we describe the gene structure and novel somatic mutations of the activin type I receptor, ACVR1B, in pancreatic cancer. ACVR1B has not been described previously as a mutated tumor-suppressor gene. PMID:11248065

  19. A Membrane-Bound NAC Transcription Factor, ANAC017, Mediates Mitochondrial Retrograde Signaling in Arabidopsis[W][OPEN

    PubMed Central

    Ng, Sophia; Ivanova, Aneta; Duncan, Owen; Law, Simon R.; Van Aken, Olivier; De Clercq, Inge; Wang, Yan; Carrie, Chris; Xu, Lin; Kmiec, Beata; Walker, Hayden; Van Breusegem, Frank; Whelan, James; Giraud, Estelle

    2013-01-01

    Plants require daily coordinated regulation of energy metabolism for optimal growth and survival and therefore need to integrate cellular responses with both mitochondrial and plastid retrograde signaling. Using a forward genetic screen to characterize regulators of alternative oxidase1a (rao) mutants, we identified RAO2/Arabidopsis NAC domain-containing protein17 (ANAC017) as a direct positive regulator of AOX1a. RAO2/ANAC017 is targeted to connections and junctions in the endoplasmic reticulum (ER) and F-actin via a C-terminal transmembrane (TM) domain. A consensus rhomboid protease cleavage site is present in ANAC017 just prior to the predicted TM domain. Furthermore, addition of the rhomboid protease inhibitor N-p-Tosyl-l-Phe chloromethyl abolishes the induction of AOX1a upon antimycin A treatment. Simultaneous fluorescent tagging of ANAC017 with N-terminal red fluorescent protein (RFP) and C-terminal green fluorescent protein (GFP) revealed that the N-terminal RFP domain migrated into the nucleus, while the C-terminal GFP tag remained in the ER. Genome-wide analysis of the transcriptional network regulated by RAO2/ANAC017 under stress treatment revealed that RAO2/ANAC017 function was necessary for >85% of the changes observed as a primary response to cytosolic hydrogen peroxide (H2O2), but only ∼33% of transcriptional changes observed in response to antimycin A treatment. Plants with mutated rao2/anac017 were more stress sensitive, whereas a gain-of-function mutation resulted in plants that had lower cellular levels of H2O2 under untreated conditions. PMID:24045017

  20. Activin B promotes BMSC-mediated cutaneous wound healing by regulating cell migration via the JNK-ERK signaling pathway.

    PubMed

    Zhang, Min; Sun, Li; Wang, Xueer; Chen, Shixuan; Kong, Yanan; Liu, Nuyun; Chen, Yinghua; Jia, Qin; Zhang, Lu; Zhang, Lin

    2014-01-01

    Bone marrow-derived mesenchymal stem cells (BMSCs) are able to differentiate into various types of skin cells and participate in skin regeneration and repair. Activin signaling can regulate wound healing and reepithelialization. The present study assessed the impact of activin B on BMSC-mediated cutaneous wound healing in rats and explored the possible mechanism involved. We found that CFSE-labeled BMSCs participated in wound healing in vivo, and compared to administration with PBS, activin B, or BMSCs, activin B plus BMSCs significantly promoted wound healing and hair follicle regeneration. Activin B induced actin stress fiber formation and cell migration in BMSCs in vitro. Activation of JNK and ERK, but not p38, was required for activin B-induced actin stress fiber formation and BMSC migration. These results show that activin B may promote BMSC-mediated wound healing by inducing actin stress fiber formation and BMSC migration via the ERK and JNK signal pathways. Combined administration of BMSCs and cytokines may be a promising therapeutic strategy for the management of skin wounds.

  1. Pegylated Interferon-α Modulates Liver Concentrations of Activin-A and Its Related Proteins in Normal Wistar Rat

    PubMed Central

    Refaat, Bassem; El-Shemi, Adel Galal; Ashshi, Ahmed Mohamed; Mahamid, Elaf Wael; Al-Qadi, Noha Mohammed

    2015-01-01

    Aims. To measure the expression of activin βA-subunit, activin IIA and IIB receptors, Smad4, Smad7, and follistatin in the liver and the liver and serum concentrations of mature activin-A and follistatin in normal rat following treatment with pegylated interferon-α (Peg-INF-α) and ribavirin (RBV). Materials and Methods. 40 male Wistar rats were divided equally into 4 groups: “control,” “Peg-only” receiving 4 injections of Peg-INF-α (6 µg/rat/week), “RBV-only” receiving ribavirin (4 mg/rat/day) orally, and “Peg & RBV” group receiving both drugs. The expression of candidate molecules in liver was measured by immunohistochemistry and quantitative PCR. The concentrations of mature proteins in serum and liver homogenate samples were measured using ELISA. Results. Peg-INF-α  ± RBV altered the expression of all candidate molecules in the liver at the gene and protein levels (P < 0.05) and decreased activin-A and increased follistatin in serum and liver homogenates compared with the other groups (P < 0.05). There were also significant correlations between serum and liver activin-A and follistatin. Conclusion. Peg-INF-α modulates the hepatic production of activin-A and follistatin, which can be detected in serum. Further studies are needed to explore the role of Peg-INF-α on the production of activins and follistatin by the liver and immune cells. PMID:26236109

  2. Mutagenesis and biochemical studies on AuaA confirmed the importance of the two conserved aspartate-rich motifs and suggested difference in the amino acids for substrate binding in membrane-bound prenyltransferases.

    PubMed

    Stec, Edyta; Li, Shu-Ming

    2012-07-01

    AuaA is a membrane-bound farnesyltransferase from the myxobacterium Stigmatella aurantiaca involved in the biosynthesis of aurachins. Like other known membrane-bound aromatic prenyltransferases, AuaA contains two conserved aspartate-rich motifs. Several amino acids in the first motif NXxxDxxxD were proposed to be responsible for prenyl diphosphate binding via metal ions like Mg(2+). Site-directed mutagenesis experiments demonstrated in this study that asparagine, but not the arginine residue in NRxxDxxxD, is important for the enzyme activity of AuaA, differing from the importance of NQ or ND residues in the NQxxDxxxD or NDxxDxxxD motifs observed in some membrane-bound prenyltransferases. The second motif of known membrane-bound prenyltransferases was proposed to be involved in the binding of their aromatic substrates. KDIxDxEGD, also found in AuaA, had been previously speculated to be characteristic for binding of flavonoids or homogenisate. Site-directed mutagenesis experiments with AuaA showed that KDIxDxEGD was critical for the enzyme activity. However, this motif is very likely not specific for flavonoid or homogenisate prenyltransferases, because none of the tested flavonoids was accepted by AuaA or its mutant R53A in the presence of farnesyl, geranyl or dimethylallyl diphosphate.

  3. Increased soluble and membrane-bound PD-L1 contributes to immune regulation and disease progression in patients with tuberculous pleural effusion

    PubMed Central

    Pan, Xue; Zhong, Anyuan; Xing, Yufei; Shi, Minhua; Qian, Bin; Zhou, Tong; Chen, Yongjing; Zhang, Xueguang

    2016-01-01

    Soluble and membrane-bound programmed death ligand-1 (sPD-L1 and mPD-L1, respectively) have been demonstrated to participate in the immune suppression of non-small cell lung cancer. However, the contribution of sPD-L1 and mPD-L1 to immune regulation and disease progression in patients with pleural effusions remains unknown. The present study evaluated the levels of sPD-L1 and membrane-bound PD-1/PD-L1 in the peripheral blood and pleural effusions of patients with tuberculous pleural effusion (TPE), malignant pleural effusion (MPE) and non-tuberculous non-malignant pleural effusion (n-TB n-M). Furthermore, selected T lymphocytes and cluster of differentiation (CD)14+ monocytes were co-cultured to investigate the potential effect of the PD-1/PD-L1 pathway in TPE. Levels of sPD-L1 and PD-L1 on CD14+ monocytes were increased in the TPE group, as compared with the MPE and n-TB n-M groups. Furthermore, sPD-L1 levels and the expression levels of PD-L1 on CD14+ monocytes were demonstrated to be positively correlated with interferon (IFN)-γ concentration in pleural effusions. Therefore, IFN-γ may increase the expression of PD-L1 on CD14+ monocytes in vitro. Cell counting kit-8 analysis demonstrated that anti-PD-L1 antibody was able to partially reverse the proliferation of T lymphocytes in the co-culture system. The results of the present study indicated that sPD-L1 or mPD-L1 are associated with the immune regulation and disease progression of TPE, and may serve as possible biomarkers of TPE. Furthermore, sPD-L1 and the PD-1/PD-L1 pathway of TPE may be associated with the Th1 immune response; therefore, an anti-PD-1/PD-L1 pathway suggests a potential immune therapy strategy for the treatment of TPE. PMID:27698705

  4. Increased soluble and membrane-bound PD-L1 contributes to immune regulation and disease progression in patients with tuberculous pleural effusion

    PubMed Central

    Pan, Xue; Zhong, Anyuan; Xing, Yufei; Shi, Minhua; Qian, Bin; Zhou, Tong; Chen, Yongjing; Zhang, Xueguang

    2016-01-01

    Soluble and membrane-bound programmed death ligand-1 (sPD-L1 and mPD-L1, respectively) have been demonstrated to participate in the immune suppression of non-small cell lung cancer. However, the contribution of sPD-L1 and mPD-L1 to immune regulation and disease progression in patients with pleural effusions remains unknown. The present study evaluated the levels of sPD-L1 and membrane-bound PD-1/PD-L1 in the peripheral blood and pleural effusions of patients with tuberculous pleural effusion (TPE), malignant pleural effusion (MPE) and non-tuberculous non-malignant pleural effusion (n-TB n-M). Furthermore, selected T lymphocytes and cluster of differentiation (CD)14+ monocytes were co-cultured to investigate the potential effect of the PD-1/PD-L1 pathway in TPE. Levels of sPD-L1 and PD-L1 on CD14+ monocytes were increased in the TPE group, as compared with the MPE and n-TB n-M groups. Furthermore, sPD-L1 levels and the expression levels of PD-L1 on CD14+ monocytes were demonstrated to be positively correlated with interferon (IFN)-γ concentration in pleural effusions. Therefore, IFN-γ may increase the expression of PD-L1 on CD14+ monocytes in vitro. Cell counting kit-8 analysis demonstrated that anti-PD-L1 antibody was able to partially reverse the proliferation of T lymphocytes in the co-culture system. The results of the present study indicated that sPD-L1 or mPD-L1 are associated with the immune regulation and disease progression of TPE, and may serve as possible biomarkers of TPE. Furthermore, sPD-L1 and the PD-1/PD-L1 pathway of TPE may be associated with the Th1 immune response; therefore, an anti-PD-1/PD-L1 pathway suggests a potential immune therapy strategy for the treatment of TPE.

  5. Serum Activin A and Follistatin Levels in Gestational Diabetes and the Association of the Activin A-Follistatin System with Anthropometric Parameters in Offspring

    PubMed Central

    Näf, Silvia; Escote, Xavier; Ballesteros, Mónica; Yañez, Rosa Elena; Simón-Muela, Inmaculada; Gil, Pilar; Albaiges, Gerard

    2014-01-01

    Context The Activin A-Follistatin system has emerged as an important regulator of lipid and glucose metabolism with possible repercussions on fetal growth. Objective To analyze circulating activin A, follistatin and follistatin-like-3 (FSTL3) levels and their relationship with glucose metabolism in pregnant women and their influence on fetal growth and neonatal adiposity. Design and methods A prospective cohort was studied comprising 207 pregnant women, 129 with normal glucose tolerance (NGT) and 78 with gestational diabetes mellitus (GDM) and their offspring. Activin A, follistatin and FSTL3 levels were measured in maternal serum collected in the early third trimester of pregnancy. Serial fetal ultrasounds were performed during the third trimester to evaluate fetal growth. Neonatal anthropometry was measured to assess neonatal adiposity. Results Serum follistatin levels were significantly lower in GDM than in NGT pregnant women (8.21±2.32 ng/mL vs 9.22±3.41, P = 0.012) whereas serum FSTL3 and activin A levels were comparable between the two groups. Serum follistatin concentrations were negatively correlated with HOMA-IR and positively with ultrasound growth parameters such as fractional thigh volume estimation in the middle of the third trimester and percent fat mass at birth. Also, in the stepwise multiple linear regression analysis serum follistatin levels were negatively associated with HOMA-IR (β = −0.199, P = 0.008) and the diagnosis of gestational diabetes (β = −0.138, P = 0.049). Likewise, fractional thigh volume estimation in the middle of third trimester and percent fat mass at birth were positively determined by serum follistatin levels (β = 0.214, P = 0.005 and β = 0.231, P = 0.002, respectively). Conclusions Circulating follistatin levels are reduced in GDM compared with NGT pregnant women and they are positively associated with fetal growth and neonatal adiposity. These data suggest a role of the Activin

  6. FGF9, activin and TGFβ promote testicular characteristics in an XX gonad organ culture model.

    PubMed

    Gustin, Sonja E; Stringer, Jessica M; Hogg, Kirsten; Sinclair, Andrew H; Western, Patrick S

    2016-11-01

    Testis development is dependent on the key sex-determining factors SRY and SOX9, which activate the essential ligand FGF9. Although FGF9 plays a central role in testis development, it is unable to induce testis formation on its own. However, other growth factors, including activins and TGFβs, also present testis during testis formation. In this study, we investigated the potential of FGF9 combined with activin and TGFβ to induce testis development in cultured XX gonads. Our data demonstrated differing individual and combined abilities of FGF9, activin and TGFβ to promote supporting cell proliferation, Sertoli cell development and male germ line differentiation in cultured XX gonads. FGF9 promoted proliferation of supporting cells in XX foetal gonads at rates similar to those observed in vivo during testis cord formation in XY gonads but was insufficient to initiate testis development. However, when FGF9, activin and TGFβ were combined, aspects of testicular development were induced, including the expression of Sox9, morphological reorganisation of the gonad and deposition of laminin around germ cells. Enhancing β-catenin activity diminished the testis-promoting activities of the combined growth factors. The male promoting activity of FGF9 and the combined growth factors directly or indirectly extended to the germ line, in which a mixed phenotype was observed. FGF9 and the combined growth factors promoted male germ line development, including mitotic arrest, but expression of pluripotency genes was maintained, rather than being repressed. Together, our data provide evidence that combined signalling by FGF9, activin and TGFβ can induce testicular characteristics in XX gonads.

  7. FGF9, activin and TGFβ promote testicular characteristics in an XX gonad organ culture model.

    PubMed

    Gustin, Sonja E; Stringer, Jessica M; Hogg, Kirsten; Sinclair, Andrew H; Western, Patrick S

    2016-11-01

    Testis development is dependent on the key sex-determining factors SRY and SOX9, which activate the essential ligand FGF9. Although FGF9 plays a central role in testis development, it is unable to induce testis formation on its own. However, other growth factors, including activins and TGFβs, also present testis during testis formation. In this study, we investigated the potential of FGF9 combined with activin and TGFβ to induce testis development in cultured XX gonads. Our data demonstrated differing individual and combined abilities of FGF9, activin and TGFβ to promote supporting cell proliferation, Sertoli cell development and male germ line differentiation in cultured XX gonads. FGF9 promoted proliferation of supporting cells in XX foetal gonads at rates similar to those observed in vivo during testis cord formation in XY gonads but was insufficient to initiate testis development. However, when FGF9, activin and TGFβ were combined, aspects of testicular development were induced, including the expression of Sox9, morphological reorganisation of the gonad and deposition of laminin around germ cells. Enhancing β-catenin activity diminished the testis-promoting activities of the combined growth factors. The male promoting activity of FGF9 and the combined growth factors directly or indirectly extended to the germ line, in which a mixed phenotype was observed. FGF9 and the combined growth factors promoted male germ line development, including mitotic arrest, but expression of pluripotency genes was maintained, rather than being repressed. Together, our data provide evidence that combined signalling by FGF9, activin and TGFβ can induce testicular characteristics in XX gonads. PMID:27495231

  8. Cloning, sequencing, and characterization of the gene encoding the smallest subunit of the three-component membrane-bound alcohol dehydrogenase from Acetobacter pasteurianus.

    PubMed

    Kondo, K; Beppu, T; Horinouchi, S

    1995-09-01

    The membrane-bound alcohol dehydrogenase (ADH) of Acetobacter pasteurianus NCI1452 consists of three different subunits, a 78-kDa dehydrogenase subunit, a 48-kDa cytochrome c subunit, and a 20-kDa subunit of unknown function. For elucidation of the function of the smallest subunit, this gene was cloned from this strain by the oligonucleotide-probing method, and its nucleotide sequence was determined. Comparison of the deduced amino acid sequence and the NH2-terminal sequence determined for the purified protein indicated that the smallest subunit contained a typical signal peptide of 28 amino acids, as did the larger two subunits. This gene complemented the ADH activity of a mutant strain which had lost the smallest subunit. Disruption of this gene on the chromosome resulted in loss of ADH activity in Acetobacter aceti, indicating that the smallest subunit was essential for ADH activity. Immunoblot analyses of cell lysates prepared from various ADH mutants suggested that the smallest subunit was concerned with the stability of the 78-kDa subunit and functioned as a molecular coupler of the 78-kDa subunit to the 48-kDa subunit on the cytoplasmic membrane.

  9. Molybdenum-containing membrane-bound formate dehydrogenase isolated from Citrobacter sp. S-77 having high stability against oxygen, pH, and temperature.

    PubMed

    Nguyen, Nga T; Yatabe, Takeshi; Yoon, Ki-Seok; Ogo, Seiji

    2014-10-01

    Membrane-bound formate dehydrogenase (FDH) was purified to homogeneity from a facultative anaerobic bacterium Citrobacter sp. S-77. The FDH from Citrobacter sp. S-77 (FDHS77) was a monomer with molecular mass of approximately 150 kDa. On SDS-PAGE, the purified FDHS77 showed as three different protein bands with molecular mass of approximately 95, 87, and 32 kDa, respectively. Based on the N-terminal amino acid sequence analysis, the sequence alignments observed for the 87 kDa protein band were identical to that of the large subunit of 95 kDa, indicating that the purified FDHS77 consisted of two subunits; a 95 kDa large subunit and a 32 kDa small subunit. The purified FDHS77 in this purification did not contain a heme b subunit, but the FDHS77 showed significant activity for formate oxidation, determined by the Vmax of 30.4 U/mg using benzyl viologen as an electron acceptor. The EPR and ICP-MS spectra indicate that the FDHS77 is a molybdenum-containing enzyme, displaying a remarkable O2-stability along with thermostability and pH resistance. This is the first report of the purification and characterization of a FDH from Citrobacter species.

  10. Maximal Expression of Membrane-Bound Nitrate Reductase in Paracoccus Is Induced by Nitrate via a Third FNR-Like Regulator Named NarR

    PubMed Central

    Wood, Nicholas J.; Alizadeh, Tooba; Bennett, Scott; Pearce, Joanne; Ferguson, Stuart J.; Richardson, David J.; Moir, James W. B.

    2001-01-01

    Respiratory reduction of nitrate to nitrite is the first key step in the denitrification process that leads to nitrate loss from soils. In Paracoccus pantotrophus, the enzyme system that catalyzes this reaction is encoded by the narKGHJI gene cluster. Expression of this cluster is maximal under anaerobic conditions in the presence of nitrate. Upstream from narK is narR, a gene encoding a member of the FNR family of transcriptional activators. narR is transcribed divergently from the other nar genes. Mutational analysis reveals that NarR is required for maximal expression of the membrane-bound nitrate reductase genes and narK but has no other regulatory function related to denitrification. NarR is shown to require nitrate and/or nitrite is order to activate gene expression. The N-terminal region of the protein lacks the cysteine residues that are required for formation of an oxygen-sensitive iron-sulfur cluster in some other members of the FNR family. Also, NarR lacks a crucial residue involved in interactions of this family of regulators with the ς70 subunit of RNA polymerase, indicating that a different mechanism is used to promote transcription. narR is also found in Paracoccus denitrificans, indicating that this species contains at least three FNR homologues. PMID:11371524

  11. A Quantitative Approach to Evaluate the Impact of Fluorescent Labeling on Membrane-Bound HIV-Gag Assembly by Titration of Unlabeled Proteins.

    PubMed

    Gunzenhäuser, Julia; Wyss, Romain; Manley, Suliana

    2014-01-01

    The assembly process of the human immunodeficiency virus 1 (HIV-1) is driven by the viral polyprotein Gag. Fluorescence imaging of Gag protein fusions is widely performed and has revealed important information on viral assembly. Gag fusion proteins are commonly co-transfected with an unlabeled form of Gag to prevent labeling artifacts such as morphological defects and decreased infectivity. Although viral assembly is widely studied on individual cells, the efficiency of the co-transfection rescue has never been tested at the single cell level. Here, we first develop a methodology to quantify levels of unlabeled to labeled Gag in single cells using a fluorescent reporter protein for unlabeled Gag and fluorescence correlation spectroscopy. Using super-resolution imaging based on photoactivated localization microscopy (PALM) combined with molecular counting we then study the nanoscale morphology of Gag clusters as a function of unlabeled to labeled Gag ratios in single cells. We show that for a given co-transfection ratio, individual cells express a wide range of protein ratios, necessitating a quantitative read-out for the expression of unlabeled Gag. Further, we show that monomerically labeled Gag assembles into membrane-bound clusters that are morphologically indistinguishable from mixtures of unlabeled and labeled Gag.

  12. A Quantitative Approach to Evaluate the Impact of Fluorescent Labeling on Membrane-Bound HIV-Gag Assembly by Titration of Unlabeled Proteins

    PubMed Central

    Gunzenhäuser, Julia; Wyss, Romain; Manley, Suliana

    2014-01-01

    The assembly process of the human immunodeficiency virus 1 (HIV-1) is driven by the viral polyprotein Gag. Fluorescence imaging of Gag protein fusions is widely performed and has revealed important information on viral assembly. Gag fusion proteins are commonly co-transfected with an unlabeled form of Gag to prevent labeling artifacts such as morphological defects and decreased infectivity. Although viral assembly is widely studied on individual cells, the efficiency of the co-transfection rescue has never been tested at the single cell level. Here, we first develop a methodology to quantify levels of unlabeled to labeled Gag in single cells using a fluorescent reporter protein for unlabeled Gag and fluorescence correlation spectroscopy. Using super-resolution imaging based on photoactivated localization microscopy (PALM) combined with molecular counting we then study the nanoscale morphology of Gag clusters as a function of unlabeled to labeled Gag ratios in single cells. We show that for a given co-transfection ratio, individual cells express a wide range of protein ratios, necessitating a quantitative read-out for the expression of unlabeled Gag. Further, we show that monomerically labeled Gag assembles into membrane-bound clusters that are morphologically indistinguishable from mixtures of unlabeled and labeled Gag. PMID:25493438

  13. Epithelial Sodium Channel-Mediated Sodium Transport Is Not Dependent on the Membrane-Bound Serine Protease CAP2/Tmprss4.

    PubMed

    Keppner, Anna; Andreasen, Ditte; Mérillat, Anne-Marie; Bapst, Julie; Ansermet, Camille; Wang, Qing; Maillard, Marc; Malsure, Sumedha; Nobile, Antoine; Hummler, Edith

    2015-01-01

    The membrane-bound serine protease CAP2/Tmprss4 has been previously identified in vitro as a positive regulator of the epithelial sodium channel (ENaC). To study its in vivo implication in ENaC-mediated sodium absorption, we generated a knockout mouse model for CAP2/Tmprss4. Mice deficient in CAP2/Tmprss4 were viable, fertile, and did not show any obvious histological abnormalities. Unexpectedly, when challenged with sodium-deficient diet, these mice did not develop any impairment in renal sodium handling as evidenced by normal plasma and urinary sodium and potassium electrolytes, as well as normal aldosterone levels. Despite minor alterations in ENaC mRNA expression, we found no evidence for altered proteolytic cleavage of ENaC subunits. In consequence, ENaC activity, as monitored by the amiloride-sensitive rectal potential difference (ΔPD), was not altered even under dietary sodium restriction. In summary, ENaC-mediated sodium balance is not affected by lack of CAP2/Tmprss4 expression and thus, does not seem to directly control ENaC expression and activity in vivo.

  14. Evaluation of Mut(S) and Mut⁺ Pichia pastoris strains for membrane-bound catechol-O-methyltransferase biosynthesis.

    PubMed

    Pedro, A Q; Oppolzer, D; Bonifácio, M J; Maia, C J; Queiroz, J A; Passarinha, L A

    2015-04-01

    Catechol-O-methyltransferase (COMT, EC 2.1.1.6) is an enzyme that catalyzes the methylation of catechol substrates, and while structural and functional studies of its membrane-bound isoform (MBCOMT) are still hampered by low recombinant production, Pichia pastoris has been described as an attractive host for the production of correctly folded and inserted membrane proteins. Hence, in this work, MBCOMT biosynthesis was developed using P. pastoris X33 and KM71H cells in shake flasks containing a semidefined medium with different methanol concentrations. Moreover, after P. pastoris glass beads lysis, biologically and immunologically active hMBCOMT was found mainly in the solubilized membrane fraction whose kinetic parameters were identical to its correspondent native enzyme. In addition, mixed feeds of methanol and glycerol or sorbitol were also employed, and its levels quantified using liquid chromatography coupled to refractive index detection. Overall, for the first time, two P. pastoris strains with opposite phenotypes were applied for MBCOMT biosynthesis under the control of the strongly methanol-inducible alcohol oxidase (AOX) promoter. Moreover, this eukaryotic system seems to be a promising approach to deliver MBCOMT in high quantities from fermentor cultures with a lower cost-benefit due to the cheaper cultivation media coupled with the higher titers tipically achieved in biorreactors, when compared with previously reported mammallian cell cultures. PMID:25712908

  15. Differences in the effect of phosphatidylinositol 4,5-bisphosphate on the hydrolytic and transphosphatidylation activities of membrane-bound phospholipase D from poppy seedlings.

    PubMed

    Oblozinsky, Marek; Bezakova, Lydia; Mansfeld, Johanna; Heilmann, Ingo; Ulbrich-Hofmann, Renate

    2013-08-01

    The hydrolytic activity of phospholipase D (PLD) yielding phosphatidic acid from phosphatidylcholine and other glycerophospholipids is known to be involved in many cellular processes. In contrast, it is not clear whether the competitive transphosphatidylation activity of PLD catalyzing the head group exchange of phospholipids has a natural function. In poppy seedlings (Papaver somniferum L.) where lipid metabolism and alkaloid synthesis are closely linked, five isoenzymes with different substrate and hydrolysis/transphosphatidylation selectivities have been detected hitherto. A membrane-bound PLD, found in microsomal fractions of poppy seedlings, is active at micromolar concentrations of Ca(2+) ions and needs phosphatidylinositol 4,5-bisphosphate (PIP2) as effector in the hydrolysis of phosphatidylcholine (PC). The optimum PIP2 concentration at 1.2 mol% of the concentration of the substrate PC indicates a specific activation effect. Transphosphatidylation with glycerol, ethanolamine, l-serine, or myo-inositol as acceptor alcohols is also activated by PIP2, however, with an optimum concentration at 0.6-0.9 mol%. In contrast to hydrolysis, a basic transphosphatidylation activity occurs even in the absence of PIP2, suggesting a different fine-tuning of the two competing reactions.

  16. Detection and phylogenetic analysis of the membrane-bound nitrate reductase (Nar) in pure cultures and microbial communities from deep-sea hydrothermal vents.

    PubMed

    Pérez-Rodríguez, Ileana; Bohnert, Kenneth A; Cuebas, Mariola; Keddis, Ramaydalis; Vetriani, Costantino

    2013-11-01

    Over the past few years the relevance of nitrate respiration in microorganisms from deep-sea hydrothermal vents has become evident. In this study, we surveyed the membrane-bound nitrate reductase (Nar) encoding gene in three different deep-sea vent microbial communities from the East Pacific Rise and the Mid-Atlantic Ridge. Additionally, we tested pure cultures of vent strains for their ability to reduce nitrate and for the presence of the NarG-encoding gene in their genomes. By using the narG gene as a diagnostic marker for nitrate-reducing bacteria, we showed that nitrate reductases related to Gammaproteobacteria of the genus Marinobacter were numerically prevalent in the clone libraries derived from a black smoker and a diffuse flow vent. In contrast, NarG sequences retrieved from a community of filamentous bacteria located about 50 cm above a diffuse flow vent revealed the presence of a yet to be identified group of enzymes. 16S rRNA gene-inferred community compositions, in accordance with previous studies, showed a shift from Alpha- and Gammaproteobacteria to Epsilonproteobacteria as the vent fluids become warmer and more reducing. Based on these findings, we argue that Nar-catalyzed nitrate reduction is likely relevant in temperate and less reducing environments where Alpha- and Gammaproteobacteria are more abundant and where nitrate concentrations reflect that of background deep seawater.

  17. Arabidopsis AtGPAT1, a Member of the Membrane-Bound Glycerol-3-Phosphate Acyltransferase Gene Family, Is Essential for Tapetum Differentiation and Male Fertility

    PubMed Central

    Zheng, Zhifu; Xia, Qun; Dauk, Melanie; Shen, Wenyun; Selvaraj, Gopalan; Zou, Jitao

    2003-01-01

    Membrane-bound glycerol-3-phosphate acyltransferase (GPAT; EC 2.3.1.15) mediates the initial step of glycerolipid biosynthesis in the extraplastidic compartments of plant cells. Here, we report the molecular characterization of a novel GPAT gene family from Arabidopsis, designated AtGPAT. The corresponding polypeptides possess transmembrane domains and GPAT activity when expressed heterologously in a yeast lipid mutant. The functional significance of one isoform, AtGPAT1, is the focus of the present study. Disruption of the AtGPAT1 gene causes a massive pollen development arrest, and subsequent introduction of the gene into the mutant plant rescues the phenotype, illustrating a pivotal role for AtGPAT1 in pollen development. Microscopic examinations revealed that the gene lesion results in a perturbed degeneration of the tapetum, which is associated with altered endoplasmic reticulum profiles and reduced secretion. In addition to the sporophytic effect, AtGPAT1 also exerts a gametophytic effect on pollen performance, as the competitive ability of a pollen grain to pollinate is dependent on the presence of an AtGPAT1 gene. Deficiency in AtGPAT1 correlates with several fatty acid composition changes in flower tissues and seeds. Unexpectedly, however, a loss of AtGPAT1 causes no significant change in seed oil content. PMID:12897259

  18. CD4+CD25+ Regulatory T Cells Inhibit Natural Killer Cell Hepatocytotoxicity of Hepatitis B Virus Transgenic Mice via Membrane-Bound TGF-β and OX40.

    PubMed

    Chen, Yongyan; Sun, Rui; Wu, Xunyao; Cheng, Min; Wei, Haiming; Tian, Zhigang

    2016-01-01

    CD4+CD25+ regulatory T cells (Tregs) are involved in the regulation of physiological and pathological hepatic immune responses, but the roles are not well explored in natural killer (NK) cell-mediated liver diseases. In this study, using the NK cell-mediated oversensitive liver injury model of hepatitis B virus transgenic (HBs-Tg) mice triggered by a low dose of concanavalin A, it was observed that an increased number of CD4+CD25+Foxp3+ Tregs were accumulated in the liver, along with the recovery of liver injury. Adoptive transfer of hepatic Tregs from HBs-Tg mice but not wild B6 mice could significantly attenuate the oversensitive liver injury via inhibiting liver accumulation and decreasing NK cell group 2D-mediated activation of NK cells in the recipient HBs-Tg mice. Furthermore, upregulated expression of membrane-bound TGF-β (mTGF-β) and OX40 on hepatic Tregs were demonstrated to account for inhibiting the NK cell-mediated hepatic injury in HBs-Tg mice through cell-cell contact, confirmed by antibody blockade and cell Transwell experiments in vivo and in vitro. Our findings for the first time indicated that CD4+CD25+ Tregs directly suppressed NK cell-mediated hepatocytotoxicity through mTGF-β and OX40/OX40L interaction in a cell-cell contact manner in HBV-associated liver disease. PMID:26067079

  19. Electroosmotic perfusion of tissue: sampling the extracellular space and quantitative assessment of membrane-bound enzyme activity in organotypic hippocampal slice cultures

    PubMed Central

    Ou, Yangguang; Wu, Juanfang; Sandberg, Mats

    2014-01-01

    This review covers recent advances in sampling fluid from the extracellular space of brain tissue by electroosmosis (EO). Two techniques, EO sampling with a single fused-silica capillary and EO push–pull perfusion, have been developed. These tools were used to investigate the function of membrane-bound enzymes with outward-facing active sites, or ectoenzymes, in modulating the activity of the neuropeptides leu-enkephalin and galanin in organotypic-hippocampal-slice cultures (OHSCs). In addition, the approach was used to determine the endogenous concentration of a thiol, cysteamine, in OHSCs. We have also investigated the degradation of coenzyme A in the extracellular space. The approach provides information on ectoenzyme activity, including Michaelis constants, in tissue, which, as far as we are aware, has not been done before. On the basis of computational evidence, EO push–pull perfusion can distinguish ectoenzyme activity with a ~100 µm spatial resolution, which is important for studies of enzyme kinetics in adjacent regions of the rat hippocampus. PMID:25168111

  20. Escherichia coli FtsH is a membrane-bound, ATP-dependent protease which degrades the heat-shock transcription factor sigma 32.

    PubMed Central

    Tomoyasu, T; Gamer, J; Bukau, B; Kanemori, M; Mori, H; Rutman, A J; Oppenheim, A B; Yura, T; Yamanaka, K; Niki, H

    1995-01-01

    Escherichia coli FtsH is an essential integral membrane protein that has an AAA-type ATPase domain at its C-terminal cytoplasmic part, which is homologous to at least three ATPase subunits of the eukaryotic 26S proteasome. We report here that FtsH is involved in degradation of the heat-shock transcription factor sigma 32, a key element in the regulation of the E. coli heat-shock response. In the temperature-sensitive ftsH1 mutant, the amount of sigma 32 at a non-permissive temperature was higher than in the wild-type under certain conditions due to a reduced rate of degradation. In an in vitro system with purified components, FtsH catalyzed ATP-dependent degradation of biologically active histidine-tagged sigma 32. FtsH has a zinc-binding motif similar to the active site of zinc-metalloproteases. Protease activity of FtsH for histidine-tagged sigma 32 was stimulated by Zn2+ and strongly inhibited by the heavy metal chelating agent o-phenanthroline. We conclude that FtsH is a novel membrane-bound, ATP-dependent metalloprotease with activity for sigma 32. These findings indicate a new mechanism of gene regulation in E. coli. Images PMID:7781608

  1. Responsiveness to transforming growth factor-beta (TGF-beta)-mediated growth inhibition is a function of membrane-bound TGF-beta type II receptor in human breast cancer cells.

    PubMed

    Lynch, M A; Petrel, T A; Song, H; Knobloch, T J; Casto, B C; Ramljak, D; Anderson, L M; DeGroff, V; Stoner, G D; Brueggemeier, R W; Weghorst, C M

    2001-01-01

    Transforming growth factor-beta (TGF-beta) is a potent inhibitor of growth and proliferation of breast epithelial cells, and loss of sensitivity to its effects has been associated with malignant transformation and tumorigenesis. The biological effects of TGF-beta are mediated by the TGF-beta receptor complex, a multimer composed of TGF-beta receptor type I (TbetaR-I) and TGF-beta receptor type II (TbetaR-II) subunits. Evidence suggests that loss of expression of Tbeta3R-II is implicated in the loss of sensitivity of tumorigenic breast cell lines to TGF-beta-mediated growth inhibition. A panel of human breast cell lines, including the immortalized MCF-10F and tumorigenic MCF-7, ZR75-1, BT474, T47-D, MDA-MB231, BT20, and SKBR-3 cell lines, was characterized for responsiveness to TGF-beta-induced G1 growth arrest. Only the nontumorigenic MCF-10F and the tumorigenic MDA-MB231 cell lines demonstrated a significant inhibitory response to TGF-beta1 and a significant binding of 125I-labeled TGF-beta ligand. While expression of TbetaR-I mRNA was similar across the panel of cell lines, TbetaR-II mRNA expression was decreased significantly in all seven tumorigenic cell lines in comparison with the nontumorigenic MCF- 10F cell line. When total cellular protein was fractionated by centrifugation, TbetaR-I protein was observed in both the cytosolic and membrane fractions at similar levels in all cell lines; however, TbetaR-II protein was present in the cytosolic fraction in all cell lines, but was observed in the membrane fraction of only the TGF-beta-responsive MCF-10F and MDA-MB231 cells. Thus, lack of membrane-bound TbetaR-II protein appears to be an important determinant of resistance to TGF-beta-mediated growth inhibition in this group of breast cell lines. PMID:11444526

  2. ActivinB Is Induced in Insulinoma To Promote Tumor Plasticity through a β-Cell-Induced Dedifferentiation

    PubMed Central

    Ripoche, Doriane; Charbord, Jérémie; Hennino, Ana; Teinturier, Romain; Bonnavion, Rémy; Jaafar, Rami; Goehrig, Delphine; Cordier-Bussat, Martine; Ritvos, Olli; Zhang, Chang X.; Andersson, Olov

    2015-01-01

    Loss of pancreatic β-cell maturity occurs in diabetes and insulinomas. Although both physiological and pathological stresses are known to promote β-cell dedifferentiation, little is known about the molecules involved in this process. Here we demonstrate that activinB, a transforming growth factor β (TGF-β)-related ligand, is upregulated during tumorigenesis and drives the loss of insulin expression and β-cell maturity in a mouse insulinoma model. Our data further identify Pax4 as a previously unknown activinB target and potent contributor to the observed β-cell dedifferentiation. More importantly, using compound mutant mice, we found that deleting activinB expression abolishes tumor β-cell dedifferentiation and, surprisingly, increases survival without significantly affecting tumor growth. Hence, this work reveals an unexpected role for activinB in the loss of β-cell maturity, islet plasticity, and progression of insulinoma through its participation in β-cell dedifferentiation. PMID:26711255

  3. [Molecular cloning of activin betaA subunit mature peptide from peafowl and its application in taxonomy and phylogeny].

    PubMed

    Zou, Fang-Dong; Tong, Xin-Xin; Yue, Bi-Song

    2005-03-01

    The sequences of activin gene betaA subunit mature peptide have been amplified from white peafowl, blue peafowl (pavo cristatus) and green peafowl (pavo muticus) genomic DNA by polymerase chain reaction (PCR) with a pair of degenerate primers. The target fragments were cloned into the vector pMD18-T and sequenced. The length of activin gene betaA subunit mature peptide is 345bp, which encoded a peptide of 115 amino acid residues. Sequence analysis of activin gene betaA subunit mature peptide demonstrated that the identity of nucleotide is 98.0% between blue peaflowl and green peafowl, and the identity of that is 98.8% between blue peaflowl and white peafow. Sequences comparison in NCBI revealed that the sequences of activin gene betaA subunit mature peptides of different species are highly conserved during evolution process. In addition, the restriction enzyme map of activins is high similar between white peafowl and blue peafowl. Phylogenetic tree was constructed with Mega 2 and Clustalxldx software. The result showed that white peafowl has a closer relationship to blue peafowl than to green peafowl. Considered the nucleotide differences of peafowls' activin gene betaA subunit mature peptides, a highly conserved region, we supported that white peafowl was derived from blue peafowl, and it is more possible the hybrid but just the product of color mutation, or maybe as a subspecies of Pavo genus.

  4. Blood cell induction in Xenopus animal cap explants: effects of fibroblast growth factor, bone morphogenetic proteins, and activin.

    PubMed

    Miyanaga, Y; Shiurba, R; Asashima, M

    1999-02-01

    Cultures of Xenopus blastula animal caps were used to explore the haematopoietic effects of three candidate inducers of mesoderm: basic fibroblast growth factor (bFGF), bone morphogenetic proteins (BMPs) and activin A. In response to either bFGF or activin A, explants expanded into egg-shaped structures, and beneath an outer layer of epidermis, a ventral mesodermal lining surrounded a fluid-filled cavity containing "blood-like cells". Immunocytochemistry identified some of these cells as early leukocytes, but erythrocytes were rare. BMP-2 or BMP-4 induced primitive erythrocytes as well as leukocytes, and a high concentration was required for these cells to differentiate in only a small proportion of explants. BMP-2 but not BMP-4 induced ventral mesoderm concomitantly. High concentrations of activin A dorsalized explants, which contained infrequent leukocytes, and an optimal combination of activin A and bFGF caused differentiation of muscle with few blood cells. By contrast, BMP-2 or BMP-4 plus activin A synergistically increased the numbers of both leukocytes and erythrocytes. Explants treated with BMPs plus activin contained a well organized cell mass in which yolk-rich cells mixed with blood cells and pigmented cells did not. BMP-2 plus bFGF also induced numerous leukocytes and fewer erythrocytes, but BMP-4 antagonized the leukopoietic effect of bFGF. The data suggest that the signalling pathways these three factors use to induce leukopoiesis overlap and that erythropoiesis may be activated when inducers are present in combination.

  5. [Molecular cloning of activin betaA subunit mature peptide from peafowl and its application in taxonomy and phylogeny].

    PubMed

    Zou, Fang-Dong; Tong, Xin-Xin; Yue, Bi-Song

    2005-03-01

    The sequences of activin gene betaA subunit mature peptide have been amplified from white peafowl, blue peafowl (pavo cristatus) and green peafowl (pavo muticus) genomic DNA by polymerase chain reaction (PCR) with a pair of degenerate primers. The target fragments were cloned into the vector pMD18-T and sequenced. The length of activin gene betaA subunit mature peptide is 345bp, which encoded a peptide of 115 amino acid residues. Sequence analysis of activin gene betaA subunit mature peptide demonstrated that the identity of nucleotide is 98.0% between blue peaflowl and green peafowl, and the identity of that is 98.8% between blue peaflowl and white peafow. Sequences comparison in NCBI revealed that the sequences of activin gene betaA subunit mature peptides of different species are highly conserved during evolution process. In addition, the restriction enzyme map of activins is high similar between white peafowl and blue peafowl. Phylogenetic tree was constructed with Mega 2 and Clustalxldx software. The result showed that white peafowl has a closer relationship to blue peafowl than to green peafowl. Considered the nucleotide differences of peafowls' activin gene betaA subunit mature peptides, a highly conserved region, we supported that white peafowl was derived from blue peafowl, and it is more possible the hybrid but just the product of color mutation, or maybe as a subspecies of Pavo genus. PMID:15843351

  6. Structural basis for specificity of TGF[beta] family receptor small molecule inhibitors

    SciTech Connect

    Ogunjimi, Abiodun A.; Zeqiraj, Elton; Ceccarelli, Derek F.; Sicheri, Frank; Wrana, Jeffrey L.; David, Laurent

    2012-07-24

    Transforming growth factor-{beta} (TGF{beta}) receptor kinase inhibitors have a great therapeutic potential. SB431542 is one of the mainly used kinase inhibitors of the TGF{beta}/Activin pathway receptors, but needs improvement of its EC{sub 50} (EC{sub 50} = 1 {mu}M) to be translated to clinical use. A key feature of SB431542 is that it specifically targets receptors from the TGF{beta}/Activin pathway but not the closely related receptors from the bone morphogenic proteins (BMP) pathway. To understand the mechanisms of this selectivity, we solved the crystal structure of the TGF{beta} type I receptor (T{beta}RI) kinase domain in complex with SB431542. We mutated T{beta}RI residues coordinating SB431542 to their counterparts in activin-receptor like kinase 2 (ALK2), a BMP receptor kinase, and tested the kinase activity of mutated T{beta}RI. We discovered that a Ser280Thr mutation yielded a T{beta}RI variant that was resistant to SB431542 inhibition. Furthermore, the corresponding Thr283Ser mutation in ALK2 yielded a BMP receptor sensitive to SB431542. This demonstrated that Ser280 is the key determinant of selectivity for SB431542. This work provides a framework for optimising the SB431542 scaffold to more potent and selective inhibitors of the TGF{beta}/Activin pathway.

  7. Human liver long-chain 3-hydroxyacyl-coenzyme A dehydrogenase is a multifunctional membrane-bound beta-oxidation enzyme of mitochondria.

    PubMed

    Carpenter, K; Pollitt, R J; Middleton, B

    1992-03-16

    We have purified to homogeneity the long-chain specific 3-hydroxyacyl-CoA dehydrogenase from mitochondrial membranes of human infant liver. The enzyme is composed of non-identical subunits of 71 kDa and 47 kDa within a native structure of 230 kDa. The pure enzyme is active with 3-ketohexanoyl-CoA and gives maximum activity with 3-ketoacyl-CoA substrates of C10 to C16 acyl-chain length but is inactive with acetoacetyl-CoA. In addition to 3-hydroxyacyl-CoA dehydrogenase activity, the enzyme possesses 2-enoyl-CoA hydratase and 3-ketoacyl-CoA thiolase activities which cannot be separated from the dehydrogenase. None of these enzymes show activity with C4 substrates but all are active with C6 and longer acyl-chain length substrates. They are thus distinct from any described previously. This human liver mitochondrial membrane-bound enzyme catalyses the conversion of medium- and long-chain 2-enoyl-CoA compounds to: 1) 3-ketoacyl-CoA in the presence of NAD alone and 2) to acetyl-CoA (plus the corresponding acyl-CoA derivatives) in the presence of NAD and CoASH. It is therefore a multifunctional enzyme, resembling the beta-oxidation enzyme of E. coli, but unique in its membrane location and substrate specificity. We propose that its existence explains the repeated failure to detect any intermediates of mitochondrial beta-oxidation.

  8. Widespread occurrence of N-terminal acylation in animal globins and possible origin of respiratory globins from a membrane-bound ancestor.

    PubMed

    Blank, Miriam; Burmester, Thorsten

    2012-11-01

    Proteins of the (hemo-)globin superfamily have been identified in many different animals but also occur in plants, fungi, and bacteria. Globins are renowned for their ability to store and to transport oxygen, but additional globin functions such as sensing, signaling, and detoxification have been proposed. Recently, we found that the zebrafish globin X protein is myristoylated and palmitoylated at its N-terminus. The addition of fatty acids results in an association with the cellular membranes, suggesting a previously unrecognized globin function. In this study, we show that N-terminal acylation likely occurs in globin proteins from a broad range of phyla. An N-terminal myristoylation site was identified in 90 nonredundant globins from Chlorophyta, Heterokontophyta, Cnidaria, Mollusca, Arthropoda, Nematoda, Echinodermata, Hemichordata, and Chordata (including Cephalochordata), of which 66 proteins carry an additional palmitoylation site. Bayesian phylogenetic analyses identified five major globin families, which may mirror the ancient globin diversity of the Metazoa. Globin X-like proteins form two related clades, which diverged before the radiation of the Eumetazoa. Vertebrate hemoglobin (Hb), myoglobin, cytoglobin, globin E, and globin Y form a strongly supported common clade, which is the sister group of a clade consisting of invertebrate Hbs and relatives. The N-terminally acylated globins do not form a single monophyletic group but are distributed to four distinct clades. This pattern may be either explained by multiple introduction of an N-terminal acylation site into distinct globin lineages or by the origin of animal respiratory globins from a membrane-bound ancestor. Similarly, respiratory globins were not monophyletic. This suggests that respiratory globins might have emerged independently several times and that the early metazoan globins might have been associated with a membrane and carried out a function that was related to lipid protection or

  9. Membrane-bound quinoprotein D-arabitol dehydrogenase of Gluconobacter suboxydans IFO 3257: a versatile enzyme for the oxidative fermentation of various ketoses.

    PubMed

    Adachi, O; Fujii, Y; Ghaly, M F; Toyama, H; Shinagawa, E; Matsushita, K

    2001-12-01

    Solubilization of membrane-bound quinoprotein D-arabitol dehydrogenase (ARDH) was done successfully with the membrane fraction of Gluconobacter suboxydans IFO 3257. In enzyme solubilization and subsequent enzyme purification steps, special care was taken to purify ARDH as active as it was in the native membrane, after many disappointing trials. Selection of the best detergent, keeping ARDH as the holoenzyme by the addition of PQQ and Ca2+, and of a buffer system involving acetate buffer supplemented with Ca2+, were essential to treat the highly hydrophobic and thus labile enzyme. Purification of the enzyme was done by two steps of column chromatography on DEAE-Toyopearl and CM-Toyopearl in the presence of detergent and Ca2+. ARDH was homogenous and showed a single sedimentation peak in analytical ultracentrifugation. ARDH was dissociated into two different subunits upon SDS-PAGE with molecular masses of 82 kDa (subunit I) and 14 kDa (subunit II), forming a heterodimeric structure. ARDH was proven to be a quinoprotein by detecting a liberated PQQ from SDS-treated ARDH in HPLC chromatography. More preliminarily, an EDTA-treated membrane fraction lost the enzyme activity and ARDH activity was restored to the original level by the addition of PQQ and Ca2+. The most predominant unique character of ARDH, the substrate specificity, was highly versatile and many kinds of substrates were oxidized irreversibly by ARDH, not only pentitols but also other polyhydroxy alcohols including D-sorbitol, D-mannitol, glycerol, meso-erythritol, and 2,3-butanediol. ARDH may have its primary function in the oxidative fermentation of ketose production by acetic acid bacteria. ARDH contained no heme component, unlike the type II or type III quinoprotein alcohol dehydrogenase (ADH) and did not react with primary alcohols.

  10. Structural basis for a [4Fe-3S] cluster in the oxygen-tolerant membrane-bound [NiFe]-hydrogenase.

    PubMed

    Shomura, Yasuhito; Yoon, Ki-Seok; Nishihara, Hirofumi; Higuchi, Yoshiki

    2011-11-10

    Membrane-bound respiratory [NiFe]-hydrogenase (MBH), a H(2)-uptake enzyme found in the periplasmic space of bacteria, catalyses the oxidation of dihydrogen: H(2) → 2H(+) + 2e(-) (ref. 1). In contrast to the well-studied O(2)-sensitive [NiFe]-hydrogenases (referred to as the standard enzymes), MBH has an O(2)-tolerant H(2) oxidation activity; however, the mechanism of O(2) tolerance is unclear. Here we report the crystal structures of Hydrogenovibrio marinus MBH in three different redox conditions at resolutions between 1.18 and 1.32 Å. We find that the proximal iron-sulphur (Fe-S) cluster of MBH has a [4Fe-3S] structure coordinated by six cysteine residues--in contrast to the [4Fe-4S] cubane structure coordinated by four cysteine residues found in the proximal Fe-S cluster of the standard enzymes--and that an amide nitrogen of the polypeptide backbone is deprotonated and additionally coordinates the cluster when chemically oxidized, thus stabilizing the superoxidized state of the cluster. The structure of MBH is very similar to that of the O(2)-sensitive standard enzymes except for the proximal Fe-S cluster. Our results give a reasonable explanation why the O(2) tolerance of MBH is attributable to the unique proximal Fe-S cluster; we propose that the cluster is not only a component of the electron transfer for the catalytic cycle, but that it also donates two electrons and one proton crucial for the appropriate reduction of O(2) in preventing the formation of an unready, inactive state of the enzyme. PMID:22002607

  11. Membrane-bound hemagglutinin mediates antibody and complement-dependent lysis of influenza virus-treated human platelets in autologous serum.

    PubMed Central

    Kazatchkine, M D; Lambré, C R; Kieffer, N; Maillet, F; Nurden, A T

    1984-01-01

    Influenza A virus-treated human platelets were lyzed in autologous serum. Lysis required the presence of antibody and occurred predominantly through activation of the classical complement pathway. Binding of the virus followed by its elution at 37 degrees C resulted in a dose-dependent desialation of the cells with a maximal release of 45% of total platelet sialic acid. In contrast, platelets that had been treated with Vibrio cholerae neuraminidase and from which 55% of total sialic acid had been removed were not lyzed in autologous serum and did not bind C3 as shown in binding assays using radiolabeled monoclonal anti-C3 antibody. Thus, the immune-mediated lysis of virus-treated platelets in autologous serum did not involve neoantigens expressed by desialated cells. To assess the effect of viruses on the platelet surface, treated platelets were incubated with galactose oxidase and sodium [3H]borohydride prior to separation and analysis of the labeled glycoproteins by SDS-PAGE. Viral treatment resulted in a desialation of each of the surface glycoproteins. At the same time, a labeled component of Mr 72,000 (nonreduced) and Mr 55,000 (reduced) was observed that was not present when V. cholerae-desialated platelets were examined in the same way. Immunoblotting experiments performed using antiwhole virus and anti-hemagglutinin antibodies demonstrated this component to be viral hemagglutinin. Involvement of membrane-bound hemagglutinin in antibody and in complement-mediated lysis of virus-treated platelets in autologous serum was supported by the increased lytic activity of a postvaccinal serum containing an elevated titer of complement fixing anti-hemagglutinin antibodies. Binding of a viral protein to the platelet surface provides a model for immune thrombocytopenias occurring during acute viral infections at the time of the specific immune response. Images PMID:6470149

  12. The MUC4 membrane-bound mucin regulates esophageal cancer cell proliferation and migration properties: Implication for S100A4 protein

    SciTech Connect

    Bruyere, Emilie; Jonckheere, Nicolas; Frenois, Frederic; Mariette, Christophe; Van Seuningen, Isabelle

    2011-09-23

    Highlights: {yields} Loss of MUC4 reduces proliferation of esophageal cancer cells. {yields} MUC4 inhibition impairs migration of esophageal cancer cells but not their invasion. {yields} Loss of MUC4 significantly reduces in vivo tumor growth. {yields} Decrease of S100A4 induced by MUC4 inhibition impairs proliferation and migration. -- Abstract: MUC4 is a membrane-bound mucin known to participate in tumor progression. It has been shown that MUC4 pattern of expression is modified during esophageal carcinogenesis, with a progressive increase from metaplastic lesions to adenocarcinoma. The principal cause of development of esophageal adenocarcinoma is the gastro-esophageal reflux, and MUC4 was previously shown to be upregulated by several bile acids present in reflux. In this report, our aim was thus to determine whether MUC4 plays a role in biological properties of human esophageal cancer cells. For that stable MUC4-deficient cancer cell lines (shMUC4 cells) were established using a shRNA approach. In vitro (proliferation, migration and invasion) and in vivo (tumor growth following subcutaneous xenografts in SCID mice) biological properties of shMUC4 cells were analyzed. Our results show that shMUC4 cells were less proliferative, had decreased migration properties and did not express S100A4 protein when compared with MUC4 expressing cells. Absence of MUC4 did not impair shMUC4 invasiveness. Subcutaneous xenografts showed a significant decrease in tumor size when cells did not express MUC4. Altogether, these data indicate that MUC4 plays a key role in proliferative and migrating properties of esophageal cancer cells as well as is a tumor growth promoter. MUC4 mucin appears thus as a good therapeutic target to slow-down esophageal tumor progression.

  13. A Heteromeric Membrane-Bound Prenyltransferase Complex from Hop Catalyzes Three Sequential Aromatic Prenylations in the Bitter Acid Pathway1[OPEN

    PubMed Central

    Li, Haoxun; Ban, Zhaonan; Qin, Hao; Ma, Liya; King, Andrew J.

    2015-01-01

    Bitter acids (α and β types) account for more than 30% of the fresh weight of hop (Humulus lupulus) glandular trichomes and are well known for their contribution to the bitter taste of beer. These multiprenylated chemicals also show diverse biological activities, some of which have potential benefits to human health. The bitter acid biosynthetic pathway has been investigated extensively, and the genes for the early steps of bitter acid synthesis have been cloned and functionally characterized. However, little is known about the enzyme(s) that catalyze three sequential prenylation steps in the β-bitter acid pathway. Here, we employed a yeast (Saccharomyces cerevisiae) system for the functional identification of aromatic prenyltransferase (PT) genes. Two PT genes (HlPT1L and HlPT2) obtained from a hop trichome-specific complementary DNA library were functionally characterized using this yeast system. Coexpression of codon-optimized PT1L and PT2 in yeast, together with upstream genes, led to the production of bitter acids, but no bitter acids were detected when either of the PT genes was expressed by itself. Stepwise mutation of the aspartate-rich motifs in PT1L and PT2 further revealed the prenylation sequence of these two enzymes in β-bitter acid biosynthesis: PT1L catalyzed only the first prenylation step, and PT2 catalyzed the two subsequent prenylation steps. A metabolon formed through interactions between PT1L and PT2 was demonstrated using a yeast two-hybrid system, reciprocal coimmunoprecipitation, and in vitro biochemical assays. These results provide direct evidence of the involvement of a functional metabolon of membrane-bound prenyltransferases in bitter acid biosynthesis in hop. PMID:25564559

  14. In vitro assay of the chlorophyll biosynthetic enzyme Mg-chelatase: Resolution of the activity into soluble and membrane-bound fractions

    SciTech Connect

    Walker, C.J.; Weinstein, J.D. )

    1991-07-01

    The first committed step in chlorophyll synthesis is the Mg-chelatase-catalyzed insertion of magnesium into protoporphyrin IX. Since iron insertion into protoporphyrin leads to heme formation, Mg-chelatase lies at the branch point of heme and chlorophyll synthesis in chloroplasts. Little is known about the enzymology or regulation of Mg-chelatase, as it has been assayed only in intact cucumber chloroplasts. In this report we describe an in vitro assay for Mg-chelatase. Mg-chelatase activity in intact pea chloroplasts was 3- to 4-fold higher than in cucumber chloroplasts. This activity survived chloroplast lysis and could be fractionated by centrifugation into supernatant and pellet components. Both of these fractions were required to reconstitute Mg-chelatase activity, and both were inactivated by boiling indicating that the enzyme is composed of soluble and membrane-bound protein(s). The product of the reaction was confirmed fluorometrically as the magnesium chelate of the porphyrin substrate. The specific activity of the reconstituted system was typically 1 nmol of Mg-deuteroporphyrin per h per mg of protein, and activity was linear for at least 60 min under our assay conditions. ATP and magnesium were required for Mg-chelatase activity and the enzymen was sensitive to the sulfhydryl reagent N-ethylmaleimide (I{sub 50}, 20 {mu}M). Broken and reconstituted cucumber chloroplasts were unable to maintain Mg-chelatase activity. However, the cucumber supernatant fraction was active when combined with the pellet fraction of peas; the converse was not true, which suggested that the cucumber pellet was the component that lost activity during lysis.

  15. Membrane-bound and soluble Fas ligands have opposite functions in photoreceptor cell death following separation from the retinal pigment epithelium

    PubMed Central

    Matsumoto, H; Murakami, Y; Kataoka, K; Notomi, S; Mantopoulos, D; Trichonas, G; Miller, J W; Gregory, M S; Ksander, B R; Marshak-Rothstein, A; Vavvas, D G

    2015-01-01

    Fas ligand (FasL) triggers apoptosis of Fas-positive cells, and previous reports described FasL-induced cell death of Fas-positive photoreceptors following a retinal detachment. However, as FasL exists in membrane-bound (mFasL) and soluble (sFasL) forms, and is expressed on resident microglia and infiltrating monocyte/macrophages, the current study examined the relative contribution of mFasL and sFasL to photoreceptor cell death after induction of experimental retinal detachment in wild-type, knockout (FasL−/−), and mFasL-only knock-in (ΔCS) mice. Retinal detachment in FasL−/− mice resulted in a significant reduction of photoreceptor cell death. In contrast, ΔCS mice displayed significantly more apoptotic photoreceptor cell death. Photoreceptor loss in ΔCS mice was inhibited by a subretinal injection of recombinant sFasL. Thus, Fas/FasL-triggered cell death accounts for a significant amount of photoreceptor cell loss following the retinal detachment. The function of FasL was dependent upon the form of FasL expressed: mFasL triggered photoreceptor cell death, whereas sFasL protected the retina, indicating that enzyme-mediated cleavage of FasL determines, in part, the extent of vision loss following the retinal detachment. Moreover, it also indicates that treatment with sFasL could significantly reduce photoreceptor cell loss in patients with retinal detachment. PMID:26583327

  16. Formation of membrane-bound inclusions and their associations with cytoplasmic channels in early prophase male meiocytes of Althaea rosea (L.) Cavan.

    PubMed

    Luo, Xin Juan; Liu, Xu Hao; Wang, Chong Ying; Wang, Xin Yu

    2008-04-01

    To characterize the cytoplasmic structure reorganization during plant meiosis, the male meiocytes of Althaea rosea (L.) Cavan were examined under the combination of light and electron microscopy. Light microscopic observation of the toluidine blue-stained thick resin sections of young anthers revealed that the meiocytes of sporogenous cell stage were extremely voluminous and variable in shape and division plane. The cell walls (CWs) between some meiocytes were discontinuous at one or several site(s). These discontinuous portions varied between 0.2 and 3.0 microm in length. In addition, it was found that some meiocytes were able to produce protuberances that extended into another meiocyte. When transversally sectioned, the protuberance extending to another cell looked like a small cell lying in another cell. Transmission electron microscopy (TEM) showed that there were many long flat ER cisternae that were actively wrapping around a portion of cytoplasm in the male meiocytes at the sporogenous cell stage. During pre-meiosis interphase and early prophase I, a number of huge (0.5-1.0 microm diameter) spherical membrane-bound inclusions (MBIs) lined by single or double layer(s) of membrane were formed, each membrane actually representing one tightly appressed endoplasmic reticulum (ER) cisterna. The MBIs contained many granular, lamellar and fibrillar structures, and even small MBIs. Moreover, it was found that the MBIs could associate with the cytoplasmic channels (CCs) on CWs to release their contents into the cytoplasm of the opposite cell or directly extend from one cell to another through the CC. Taking all the data together, it is suggested that association of the MBIs and other organelles with CCs possibly functions in eliminating the non-identity of cytoplasm of the male meiocytes caused probably by the random asymmetric division observed at sporogenous cell phase, so as to ensure production of a large number of identical functional male gametes required for

  17. Widespread occurrence of N-terminal acylation in animal globins and possible origin of respiratory globins from a membrane-bound ancestor.

    PubMed

    Blank, Miriam; Burmester, Thorsten

    2012-11-01

    Proteins of the (hemo-)globin superfamily have been identified in many different animals but also occur in plants, fungi, and bacteria. Globins are renowned for their ability to store and to transport oxygen, but additional globin functions such as sensing, signaling, and detoxification have been proposed. Recently, we found that the zebrafish globin X protein is myristoylated and palmitoylated at its N-terminus. The addition of fatty acids results in an association with the cellular membranes, suggesting a previously unrecognized globin function. In this study, we show that N-terminal acylation likely occurs in globin proteins from a broad range of phyla. An N-terminal myristoylation site was identified in 90 nonredundant globins from Chlorophyta, Heterokontophyta, Cnidaria, Mollusca, Arthropoda, Nematoda, Echinodermata, Hemichordata, and Chordata (including Cephalochordata), of which 66 proteins carry an additional palmitoylation site. Bayesian phylogenetic analyses identified five major globin families, which may mirror the ancient globin diversity of the Metazoa. Globin X-like proteins form two related clades, which diverged before the radiation of the Eumetazoa. Vertebrate hemoglobin (Hb), myoglobin, cytoglobin, globin E, and globin Y form a strongly supported common clade, which is the sister group of a clade consisting of invertebrate Hbs and relatives. The N-terminally acylated globins do not form a single monophyletic group but are distributed to four distinct clades. This pattern may be either explained by multiple introduction of an N-terminal acylation site into distinct globin lineages or by the origin of animal respiratory globins from a membrane-bound ancestor. Similarly, respiratory globins were not monophyletic. This suggests that respiratory globins might have emerged independently several times and that the early metazoan globins might have been associated with a membrane and carried out a function that was related to lipid protection or

  18. Tracking of proton flow during transition from anaerobiosis to steady state. 2. Effect of cation uptake on the response of a hydrophobic membrane bound pH indicator.

    PubMed

    Luvisetto, S; Cola, C; Schmehl, I; Azzone, G F

    1991-11-15

    1. During aerobic cation uptake in liver mitochondria, the hydrophobic pH indicator bromothymol blue undergoes a multiphase response: phase 1 (rapid acidification), phase 2 (slow alkalinization), phase 3 (rapid alkalinization) and phase 4 (reacidification). 2. Titrations with ruthenium red and malonate indicate that the various phases depend on the relative rates of cation uptake and proton translocation: at high rates of cation uptake, phase 1 disappears and phases 2 and 3 are transformed in a monotonic process of alkalinization. 3. The comparison of the bromothymol blue response with the arsenazo III, 2',7'-bis(carboxyethyl)-5(6)carboxyfluorescein (BCECF) and safranine responses indicates that: (a) phase 2 (slow alkalinization) corresponds to a slow rise of matrix pH and a parallel decline of membrane potential; (b) phase 3 (rapid alkalinization) corresponds to termination of proton translocation and initiation of the processes of cation efflux and proton reuptake. All the above processes reach completion during phase 4. 4. Although bromothymol blue always behaves as a membrane-bound indicator, the extent to which it reflects the matrix or the cytosolic pH is a function of the membrane-potential-determined asymmetric distribution: in parallel with the lowering of the membrane potential, the dye chromophore is shifted from the cytosolic to the matrix side membrane layer. 5. A model is discussed which describes the behaviour of bromothymol blue as pH indicator recording the changes in membrane layers facing either the matrix or the cytosolic side. The complex response of the dye during cation uptake is due to two independent processes, one of pH change and another of dye intramembrane shift. Computer simulations of the dye response, based on the conversion of a kinetic model into an electrical network and closely reproducing the experimental observations, are reported. PMID:1718751

  19. Menaquinone as Well as Ubiquinone as a Bound Quinone Crucial for Catalytic Activity and Intramolecular Electron Transfer in Escherichia coli Membrane-bound Glucose Dehydrogenase*

    PubMed Central

    Mustafa, Golam; Migita, Catharina T.; Ishikawa, Yoshinori; Kobayashi, Kazuo; Tagawa, Seiichi; Yamada, Mamoru

    2008-01-01

    Escherichia coli membrane-bound glucose dehydrogenase (mGDH), which is one of quinoproteins containing pyrroloquinoline quinone (PQQ) as a coenzyme, is a good model for elucidating the function of bound quinone inside primary dehydrogenases in respiratory chains. Enzymatic analysis of purified mGDH from cells defective in synthesis of ubiquinone (UQ) and/or menaquinone (MQ) revealed that Q-free mGDH has very low levels of activity of glucose dehydrogenase and UQ2 reductase compared with those of UQ-bearing mGDH, and both activities were significantly increased by reconstitution with UQ1. On the other hand, MQ-bearing mGDH retains both catalytic abilities at the same levels as those of UQ-bearing mGDH. A radiolytically generated hydrated electron reacted with the bound MQ to form a semiquinone anion radical with an absorption maximum at 400 nm. Subsequently, decay of the absorbance at 400 nm was accompanied by an increase in the absorbance at 380 nm with a first order rate constant of 5.7 × 103 s–1. This indicated that an intramolecular electron transfer from the bound MQ to the PQQ occurred. EPR analysis revealed that characteristics of the semiquinone radical of bound MQ are similar to those of the semiquinone radical of bound UQ and indicated an electron flow from PQQ to MQ as in the case of UQ. Taken together, the results suggest that MQ is incorporated into the same pocket as that for UQ to perform a function almost equivalent to that of UQ and that bound quinone is involved at least partially in the catalytic reaction and primarily in the intramolecular electron transfer of mGDH. PMID:18708350

  20. Identification of a potent Xenopus mesoderm-inducing factor as a homologue of activin A.

    PubMed

    Smith, J C; Price, B M; Van Nimmen, K; Huylebroeck, D

    1990-06-21

    The first inductive interaction in amphibian development is mesoderm induction, when a signal from the vegetal hemisphere of the blastula induces mesoderm from overlying equatorial cells. Recently, several 'mesoderm-inducing factors' (MIFs) have been discovered. These cause isolated Xenopus animal caps to form mesodermal cell types such as muscle, instead of their normal fate of epidermis. The MIFs fall into two classes. One comprises members of the fibroblast growth factor (FGF) family, and the other members of the transforming growth factor type beta (TGF-beta) family. Of the latter group, the most potent is XTC-MIF, a protein produced by Xenopus XTC cells. Here we show that XTC-MIF is the homologue of mammalian activin A. Activins modulate the release of follicle-stimulating hormone from cultured anterior pituitary cells and cause the differentiation of two erythroleukaemia cell lines. Our results indicate that these molecules may also act in early development during formation of the mesoderm.

  1. Serum activin A and B levels predict outcome in patients with acute respiratory failure: a prospective cohort study

    PubMed Central

    2013-01-01

    Introduction 30 day mortality in patients with Acute Respiratory Failure (ARF) is approximately 30%, defined as patients requiring ventilator support for more than 6 hours. Novel biomarkers are needed to predict patient outcomes and to guide potential future therapies. The activins A and B, members of the Transforming Growth Factor β family of proteins, and their binding protein, follistatin, have recently been shown to be important regulators of inflammation and fibrosis but no substantial data are available concerning their roles in ARF. Our objectives were to evaluate whether the serum levels of activin A, B and follistatin are elevated in 518 patients with ARF from the FINNALI study compared the concentrations in 138 normal subjects that form a reference range. Methods Specific assays for activin A, B and follistatin were used and the results analyzed according to diagnostic groups as well as according to standard measures in intensive care. Multivariable logistic regression was used to create a model to predict death at 90 days and 12 months from the onset of the ARF. Results Serum activin A and B were significantly elevated in most patients and in most of the diagnostic groups. Patients who had activin A and/or B concentrations above the reference maximum were significantly more likely to die in the 12 months following admission [either activin A or B above reference maximum: Positive Likelihood Ratio [LR+] 1.65 [95% CI 1.28-2.12, P = 0.00013]; both activin A and B above reference maximum: LR + 2.78 [95% CI 1.96-3.95, P < 0.00001]. The predictive model at 12 months had an overall accuracy of 80.2% [95% CI 76.6-83.3%]. Conclusions The measurement of activin A and B levels in these patients with ARF would have assisted in predicting those at greatest risk of death. Given the existing data from animal studies linking high activin A levels to significant inflammatory challenges, the results from this study suggest that approaches to modulate

  2. Activin A accelerates the progression of fetal oocytes throughout meiosis and early oogenesis in the mouse.

    PubMed

    Liang, Gui-Jin; Zhang, Xi-Feng; Wang, Jun-Jie; Sun, Yuan-Chao; Sun, Xiao-Feng; Cheng, Shun-Feng; Li, Lan; De Felici, Massimo; Shen, Wei

    2015-10-15

    Activins can exert several roles in ovary development. However, little is known about their involvement in early mammalian oogenesis. In this study, we reported that activin receptors (including ActRIA, ActRIB, ActRIIA, and ActRIIB) are expressed throughout the development of the mouse ovaries from 12.5 days postcoitum (dpc) to 21 days postparturition (dpp). Moreover, we found that in vitro, the addition of activin A (ActA) to the culture medium of 12.5 dpc ovarian tissues accelerated the progression of oocytes throughout meiotic prophase I stages. This result was reproduced in vivo following administration of ActA to pregnant mice. The in vitro effect of ActA was associated with increased expression of premeiotic and meiotic genes (including Dazl, Spo11, Stra8, Scp3, and Rec8) in the ovarian tissues. Mechanistically, ActA-dependent SMAD3 signaling modulated the expression of members of the retinoic acid (RA) system, including the RA degradation CYP26B1 enzyme and the RA receptors. Finally, ActA promoted the survival and growth of fetal and early postnatal oocytes and primordial follicle assembly both in vitro and in vivo. In conclusion, the present study identifies new roles of ActA in early oogenesis and suggested that ActA and RA might cooperate in promoting meiosis in female germ cells.

  3. Overexpression of activin-A and -B in malignant mesothelioma – Attenuated Smad3 signaling responses and ERK activation promote cell migration and invasive growth

    SciTech Connect

    Tamminen, Jenni A.; Yin, Miao; Rönty, Mikko; Sutinen, Eva; Pasternack, Arja; Ritvos, Olli; Myllärniemi, Marjukka; Koli, Katri

    2015-03-01

    Activin-A and activin-B, members of the TGF-β superfamily, are regulators of reproductive functions, inflammation and wound healing. These dimeric molecules regulate various cellular activities such as proliferation, migration and suvival. Malignant mesothelioma is an asbestos exposure related tumor affecting mainly pleura and it usually has a dismal prognosis. Here, we demonstrate that both activin-A and -B are abundantly expressed in mesothelioma tumor tissue as well as in cultured primary and established mesothelioma cells. Migratory and invasive mesothelioma cells were also found to have attenuated activation of the Smad2/3 pathway in response to activins. Migration and invasive growth of the cells in three-dimentional matrix was prevented by inhibition of activin activity using a soluble activin receptor 2B (sActR2B-Fc). This was associated with decreased ERK activity. Furthermore, migration and invasive growth was significantly inhibited by blocking ERK phosphorylation. Mesothelioma tumors are locally invasive and our results clearly suggest that acivins have a tumor-promoting function in mesothelioma through increasing expression and switching from canonical Smad3 pathway to non-canonical ERK pathway signaling. Blocking activin activity offers a new therapeutic approach for inhibition of mesothelioma invasive growth. - Highlights: • Activin-A and activin-B are highly expressed in mesothelioma. • Mesothelioma cell migration and invasive growth can be blocked with sActR2B. • Activin induced Smad3 activity is attenuated in invasive mesothelioma cells. • Activins induce ERK activity in mesothelioma cells.

  4. Growth of the Obligate Anaerobe Desulfovibrio vulgaris Hildenborough under Continuous Low Oxygen Concentration Sparging: Impact of the Membrane-Bound Oxygen Reductases

    PubMed Central

    Ramel, Fanny; Brasseur, Gael; Pieulle, Laetitia; Valette, Odile; Hirschler-Réa, Agnès; Fardeau, Marie Laure; Dolla, Alain

    2015-01-01

    Although obligate anaerobe, the sulfate-reducing bacterium Desulfovibrio vulgaris Hildenborough (DvH) exhibits high aerotolerance that involves several enzymatic systems, including two membrane-bound oxygen reductases, a bd-quinol oxidase and a cc(b/o)o3 cytochrome oxidase. Effect of constant low oxygen concentration on growth and morphology of the wild-type, single (Δbd, Δcox) and double deletion (Δcoxbd) mutant strains of the genes encoding these oxygen reductases was studied. When both wild-type and deletion mutant strains were cultured in lactate/sulfate medium under constant 0.02% O2 sparging, they were able to grow but the final biomasses and the growth yield were lower than that obtained under anaerobic conditions. At the end of the growth, lactate was not completely consumed and when conditions were then switched to anaerobic, growth resumed. Time-lapse microscopy revealed that a large majority of the cells were then able to divide (over 97%) but the time to recover a complete division event was longer for single deletion mutant Δbd than for the three other strains. Determination of the molar growth yields on lactate suggested that a part of the energy gained from lactate oxidation was derived toward cells protection/repairing against oxidative conditions rather than biosynthesis, and that this part was higher in the single deletion mutant Δbd and, to a lesser extent, Δcox strains. Our data show that when DvH encounters oxidative conditions, it is able to stop growing and to rapidly resume growing when conditions are switched to anaerobic, suggesting that it enters active dormancy sate under oxidative conditions. We propose that the pyruvate-ferredoxin oxidoreductase (PFOR) plays a central role in this phenomenon by reversibly switching from an oxidative-sensitive fully active state to an oxidative-insensitive inactive state. The oxygen reductases, and especially the bd-quinol oxidase, would have a crucial function by maintaining reducing conditions

  5. Amino acid residues interacting with both the bound quinone and coenzyme, pyrroloquinoline quinone, in Escherichia coli membrane-bound glucose dehydrogenase.

    PubMed

    Mustafa, Golam; Ishikawa, Yoshinori; Kobayashi, Kazuo; Migita, Catharina T; Elias, M D; Nakamura, Satsuki; Tagawa, Seiichi; Yamada, Mamoru

    2008-08-01

    The Escherichia coli membrane-bound glucose dehydrogenase (mGDH) as the primary component of the respiratory chain possesses a tightly bound ubiquinone (UQ) flanking pyrroloquinoline quinone (PQQ) as a coenzyme. Several mutants for Asp-354, Asp-466, and Lys-493, located close to PQQ, that were constructed by site-specific mutagenesis were characterized by enzymatic, pulse radiolysis, and EPR analyses. These mutants retained almost no dehydrogenase activity or ability of PQQ reduction. CD and high pressure liquid chromatography analyses revealed that K493A, D466N, and D466E mutants showed no significant difference in molecular structure from that of the wild-type mGDH but showed remarkably reduced content of bound UQ. A radiolytically generated hydrated electron (e(aq)(-)) reacted with the bound UQ of the wild enzyme and K493R mutant to form a UQ neutral semiquinone with an absorption maximum at 420 nm. Subsequently, intramolecular electron transfer from the bound UQ semiquinone to PQQ occurred. In K493R, the rate of UQ to PQQ electron transfer is about 4-fold slower than that of the wild enzyme. With D354N and D466N mutants, on the other hand, transient species with an absorption maximum at 440 nm, a characteristic of the formation of a UQ anion radical, appeared in the reaction of e(aq)(-), although the subsequent intramolecular electron transfer was hardly affected. This indicates that D354N and D466N are prevented from protonation of the UQ semiquinone radical. Moreover, EPR spectra showed that mutations on Asp-466 or Lys-493 residues changed the semiquinone state of bound UQ. Taken together, we reported here for the first time the existence of a semiquinone radical of bound UQ in purified mGDH and the difference in protonation of ubisemiquinone radical because of mutations in two different amino acid residues, located around PQQ. Furthermore, based on the present results and the spatial arrangement around PQQ, Asp-466 and Lys-493 are suggested to interact both

  6. Growth of the obligate anaerobe Desulfovibrio vulgaris Hildenborough under continuous low oxygen concentration sparging: impact of the membrane-bound oxygen reductases.

    PubMed

    Ramel, Fanny; Brasseur, Gael; Pieulle, Laetitia; Valette, Odile; Hirschler-Réa, Agnès; Fardeau, Marie Laure; Dolla, Alain

    2015-01-01

    Although obligate anaerobe, the sulfate-reducing bacterium Desulfovibrio vulgaris Hildenborough (DvH) exhibits high aerotolerance that involves several enzymatic systems, including two membrane-bound oxygen reductases, a bd-quinol oxidase and a cc(b/o)o3 cytochrome oxidase. Effect of constant low oxygen concentration on growth and morphology of the wild-type, single (Δbd, Δcox) and double deletion (Δcoxbd) mutant strains of the genes encoding these oxygen reductases was studied. When both wild-type and deletion mutant strains were cultured in lactate/sulfate medium under constant 0.02% O2 sparging, they were able to grow but the final biomasses and the growth yield were lower than that obtained under anaerobic conditions. At the end of the growth, lactate was not completely consumed and when conditions were then switched to anaerobic, growth resumed. Time-lapse microscopy revealed that a large majority of the cells were then able to divide (over 97%) but the time to recover a complete division event was longer for single deletion mutant Δbd than for the three other strains. Determination of the molar growth yields on lactate suggested that a part of the energy gained from lactate oxidation was derived toward cells protection/repairing against oxidative conditions rather than biosynthesis, and that this part was higher in the single deletion mutant Δbd and, to a lesser extent, Δcox strains. Our data show that when DvH encounters oxidative conditions, it is able to stop growing and to rapidly resume growing when conditions are switched to anaerobic, suggesting that it enters active dormancy sate under oxidative conditions. We propose that the pyruvate-ferredoxin oxidoreductase (PFOR) plays a central role in this phenomenon by reversibly switching from an oxidative-sensitive fully active state to an oxidative-insensitive inactive state. The oxygen reductases, and especially the bd-quinol oxidase, would have a crucial function by maintaining reducing conditions

  7. Membrane-Bound CYB5R3 Is a Common Effector of Nutritional and Oxidative Stress Response Through FOXO3a and Nrf2

    PubMed Central

    Siendones, Emilio; SantaCruz-Calvo, Sara; Martín-Montalvo, Alejandro; Cascajo, María V.; Ariza, Julia; López-Lluch, Guillermo; Villalba, José M.; Acquaviva-Bourdain, Cécile; Roze, Emmanuel; Bernier, Michel; de Cabo, Rafael

    2014-01-01

    Abstract Aims: Membrane-bound CYB5R3 deficiency in humans causes recessive hereditary methaemoglobinaemia (RHM), an incurable disease that is characterized by severe neurological disorders. CYB5R3 encodes for NADH-dependent redox enzyme that contributes to metabolic homeostasis and stress protection; however, how it is involved in the neurological pathology of RHM remains unknown. Here, the role and transcriptional regulation of CYB5R3 was studied under nutritional and oxidative stress. Results: CYB5R3-deficient cells exhibited a decrease of the NAD+/NADH ratio, mitochondrial respiration rate, ATP production, and mitochondrial electron transport chain activities, which were associated with higher sensitivity to oxidative stress, and an increase in senescence-associated β-galactosidase activity. Overexpression of either forkhead box class O 3a (FOXO3a) or nuclear factor (erythroid-derived 2)-like2 (Nrf2) was associated with increased CYB5R3 levels, and genetic ablation of Nrf2 resulted in lower CYB5R3 expression. The presence of two antioxidant response element sequences in the CYB5R3 promoter led to chromatin immunoprecipitation studies, which showed that cellular stressors enhanced the binding of Nrf2 and FOXO3a to the CYB5R3 promoter. Innovation: Our findings demonstrate that CYB5R3 contributes to regulate redox homeostasis, aerobic metabolism, and cellular senescence, suggesting that CYB5R3 might be a key effector of oxidative and nutritional stress pathways. The expression of CYB5R3 is regulated by the cooperation of Nrf2 and FOXO3a. Conclusion: CYB5R3 is an essential gene that appears as a final effector for both nutritional and oxidative stress responses through FOXO3a and Nrf2, respectively, and their interaction promotes CYB5R3 expression. These results unveil a potential mechanism of action by which CYB5R3 deficiency contributes to the pathophysiological underpinnings of neurological disorders in RHM patients. Antioxid. Redox Signal. 21, 1708–1725. PMID

  8. Activin A-induced increase in LOX activity in human granulosa-lutein cells is mediated by CTGF.

    PubMed

    Chang, Hsun-Ming; Cheng, Jung-Chien; Liu, Yingtao; Klausen, Christian; Xu, Congjian; Leung, Peter C K

    2016-10-01

    Lysyl oxidase (LOX) is the key enzyme involved in the crosslinking of collagen and elastin that is essential for the formation of extracellular matrix (ECM). LOX-mediated ECM remodeling plays a critical role in follicle development, oocyte maturation and corpus luteum formation. To date, the regulation of LOX in human ovary has never been elucidated. Activin A and its functional receptors are highly expressed in ovarian follicles from an early developmental stage. They locally regulate follicle progression. The aim of this study was to investigate the effects of activin A on the expression of LOX and its extracellular enzyme activity in primary and immortalized human granulosa-lutein cells obtained from patients undergoing an in vitro fertilization procedure. We demonstrated that activin A significantly upregulated the expression of connective tissue growth factor (CTGF) and LOX via an activin/TGF-β type I receptor mediated-signaling pathway. Using a target depletion small interfering RNA knockdown approach, we further confirmed that the upregulation of CTGF expression resulted in an activin-A-induced increases in LOX expression and activity. These findings may provide insight into the mechanisms by which intrafollicular growth factors regulate the expression of LOX for ECM formation and tissue remodeling in the human ovary. PMID:27530347

  9. Activin Regulates Self-renewal and Differentiation of Trophoblast Stem Cells by Down-regulating the X Chromosome Gene Bcor*

    PubMed Central

    Zhu, Gaoyang; Fei, Teng; Li, Zhongwei; Yan, Xiaohua; Chen, Ye-Guang

    2015-01-01

    The development of a functional placenta is largely dependent upon proper proliferation and differentiation of trophoblast stem cells (TSCs). Activin signaling has long been regarded to play important roles during this process, but the exact mechanism is largely unknown. Here, we demonstrate that the X chromosome gene BCL-6 corepressor (Bcor) is a critical downstream effector of activin to fine-tune mouse TSC fate decision. Bcor was specifically down-regulated by activin A in TSCs in a dose-dependent manner, and immediately up-regulated upon TSC differentiation. Knockdown of Bcor partially compensated for the absence of activin A in maintaining the self-renewal of TSCs together with FGF4, while promoting syncytiotrophoblast differentiation in the absence of FGF4. Moreover, the impaired trophoblast giant cell and spongiotrophoblast differentiation upon Bcor knockdown also resembled the function of activin. Reporter analysis showed that BCOR inhibited the expression of the key trophoblast regulator genes Eomes and Cebpa by binding to their promoter regions. Our findings provide us with a better understanding of placental development and placenta-related diseases. PMID:26221038

  10. Seasonal changes in spermatogenesis and immunolocalization of inhibin/activin subunits in the wild male ground squirrel (Citellus dauricus Brandt).

    PubMed

    Sheng, Xia; Zhang, Haolin; Zhang, Wei; Song, Moshi; Zhang, Mengyuan; Li, Ben; Weng, Qiang; Watanabe, Gen; Taya, Kazuyoshi

    2008-12-01

    The objective of this study was to investigate the seasonal changes in spermatogenesis and the immunolocalization of the inhibin alpha and inhibin/activin (betaA and betaB) subunits during the breeding and non-breeding seasons in the wild male ground squirrel. The testicular weight and size and seminiferous tubule diameter were measured, and histological observations of testes were performed. The sections of the testes were immunostained by the avidin-biotin-peroxidase complex method (ABC) using polyclonal antisera raised against porcine inhibin alpha, inhibin/activin betaA and inhibin/activin betaB during the breeding and non-breeding seasons. There were marked variations in testicular weight and size and seminiferous tubule diameter between the breeding and non-breeding seasons, and all types of spermatogenic cells, including spermatozoa, were found in the breeding season. In addition, immunoreactivity was also detected for the inhibin alpha, betaA and betaB subunits in Sertoli and Leydig cells during the breeding season, but immunostaining was only present for the inhibin alpha and inhibin/activin betaB subunits in Sertoli cells during the non-breeding season. These results suggest that seasonal changes in testicular weight and size and seminiferous tubule diameter of wild ground squirrels are correlated with changes in spermatogenesis, and the cellular localization of the inhibin/activin subunits showed season related changes in the breeding and non-breeding seasons.

  11. Activin signaling balances proliferation and differentiation of ovarian niche precursors and enables adjustment of niche numbers.

    PubMed

    Lengil, Tamar; Gancz, Dana; Gilboa, Lilach

    2015-03-01

    How the numbers of niches and resident stem cells within a particular organ are determined during development and how they may be modulated or corrected is a question with significant medical implications. In the larval ovary of Drosophila melanogaster, somatic precursors for niches, and germ cells that will become germline stem cells, co-develop. Somatic precursors proliferate during the first 3 days of larval development. By mid-third instar, adult terminal filament (TF) (part of the germline stem cell niche) cells first appear, and differentiation terminates 24 h later when 16-20 TFs fully form. The developmental sequence responsible for TF cell determination and final TF numbers is only partially understood. We show that TF formation proceeds through several, hitherto uncharacterized stages, which include an early exit from the cell cycle to form TF precursors and two steps of cell shape change to form the mature TF cells. The Activin receptor Baboon (Babo) is required for somatic precursor cell proliferation and therefore determines the pool of TF precursors available for TF differentiation. During the final differentiation stage, Babo facilitates TF and germ cell differentiation, and promotes the accumulation of Broad-Z1, which is also a target of the steroid hormone ecdysone. Epistasis analysis shows that Activin controls cell proliferation in an ecdysone-independent manner and TF differentiation by affecting ecdysone targets. We propose that this mode of function allows Activin to balance proliferation and differentiation, and to equilibrate niche numbers. These results suggest a novel model for how niche numbers are corrected during development.

  12. Neuropoietic cytokines and activin A differentially regulate the phenotype of cultured sympathetic neurons.

    PubMed Central

    Fann, M J; Patterson, P H

    1994-01-01

    A number of cytokines sharing limited sequence homology have been grouped as a family because of partially overlapping biological activities, receptor subunit promiscuity, and the prediction of a shared secondary structure. Since several of these cytokines regulate gene expression and cell number in the nervous and hematopoietic systems, this specific group is termed the neuropoietic cytokine family. Using a reverse transcription-polymerase chain reaction-based assay system for monitoring the expression of multiple phenotypic markers in cultured sympathetic neurons, we present further evidence that, in addition to cholinergic differentiation factor/leukemia inhibitory factor and ciliary neurotrophic factor, oncostatin M, growth promoting activity, interleukin 6, and interleukin 11 belong in this family. In addition, one member of the transforming growth factor beta superfamily, activin A, shares a selective overlap with the neuropoietic family in the spectrum of neuropeptides that it induces in sympathetic neurons. The particular neuropeptides induced by activin A, however, demonstrate that the activity of this cytokine is distinct from that of the neuropoietic family. Twenty-six other cytokines and growth factors were without detectable activity in this assay. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 PMID:7904069

  13. Clinical Relevance and Mechanisms of Antagonism Between the BMP and Activin/TGF-β Signaling Pathways.

    PubMed

    Hudnall, Aaron M; Arthur, Jon W; Lowery, Jonathan W

    2016-07-01

    The transforming growth factor β (TGF-β) superfamily is a large group of signaling molecules that participate in embryogenesis, organogenesis, and tissue homeostasis. These molecules are present in all animal genomes. Dysfunction in the regulation or activity of this superfamily's components underlies numerous human diseases and developmental defects. There are 2 distinct arms downstream of the TGF-β superfamily ligands-the bone morphogenetic protein (BMP) and activin/TGF-β signaling pathways-and these 2 responses can oppose one another's effects, most notably in disease states. However, studies have commonly focused on a single arm of the TGF-β superfamily, and the antagonism between these pathways is unknown in most physiologic and pathologic contexts. In this review, the authors summarize the clinically relevant scenarios in which the BMP and activin/TGF-β pathways reportedly oppose one another and identify several molecular mechanisms proposed to mediate this interaction. Particular attention is paid to experimental findings that may be informative to human pathology to highlight potential therapeutic approaches for future investigation. PMID:27367950

  14. Activation of activin type IB receptor signals in pancreatic β cells leads to defective insulin secretion through the attenuation of ATP-sensitive K+ channel activity.

    PubMed

    Nomura, Masatoshi; Morinaga, Hidetaka; Zhu, Hai-Lei; Wang, Lixiang; Hasuzawa, Nao; Takayanagi, Ryoichi; Teramoto, Noriyoshi

    2014-07-18

    In studies of gene-ablated mice, activin signaling through activin type IIB receptors (ActRIIB) and Smad2 has been shown to regulate not only pancreatic β cell mass but also insulin secretion. However, it still remains unclear whether gain of function of activin signaling is involved in the modulation of pancreatic β cell mass and insulin secretion. To identify distinct roles of activin signaling in pancreatic β cells, the Cre-loxP system was used to activate signaling through activin type IB receptor (ActRIB) in pancreatic β cells. The resultant mice (pancreatic β cell-specific ActRIB transgenic (Tg) mice; ActRIBCAβTg) exhibited a defect in glucose-stimulated insulin secretion (GSIS) and a progressive impairment of glucose tolerance. Patch-clamp techniques revealed that the activity of ATP-sensitive K(+) channels (KATP channels) was decreased in mutant β cells. These results indicate that an appropriate level of activin signaling may be required for GSIS in pancreatic β cells, and that activin signaling involves modulation of KATP channel activity.

  15. [The Role of Membrane-Bound Heat Shock Proteins Hsp90 in Migration of Tumor Cells in vitro and Involvement of Cell Surface Heparan Sulfate Proteoglycans in Protein Binding to Plasma Membrane].

    PubMed

    Snigireva, A V; Vrublevskaya, V V; Skarga, Y Y; Morenkov, O S

    2016-01-01

    Heat shock protein Hsp90, detected in the extracellular space and on the membrane of cells, plays an important role in cell motility, migration, invasion and metastasis of tumor cells. At present, the functional role and molecular mechanisms of Hsp90 binding to plasma membrane are not elucidated. Using isoform-specific antibodies against Hsp90, Hsp9α and Hsp90β, we showed that membrane-bound Hsp90α and Hsp90β play a significant role in migration of human fibrosarcoma (HT1080) and glioblastoma (A-172) cells in vitro. Disorders of sulfonation of cell heparan sulfates, cleavage of cell heparan. sulfates by heparinase I/III as well as treatment of cells with heparin lead to an abrupt reduction in the expression level of Hsp90 isoforms. Furthermore, heparin significantly inhibits tumor cell migration. The results obtained demonstrate that two isoforms of membrane-bound Hsp90 are involved in migration of tumor cells in vitro and that cell surface heparan sulfate proteoglycans play a pivotal role in the "anchoring" of Hsp90α and Hsp90β to the plasma membrane.

  16. Modulatory Effect of Taurine on 7,12-Dimethylbenz(a)Anthracene-Induced Alterations in Detoxification Enzyme System, Membrane Bound Enzymes, Glycoprotein Profile and Proliferative Cell Nuclear Antigen in Rat Breast Tissue.

    PubMed

    Vanitha, Manickam Kalappan; Baskaran, Kuppusamy; Periyasamy, Kuppusamy; Selvaraj, Sundaramoorthy; Ilakkia, Aruldoss; Saravanan, Dhiravidamani; Venkateswari, Ramachandran; Revathi Mani, Balasundaram; Anandakumar, Pandi; Sakthisekaran, Dhanapal

    2016-08-01

    The modulatory effect of taurine on 7,12-dimethylbenz(a)anthracene (DMBA)-induced breast cancer in rats was studied. DMBA (25 mg/kg body weight) was administered to induce breast cancer in rats. Protein carbonyl levels, activities of membrane bound enzymes (Na(+) /K(+) ATPase, Ca(2+) ATPase, and Mg(2+) ATPase), phase I drug metabolizing enzymes (cytochrome P450, cytochrome b5, NADPH cytochrome c reductase), phase II drug metabolizing enzymes (glutathione-S-transferase and UDP-glucuronyl transferase), glycoprotein levels, and proliferative cell nuclear antigen (PCNA) were studied. DMBA-induced breast tumor bearing rats showed abnormal alterations in the levels of protein carbonyls, activities of membrane bound enzymes, drug metabolizing enzymes, glycoprotein levels, and PCNA protein expression levels. Taurine treatment (100 mg/kg body weight) appreciably counteracted all the above changes induced by DMBA. Histological examination of breast tissue further supported our biochemical findings. The results of the present study clearly demonstrated the chemotherapeutic effect of taurine in DMBA-induced breast cancer. PMID:27091720

  17. Modulatory Effect of Taurine on 7,12-Dimethylbenz(a)Anthracene-Induced Alterations in Detoxification Enzyme System, Membrane Bound Enzymes, Glycoprotein Profile and Proliferative Cell Nuclear Antigen in Rat Breast Tissue.

    PubMed

    Vanitha, Manickam Kalappan; Baskaran, Kuppusamy; Periyasamy, Kuppusamy; Selvaraj, Sundaramoorthy; Ilakkia, Aruldoss; Saravanan, Dhiravidamani; Venkateswari, Ramachandran; Revathi Mani, Balasundaram; Anandakumar, Pandi; Sakthisekaran, Dhanapal

    2016-08-01

    The modulatory effect of taurine on 7,12-dimethylbenz(a)anthracene (DMBA)-induced breast cancer in rats was studied. DMBA (25 mg/kg body weight) was administered to induce breast cancer in rats. Protein carbonyl levels, activities of membrane bound enzymes (Na(+) /K(+) ATPase, Ca(2+) ATPase, and Mg(2+) ATPase), phase I drug metabolizing enzymes (cytochrome P450, cytochrome b5, NADPH cytochrome c reductase), phase II drug metabolizing enzymes (glutathione-S-transferase and UDP-glucuronyl transferase), glycoprotein levels, and proliferative cell nuclear antigen (PCNA) were studied. DMBA-induced breast tumor bearing rats showed abnormal alterations in the levels of protein carbonyls, activities of membrane bound enzymes, drug metabolizing enzymes, glycoprotein levels, and PCNA protein expression levels. Taurine treatment (100 mg/kg body weight) appreciably counteracted all the above changes induced by DMBA. Histological examination of breast tissue further supported our biochemical findings. The results of the present study clearly demonstrated the chemotherapeutic effect of taurine in DMBA-induced breast cancer.

  18. Identification and expression of Smads associated with TGF-beta/activin/nodal signaling pathways in the rainbow trout (Oncorhynuchus mykiss)

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The Smad proteins are essential components of the TGF-beta/activin/nodal family signaling pathway. We report the identification and characterization of transcripts representing 3 receptor Smads (Smad2a, Smad2b, Smad3), 2 common Smads (Smad4a, Smad4b) and one inhibitory Smad (Smad7). Phylogenetic an...

  19. Molecular and cellular mechanisms of bone morphogenetic proteins and activins in the skin: potential benefits for wound healing.

    PubMed

    Moura, J; da Silva, L; Cruz, M T; Carvalho, E

    2013-09-01

    Bone morphogenetic proteins (BMPs) and activins are phylogenetically conserved proteins, belonging to the transforming growth factor-β superfamily, that signal through the phosphorylation of receptor-regulated Smad proteins, activating different cell responses. They are involved in various steps of skin morphogenesis and wound repair, as can be evidenced by the fact that their expression is increased in skin injuries. BMPs play not only a role in bone regeneration but are also involved in cartilage, tendon-like tissue and epithelial regeneration, maintain vascular integrity, capillary sprouting, proliferation/migration of endothelial cells and angiogenesis, promote neuron and dendrite formation, alter neuropeptide levels and are involved in immune response modulation, at least in animal models. On the other hand, activins are involved in wound repair through the regulation of skin and immune cell migration and differentiation, re-epithelialization and granulation tissue formation, and also promote the expression of collagens by fibroblasts and modulate scar formation. This review aims at enunciating the effects of BMPs and activins in the skin, namely in skin development, as well as in crucial phases of skin wound healing, such as inflammation, angiogenesis and repair, and will focus on the effects of these proteins on skin cells and their signaling pathways, exploring the potential therapeutic approach of the application of BMP-2, BMP-6 and activin A in chronic wounds, particularly diabetic foot ulcerations.

  20. An activin A/BMP2 chimera, AB204, displays bone-healing properties superior to those of BMP2.

    PubMed

    Yoon, Byung-Hak; Esquivies, Luis; Ahn, Chihoon; Gray, Peter C; Ye, Sang-Kyu; Kwiatkowski, Witek; Choe, Senyon

    2014-09-01

    Recombinant bone morphogenetic protein 2 (rhBMP2) has been used clinically to treat bone fractures in human patients. However, the high doses of rhBMP2 required for a therapeutic response can cause undesirable side effects. Here, we demonstrate that a novel Activin A/BMP2 (AB2) chimera, AB204, promotes osteogenesis and bone healing much more potently and effectively than rhBMP2. Remarkably, 1 month of AB204 treatment completely heals tibial and calvarial defects of critical size in mice at a concentration 10-fold lower than a dose of rhBMP2 that only partially heals the defect. We determine the structure of AB204 to 2.3 Å that reveals a distinct BMP2-like fold in which the Activin A sequence segments confer insensitivity to the BMP2 antagonist Noggin and an affinity for the Activin/BMP type II receptor ActRII that is 100-fold greater than that of BMP2. The structure also led to our identification of a single Activin A-derived amino acid residue, which, when mutated to the corresponding BMP2 residue, resulted in a significant increase in the affinity of AB204 for its type I receptor BMPRIa and a further enhancement in AB204's osteogenic potency. Together, these findings demonstrate that rationally designed AB2 chimeras can provide BMP2 substitutes with enhanced potency for treating non-union bone fractures.

  1. Activin Plays a Key Role in the Maintenance of Long-Term Memory and Late-LTP

    ERIC Educational Resources Information Center

    Ageta, Hiroshi; Ikegami, Shiro; Miura, Masami; Masuda, Masao; Migishima, Rika; Hino, Toshiaki; Takashima, Noriko; Murayama, Akiko; Sugino, Hiromu; Setou, Mitsutoshi; Kida, Satoshi; Yokoyama, Minesuke; Hasegawa, Yoshihisa; Tsuchida, Kunihiro; Aosaki, Toshihiko; Inokuchi, Kaoru

    2010-01-01

    A recent study has revealed that fear memory may be vulnerable following retrieval, and is then reconsolidated in a protein synthesis-dependent manner. However, little is known about the molecular mechanisms of these processes. Activin [beta]A, a member of the TGF-[beta] superfamily, is increased in activated neuronal circuits and regulates…

  2. ACVR1R206H receptor mutation causes fibrodysplasia ossificans progressiva by imparting responsiveness to activin A.

    PubMed

    Hatsell, Sarah J; Idone, Vincent; Wolken, Dana M Alessi; Huang, Lily; Kim, Hyon J; Wang, Lili; Wen, Xialing; Nannuru, Kalyan C; Jimenez, Johanna; Xie, Liqin; Das, Nanditha; Makhoul, Genevieve; Chernomorsky, Rostislav; D'Ambrosio, David; Corpina, Richard A; Schoenherr, Christopher J; Feeley, Kieran; Yu, Paul B; Yancopoulos, George D; Murphy, Andrew J; Economides, Aris N

    2015-09-01

    Fibrodysplasia ossificans progressiva (FOP) is a rare genetic disorder characterized by episodically exuberant heterotopic ossification (HO), whereby skeletal muscle is abnormally converted into misplaced, but histologically normal bone. This HO leads to progressive immobility with catastrophic consequences, including death by asphyxiation. FOP results from mutations in the intracellular domain of the type I BMP (bone morphogenetic protein) receptor ACVR1; the most common mutation alters arginine 206 to histidine (ACVR1(R206H)) and has been thought to drive inappropriate bone formation as a result of receptor hyperactivity. We unexpectedly found that this mutation rendered ACVR1 responsive to the activin family of ligands, which generally antagonize BMP signaling through ACVR1 but cannot normally induce bone formation. To test the implications of this finding in vivo, we engineered mice to carry the Acvr1(R206H) mutation. Because mice that constitutively express Acvr1[R206H] die perinatally, we generated a genetically humanized conditional-on knock-in model for this mutation. When Acvr1[R206H] expression was induced, mice developed HO resembling that of FOP; HO could also be triggered by activin A administration in this mouse model of FOP but not in wild-type controls. Finally, HO was blocked by broad-acting BMP blockers, as well as by a fully human antibody specific to activin A. Our results suggest that ACVR1(R206H) causes FOP by gaining responsiveness to the normally antagonistic ligand activin A, demonstrating that this ligand is necessary and sufficient for driving HO in a genetically accurate model of FOP; hence, our human antibody to activin A represents a potential therapeutic approach for FOP.

  3. Ligand trap for the activin type IIA receptor protects against vascular disease and renal fibrosis in mice with chronic kidney disease.

    PubMed

    Agapova, Olga A; Fang, Yifu; Sugatani, Toshifumi; Seifert, Michael E; Hruska, Keith A

    2016-06-01

    The causes of cardiovascular mortality associated with chronic kidney disease (CKD) are partly attributed to the CKD-mineral bone disorder (CKD-MBD). The causes of the early CKD-MBD are not well known. Our discovery of Wnt (portmanteau of wingless and int) inhibitors, especially Dickkopf 1, produced during renal repair as participating in the pathogenesis of the vascular and skeletal components of the CKD-MBD implied that additional pathogenic factors are critical. In the search for such factors, we studied the effects of activin receptor type IIA (ActRIIA) signaling by using a ligand trap for the receptor, RAP-011 (a soluble extracellular domain of ActRIIA fused to a murine IgG-Fc fragment). In a mouse model of CKD that stimulated atherosclerotic calcification, RAP-011 significantly increased aortic ActRIIA signaling assessed by the levels of phosphorylated Smad2/3. Furthermore, RAP-011 treatment significantly reversed CKD-induced vascular smooth muscle dedifferentiation as assessed by smooth muscle 22α levels, osteoblastic transition, and neointimal plaque calcification. In the diseased kidneys, RAP-011 significantly stimulated αklotho levels and it inhibited ActRIIA signaling and decreased renal fibrosis and proteinuria. RAP-011 treatment significantly decreased both renal and circulating Dickkopf 1 levels, showing that Wnt activation was downstream of ActRIIA. Thus, ActRIIA signaling in CKD contributes to the CKD-MBD and renal fibrosis. ActRIIA signaling may be a potential therapeutic target in CKD. PMID:27165838

  4. Characterization of Follistatin-Type Domains and Their Contribution to Myostatin and Activin A Antagonism

    PubMed Central

    Cash, Jennifer N.; Angerman, Elizabeth B.; Keutmann, Henry T.

    2012-01-01

    Follistatin (FST)-type proteins are important antagonists of some members of the large TGF-β family of cytokines. These include myostatin, an important negative regulator of muscle growth, and the closely related activin A, which is involved in many physiological functions, including maintenance of a normal reproductive axis. FST-type proteins, including FST and FST-like 3 (FSTL3), differentially inhibit various TGF-β family ligands by binding each ligand with two FST-type molecules. In this study, we sought to examine features that are important for ligand antagonism by FST-type proteins. Previous work has shown that a modified construct consisting of the FST N-terminal domain (ND) followed by two repeating follistatin domains (FSD), herein called FST ND-FSD1-FSD1, exhibits strong specificity for myostatin over activin A. Using cell-based assays, we show that FST ND-FSD1-FSD1 is unique in its specificity for myostatin as compared with similar constructs containing domains from FSTL3 and that the ND is critical to its activity. Furthermore, we demonstrate that FSD3 of FST provides affinity to ligand inhibition and confers resistance to perturbations in the ND and FSD2, likely through the interaction of FSD3 of one FST molecule with the ND of the other FST molecule. Additionally, our data suggest that this contact provides cooperativity to ligand antagonism. Cross-linking studies show that this interaction also potentiates formation of 1:2 ligand-FST complexes, whereas lack of FSD3 allows formation of 1:1 complexes. Altogether, these studies support that domain differences generate FST-type molecules that are each uniquely suited ligand antagonists. PMID:22593183

  5. RAP-011, an activin receptor ligand trap, increases hemoglobin concentration in hepcidin transgenic mice.

    PubMed

    Langdon, Jacqueline M; Barkataki, Sangjucta; Berger, Alan E; Cheadle, Chris; Xue, Qian-Li; Sung, Victoria; Roy, Cindy N

    2015-01-01

    Over expression of hepcidin antimicrobial peptide is a common feature of iron-restricted anemia in humans. We investigated the erythroid response to either erythropoietin or RAP-011, a "murinized" ortholog of sotatercept, in C57BL/6 mice and in hepcidin antimicrobial peptide 1 over expressing mice. Sotatercept, a soluble, activin receptor type IIA ligand trap, is currently being evaluated for the treatment of anemias associated with chronic renal disease, myelodysplastic syndrome, β-thalassemia, and Diamond Blackfan anemia and acts by inhibiting signaling downstream of activin and other Transforming Growth Factor-β superfamily members. We found that erythropoietin and RAP-011 increased hemoglobin concentration in C57BL/6 mice and in hepcidin antimicrobial peptide 1 over expressing mice. While erythropoietin treatment depleted splenic iron stores in C57BL/6 mice, RAP-011 treatment did not deplete splenic iron stores in mice of either genotype. Bone marrow erythroid progenitors from erythropoietin-treated mice exhibited iron-restricted erythropoiesis, as indicated by increased median fluorescence intensity of transferrin receptor immunostaining by flow cytometry. In contrast, RAP-011-treated mice did not exhibit the same degree of iron-restricted erythropoiesis. In conclusion, we have demonstrated that RAP-011 can improve hemoglobin concentration in hepcidin antimicrobial peptide 1 transgenic mice. Our data support the hypothesis that RAP-011 has unique biologic effects which prevent or circumvent depletion of mouse splenic iron stores. RAP-011 may, therefore, be an appropriate therapeutic for trials in human anemias characterized by increased expression of hepcidin antimicrobial peptide and iron-restricted erythropoiesis.

  6. Three homologues, including two membrane-bound proteins, of the disulfide oxidoreductase DsbA in Neisseria meningitidis: effects on bacterial growth and biogenesis of functional type IV pili.

    PubMed

    Tinsley, Colin R; Voulhoux, Romé; Beretti, Jean-Luc; Tommassen, Jan; Nassif, Xavier

    2004-06-25

    Many proteins, especially membrane and exported proteins, are stabilized by intramolecular disulfide bridges between cysteine residues without which they fail to attain their native functional conformation. The formation of these bonds is catalyzed in Gram-negative bacteria by enzymes of the Dsb system. Thus, the activity of DsbA has been shown to be necessary for many phenotypes dependent on exported proteins, including adhesion, invasion, and intracellular survival of various pathogens. The Dsb system in Neisseria meningitidis, the causative agent of cerebrospinal meningitis, has not, however, been studied. In a previous work where genes specific to N. meningitidis and not present in the other pathogenic Neisseria were isolated, a meningococcus-specific dsbA gene was brought to light (Tinsley, C. R., and Nassif, X. (1996) Proc. Natl. Acad. Sci. U. S. A. 93, 11109-11114). Inactivation of this gene, however, did not result in deficits in the phenotypes commonly associated with DsbA. A search of available genome data revealed that the meningococcus contains three dsbA genes encoding proteins with different predicted subcellular locations, i.e. a soluble periplasmic enzyme and two membrane-bound lipoproteins. Cell fractionation experiments confirmed the localization in the inner membrane of the latter two, which include the previously identified meningococcus-specific enzyme. Mutational analysis demonstrated that the deletion of any single enzyme was compensated by the action of the remaining two on bacterial growth, whereas the triple mutant was unable to grow at 37 degrees C. Remarkably, however, the combined absence of the two membrane-bound enzymes led to a phenotype of sensitivity to reducing agents and loss of functionality of the pili. Although in many species a single periplasmic DsbA is sufficient for the correct folding of various proteins, in the meningococcus a membrane-associated DsbA is required for a wild type DsbA+ phenotype even in the presence of a

  7. Role of osteoclasts in heterotopic ossification enhanced by fibrodysplasia ossificans progressiva-related activin-like kinase 2 mutation in mice.

    PubMed

    Kawao, Naoyuki; Yano, Masato; Tamura, Yukinori; Okumoto, Katsumi; Okada, Kiyotaka; Kaji, Hiroshi

    2016-09-01

    Fibrodysplasia ossificans progressiva (FOP) is a disorder of skeletal malformations and progressive heterotopic ossification. The constitutively activating mutation (R206H) of the bone morphogenetic protein type 1 receptor, activin-like kinase 2 (ALK2), is responsible for the pathogenesis of FOP. Although transfection of the causal mutation of FOP into myoblasts enhances osteoclast formation by transforming growth factor-β (TGF-β), the role of osteoclasts in heterotopic ossification is unknown. We therefore examined the effects of alendronate, SB431542 and SB203580 on heterotopic ossification induced by the causal mutation of FOP. Total bone mineral content as well as numbers of tartrate-resistant acid phosphatase (TRAP)-positive multinucleated and alkaline phosphatase (ALP)-positive cells in heterotopic bone were significantly higher in muscle tissues implanted with ALK2 (R206H)-transfected mouse myoblastic C2C12 cells than in the tissues implanted with empty vector-transfected cells in nude mice. Alendronate, an aminobisphosphonate, did not affect total mineral content or numbers of TRAP-positive multinucleated and ALP-positive cells in heterotopic bone, which were enhanced by the implantation of ALK2 (R206H)-transfected C2C12 cells, although it significantly decreased serum levels of cross-linked C-telopeptide of type I collagen, a bone resorption index. Moreover, neither SB431542, an inhibitor of TGF-β receptor type I kinase, nor SB203580, an inhibitor of p38 mitogen-activated protein kinase, affected the increase in heterotopic ossification due to the implantation of ALK2 (R206H)-transfected C2C12 cells. In conclusion, the present study indicates that osteoclast inhibition does not affect heterotopic ossification enhanced by FOP-related mutation.

  8. Discovery of Pyridinyl Acetamide Derivatives as Potent, Selective, and Orally Bioavailable Porcupine Inhibitors.

    PubMed

    Cheng, Dai; Liu, Jun; Han, Dong; Zhang, Guobao; Gao, Wenqi; Hsieh, Mindy H; Ng, Nicholas; Kasibhatla, Shailaja; Tompkins, Celin; Li, Jie; Steffy, Auzon; Sun, Fangxian; Li, Chun; Seidel, H Martin; Harris, Jennifer L; Pan, Shifeng

    2016-07-14

    Blockade of aberrant Wnt signaling is an attractive therapeutic approach in multiple cancers. We developed and performed a cellular high-throughput screen for inhibitors of Wnt secretion and pathway activation. A lead structure (GNF-1331) was identified from the screen. Further studies identified the molecular target of GNF-1331 as Porcupine, a membrane bound O-acyl transferase. Structure-activity relationship studies led to the discovery of a novel series of potent and selective Porcupine inhibitors. Compound 19, GNF-6231, demonstrated excellent pathway inhibition and induced robust antitumor efficacy in a mouse MMTV-WNT1 xenograft tumor model. PMID:27437076

  9. IMMUNOLOCALIZATION OF INHIBIN/ACTIVIN SUBUNITS AND STEROIDOGENIC ENZYMES IN THE TESTES OF AN ADULT AFRICAN ELEPHANT (LOXODONTA AFRICANA).

    PubMed

    Li, Qinglin; Lu, Lu; Weng, Qiang; Kawakami, Shigehisa; Saito, Eriko; Yamamoto, Tatsuya; Yamamoto, Yuki; Kaewmanee, Saroch; Nagaoka, Kentaro; Watanabe, Gen; Taya, Kazuyoshi

    2016-06-01

    In this case report, the authors investigated immunolocalization of inhibin α and inhibin/activin βA and βB subunits, as well as steroidogenic enzymes, in the testes of an African elephant. Testes were collected from a reproductively active male African elephant (24 yr old) at autopsy. Histologically, all types of spermatogenic cells including mature-phase spermatozoa were found in the seminiferous tubules. Positive immunostaining for inhibin α and inhibin/activin βA and βB subunits was observed in Sertoli and Leydig cells. In addition, P450scc, 3βHSD, P450c17, and P450arom were also detected in the cytoplasm of Leydig cells. These results suggested that Leydig cells of adult African elephant testes have the ability to synthesize progestin, androgen, and estrogen, whereas both Sertoli and Leydig cells appear as a major source of inhibin secretion in the male African elephant.

  10. An activin receptor IIA ligand trap promotes erythropoiesis resulting in a rapid induction of red blood cells and haemoglobin

    PubMed Central

    Carrancio, Soraya; Markovics, Jennifer; Wong, Piu; Leisten, Jim; Castiglioni, Paola; Groza, Matthew C; Raymon, Heather K; Heise, Carla; Daniel, Tom; Chopra, Rajesh; Sung, Victoria

    2014-01-01

    Sotatercept (ACE-011), a recombinant human fusion protein containing the extracellular domain of the human Activin receptor IIA, binds to and inhibits activin and other members of the transforming growth factor -β (TGF-β) superfamily. Administration of sotatercept led to a rapid and sustained increase in red blood cell (RBC) count and haemoglobin (Hb) in healthy volunteers (phase I clinical trials), but the mechanism is not fully understood. Mice treated with RAP-011 (murine ortholog of ACE-011) respond with a rapid (within 24 h) increase in haematocrit, Hb, and RBC count. These effects are accompanied by an equally rapid stimulation of late-stage erythroid precursors in the bone marrow (BM). RAP-011 also induces a significant increase in erythroid burst-forming units and erythropoietin, which could contribute to additional, sustained effects on RBC production. Further in vitro co-culture studies demonstrate that BM accessory cells are required for RAP-011 effects. To better understand which TGF-β family ligand(s) mediate RAP-011 effects, we evaluated the impact of several of these ligands on erythroid differentiation. Our data suggest that RAP-011 may act to rescue growth differentiation factor 11/Activin A-induced inhibition of late-stage erythropoiesis. These data define the mechanism of action of a novel agent that regulates RBC differentiation and provide the rationale to develop sotatercept for the treatment of anaemia and ineffective erythropoiesis. PMID:24635723

  11. Activin type IB receptor signaling in prostate cancer cells promotes lymph node metastasis in a xenograft model

    SciTech Connect

    Nomura, Masatoshi; Tanaka, Kimitaka; Wang, Lixiang; Goto, Yutaka; Mukasa, Chizu; Ashida, Kenji; Takayanagi, Ryoichi

    2013-01-04

    Highlights: Black-Right-Pointing-Pointer ActRIB signaling induces Snail and S100A4 expressions in prostate cancer cells. Black-Right-Pointing-Pointer The prostate cancer cell lines expressing an active form of ActRIB were established. Black-Right-Pointing-Pointer ActRIB signaling promotes EMT and lymph node metastasis in xenograft model. -- Abstract: Activin, a member of the transforming growth factor-{beta} family, has been known to be a growth and differentiating factor. Despite its pluripotent effects, the roles of activin signaling in prostate cancer pathogenesis are still unclear. In this study, we established several cell lines that express a constitutive active form of activin type IB receptor (ActRIBCA) in human prostate cancer cells, ALVA41 (ALVA-ActRIBCA). There was no apparent change in the proliferation of ALVA-ActRIBCA cells in vitro; however, their migratory ability was significantly enhanced. In a xenograft model, histological analysis revealed that the expression of Snail, a cell-adhesion-suppressing transcription factor, was dramatically increased in ALVA-ActRIBCA tumors, indicating epithelial mesenchymal transition (EMT). Finally, mice bearing ALVA-ActRIBCA cells developed multiple lymph node metastases. In this study, we demonstrated that ActRIBCA signaling can promote cell migration in prostate cancer cells via a network of signaling molecules that work together to trigger the process of EMT, and thereby aid in the aggressiveness and progression of prostate cancers.

  12. Effect of repeated activin-A treatment on the activity of the hypothalamic-pituitary-gonadal axis of the adult male rat.

    PubMed

    Lee, S; Rivier, C

    1997-04-01

    The present study was undertaken to determine whether recombinant activin-A increases mRNA levels of the peptide GnRH, and whether this phenomenon correlates with increased FSH and/or LH release. One acute s.c. injection of activin-A (120 micrograms/kg body weight) to adult male rats was found to significantly (P < 0.01) increase plasma FSH levels, with no detectable changes in LH or testosterone (T) release. Similarly, there were no significant differences between GnRH mRNA values measured in the medial preoptic area of the hypothalamus. When activin-A was administered s.c. every 8 h for seven consecutive treatments, similar results were obtained for gonadotropin release; in addition, we observed a significant (p < 0.01) up-regulation of hypothalamic GnRH mRNA levels. The administration of activin-A intracerebroventricularly (i.c.v., 6 micrograms/rat) according to the same chronic schedule also produced a significant (p < 0.01) increase in FSH levels, as well as a modest, but detectable, elevation in LH concentrations, and a large augmentation of T secretion. In contrast, there were no changes in steady-state GnRH mRNA concentrations. Collectively, these results show that both the systemic (s.c.) and the central (i.c.v.) injection of activin-A stimulates FSH secretion. While we had originally thought that the lack of response of hypothalamic GnRH neurons to i.c.v. activin injections might have been due to increased steroid feedback, the observation that comparable results were obtained in both intact and castrated rats does not support this hypothesis. One possibility is that the previously reported stimulatory influence of i.c.v. activin-A treatment on neurons that manufacture corticotropin-releasing factor, and consequently on circulating catecholamine concentrations, may have increased testicular activity independently of changes in pituitary function.

  13. Defining a key receptor-CheA kinase contact and elucidating its function in the membrane-bound bacterial chemosensory array: a disulfide mapping and TAM-IDS Study.

    PubMed

    Piasta, Kene N; Ulliman, Caleb J; Slivka, Peter F; Crane, Brian R; Falke, Joseph J

    2013-06-01

    The three core components of the ubiquitous bacterial chemosensory array - the transmembrane chemoreceptor, the histidine kinase CheA, and the adaptor protein CheW - assemble to form a membrane-bound, hexagonal lattice in which receptor transmembrane signals regulate kinase activity. Both the regulatory domain of the kinase and the adaptor protein bind to overlapping sites on the cytoplasmic tip of the receptor (termed the protein interaction region). Notably, the kinase regulatory domain and the adaptor protein share the same fold constructed of two SH3-like domains. The present study focuses on the structural interface between the receptor and the kinase regulatory domain. Two models have been proposed for this interface: Model 1 is based on the crystal structure of a homologous Thermotoga complex between a receptor fragment and the CheW adaptor protein. This model has been used in current models of chemosensory array architecture to build the receptor-CheA kinase interface. Model 2 is based on a newly determined crystal structure of a homologous Thermotoga complex between a receptor fragment and the CheA kinase regulatory domain. Both models present unique strengths and weaknesses, and current evidence is unable to resolve which model best describes contacts in the native chemosensory arrays of Escherichia coli, Salmonella typhimurium, and other bacteria. Here we employ disulfide mapping and tryptophan and alanine mutation to identify docking sites (TAM-IDS) to test Models 1 and 2 in well-characterized membrane-bound arrays formed from E. coli and S. typhimurium components. The results reveal that the native array interface between the receptor protein interaction region and the kinase regulatory domain is accurately described by Model 2, but not by Model 1. In addition, the results show that the interface possesses both a structural function that contributes to stable CheA kinase binding in the array and a regulatory function central to transmission of the

  14. Effect of physical training on liver expression of activin A and follistatin in a nonalcoholic fatty liver disease model in rats.

    PubMed

    Silva, R N; Bueno, P G; Avó, L R S; Nonaka, K O; Selistre-Araújo, H S; Leal, A M O

    2014-09-01

    Nonalcoholic fatty liver disease (NAFLD) is characterized by fat accumulation in the liver and is associated with obesity and insulin resistance. Activin A is a member of the transforming growth factor beta (TGF)-β superfamily and inhibits hepatocyte growth. Follistatin antagonizes the biological actions of activin. Exercise is an important therapeutic strategy to reduce the metabolic effects of obesity. We evaluated the pattern of activin A and follistatin liver expression in obese rats subjected to swimming exercise. Control rats (C) and high-fat (HF) diet-fed rats were randomly assigned to a swimming training group (C-Swim and HF-Swim) or a sedentary group (C-Sed and HF-Sed). Activin βA subunit mRNA expression was significantly higher in HF-Swim than in HF-Sed rats. Follistatin mRNA expression was significantly lower in C-Swim and HF-Swim than in either C-Sed or HF-Sed animals. There was no evidence of steatosis or inflammation in C rats. In contrast, in HF animals the severity of steatosis ranged from grade 1 to grade 3. The extent of liver parenchyma damage was less in HF-Swim animals, with the severity of steatosis ranging from grade 0 to grade 1. These data showed that exercise may reduce the deleterious effects of a high-fat diet on the liver, suggesting that the local expression of activin-follistatin may be involved.

  15. Effect of physical training on liver expression of activin A and follistatin in a nonalcoholic fatty liver disease model in rats

    PubMed Central

    Silva, R.N.; Bueno, P.G.; Avó, L.R.S.; Nonaka, K.O.; Selistre-Araújo, H.S.; Leal, A.M.O.

    2014-01-01

    Nonalcoholic fatty liver disease (NAFLD) is characterized by fat accumulation in the liver and is associated with obesity and insulin resistance. Activin A is a member of the transforming growth factor beta (TGF)-β superfamily and inhibits hepatocyte growth. Follistatin antagonizes the biological actions of activin. Exercise is an important therapeutic strategy to reduce the metabolic effects of obesity. We evaluated the pattern of activin A and follistatin liver expression in obese rats subjected to swimming exercise. Control rats (C) and high-fat (HF) diet-fed rats were randomly assigned to a swimming training group (C-Swim and HF-Swim) or a sedentary group (C-Sed and HF-Sed). Activin βA subunit mRNA expression was significantly higher in HF-Swim than in HF-Sed rats. Follistatin mRNA expression was significantly lower in C-Swim and HF-Swim than in either C-Sed or HF-Sed animals. There was no evidence of steatosis or inflammation in C rats. In contrast, in HF animals the severity of steatosis ranged from grade 1 to grade 3. The extent of liver parenchyma damage was less in HF-Swim animals, with the severity of steatosis ranging from grade 0 to grade 1. These data showed that exercise may reduce the deleterious effects of a high-fat diet on the liver, suggesting that the local expression of activin-follistatin may be involved. PMID:25075578

  16. The Notch ligand Delta-like 1 integrates inputs from TGFbeta/Activin and Wnt pathways

    SciTech Connect

    Bordonaro, Michael Tewari, Shruti Atamna, Wafa Lazarova, Darina L.

    2011-06-10

    Unlike the well-characterized nuclear function of the Notch intracellular domain, it has been difficult to identify a nuclear role for the ligands of Notch. Here we provide evidence for the nuclear function of the Notch ligand Delta-like 1 in colon cancer (CC) cells exposed to butyrate. We demonstrate that the intracellular domain of Delta-like 1 (Dll1icd) augments the activity of Wnt signaling-dependent reporters and that of the promoter of the connective tissue growth factor (CTGF) gene. Data suggest that Dll1icd upregulates CTGF promoter activity through both direct and indirect mechanisms. The direct mechanism is supported by co-immunoprecipitation of endogenous Smad2/3 proteins and Dll1 and by chromatin immunoprecipitation analyses that revealed the occupancy of Dll1icd on CTGF promoter sequences containing a Smad binding element. The indirect upregulation of CTGF expression by Dll1 is likely due to the ability of Dll1icd to increase Wnt signaling, a pathway that targets CTGF. CTGF expression is induced in butyrate-treated CC cells and results from clonal growth assays support a role for CTGF in the cell growth-suppressive role of butyrate. In conclusion, integration of the Notch, Wnt, and TGFbeta/Activin signaling pathways is in part mediated by the interactions of Dll1 with Smad2/3 and Tcf4.

  17. A single membrane-bound enzyme catalyzes the conversion of 2,5-diketo-d-gluconate to 4-keto-d-arabonate in d-glucose oxidative fermentation by Gluconobacter oxydans NBRC 3292.

    PubMed

    Tazoe, Masaaki; Oishi, Hiromi; Kobayashi, Setsuko; Hoshino, Tatsuo

    2016-08-01

    4-Keto-d-arabonate synthase (4KAS), which converts 2,5-diketo-d-gluconate (DKGA) to 4-keto-d-arabonate (4KA) in d-glucose oxidative fermentation by some acetic acid bacteria, was solubilized from the Gluconobacter oxydans NBRC 3292 cytoplasmic membrane, and purified in an electrophoretically homogenous state. A single membrane-bound enzyme was found to catalyze the conversion from DKGA to 4KA. The 92-kDa 4KAS was a homodimeric protein not requiring O2 or a cofactor for the conversion, but was stimulated by Mn(2+). N-terminal amino acid sequencing of 4KAS, followed by gene homology search indicated a 1,197-bp open reading frame (ORF), corresponding to the GLS_c04240 locus, GenBank accession No. CP004373, encoding a 398-amino acid protein with a calculated molecular weight of 42,818 Da. An Escherichia coli transformant with the 4kas plasmid exhibited 4KAS activity. Furthermore, overexpressed recombinant 4KAS was purified in an electrophoretically homogenous state and had the same molecular size as the natural enzyme.

  18. Molecular characterization and expression of dipeptidase 3, a testis-specific membrane-bound dipeptidase: complex formation with TEX101, a germ-cell-specific antigen in the mouse testis.

    PubMed

    Yoshitake, Hiroshi; Yanagida, Mitsuaki; Maruyama, Mayuko; Takamori, Kenji; Hasegawa, Akiko; Araki, Yoshihiko

    2011-08-01

    We previously established an anti-sperm head auto-monoclonal antibody designated Ts4. The immunoreactivity of this antibody was also observed in other reproduction-related cells, such as testicular germ cells and early embryos, suggesting that the Ts4-recognized molecules might play a role in the reproductive process. However, the molecular characteristics and functions of the antigens warrant further clarification. In this study, we primarily attempted identification of the mAb-recognized molecules within the mouse testis. An immunoprecipitation method, together with liquid chromatography-tandem mass spectrometry, revealed that the testicular immunoprecipitants with Ts4 contained dipeptidase 3 (DPEP3), a member of the membrane-bound dipeptidase family. A Western blot analysis using an anti-DPEP3 polyclonal antibody established in this study showed that this molecule was glycosylated and formed a disulfide-linked homodimer within the testis. Expression of DPEP3 protein was observed in the testicular germ cells, but not in the Sertoli or interstitial cells, or in any other major organs. Although Western blot analysis of testicular proteins separated by two-dimensional SDS-PAGE failed to demonstrate binding of Ts4 to DPEP3, we found that DPEP3 forms complexes with Ts4-immunoreactive molecules, such as TEX101, on the surfaces of spermatocytes, spermatids, and testicular spermatozoa. Based on data showing in the present study, further studies concerning DPEP3 on the testicular germ cells may help to clarify the molecular mechanisms of testicular germ-cell development.

  19. Desensitization of membrane-bound Torpedo acetylcholine receptor by amine noncompetitive antagonists and aliphatic alcohols: studies of (/sup 3/H)acetylcholine binding and /sup 22/Na/sup +/ ion fluxes

    SciTech Connect

    Boyd, N.D.; Cohen, J.B.

    1984-08-28

    Measurements of the kinetics of binding of (/sup 3/H)acetylcholine ((/sup 3/H)AcCh) to membrane-bound nicotinic AcCh receptors from Torpedo electric tissue have been used to characterize the effects of amine and alcohol noncompetitive antagonists on receptor conformational equilibria. The receptor exists in interconvertible conformations distinguished by agonist binding affinity. The high-affinity receptor conformation stabilized by noncompetitive antagonists was characterized by (1) the rate constant (k/sub rec/) for receptor reisomerization upon removal of stabilizing ligand and (2) the rate constant (k/sub dis/) for dissociation of (/sup 3/H)AcCh-receptor complexes. On the basis of these criteria, the high-affinity receptor conformation stabilized by amine and alcohol noncompetitive blockers is the same as that stabilized by agonist. Histrionicotoxin (HTX) and adiphenine antagonized the conformational perturbation caused by proadifen, while mixtures of HTX and 2-propanol produced additive effects. Exposure to proadifen in the absence of agonist produced a reversible inhibition (desensitization) of the flux response, and recovery from desensitization occurred at the same rate as the reisomerization from the high-affinity receptor state. HTX, which did not cause desensitization of the flux response, reduced the desensitization by proadifen. These results are compatible with the hypothesis that certain noncompetitive antagonists modify receptor function by stabilizing the same high-affinity (desensitized) conformation that is stabilized by agonists, either as a consequence of binding to the allosteric site or by an alternate mechanism.

  20. Synthesis of paucimannose N-glycans by Caenorhabditis elegans requires prior actions of UDP-N-acetyl-D-glucosamine:alpha-3-D-mannoside beta1,2-N-acetylglucosaminyltransferase I, alpha3,6-mannosidase II and a specific membrane-bound beta-N-acetylglucosaminidase.

    PubMed Central

    Zhang, Wenli; Cao, Pinjiang; Chen, Shihao; Spence, Andrew M; Zhu, Shaoxian; Staudacher, Erika; Schachter, Harry

    2003-01-01

    We have previously reported three Caenorhabditis elegans genes ( gly-12, gly-13 and gly-14 ) encoding UDP- N -acetyl-D-glucosamine:alpha-3-D-mannoside beta1,2- N -acetylglucosaminyltransferase I (GnT I), an enzyme essential for hybrid and complex N-glycan synthesis. GLY-13 was shown to be the major GnT I in worms and to be the only GnT I cloned to date which can act on [Manalpha1,6(Manalpha1,3)Manalpha1,6](Manalpha1,3)Manbeta1, 4GlcNAcbeta1,4GlcNAc-R, but not on Manalpha1,6(Manalpha1,3)Manbeta1- O -R substrates. We now report the kinetic constants, bivalent-metal-ion requirements, and optimal pH, temperature and Mn(2+) concentration for this unusual enzyme. C. elegans glycoproteins are rich in oligomannose (Man(6-9)GlcNAc(2)) and 'paucimannose' Man(3-5)GlcNAc(2)(+/-Fuc) N-glycans, but contain only small amounts of complex and hybrid N-glycans. We show that the synthesis of paucimannose Man(3)GlcNAc(2) requires the prior actions of GnT I, alpha3,6-mannosidase II and a membrane-bound beta- N -acetylglucosaminidase similar to an enzyme previously reported in insects. The beta- N -acetylglucosaminidase removes terminal N -acetyl-D-glucosamine from the GlcNAcbeta1, 2Manalpha1,3Manbeta- arm of Manalpha1,6(GlcNAcbeta1,2Manalpha1,3) Manbeta1,4GlcNAcbeta1,4GlcNAc-R to produce paucimannose Man(3)GlcNAc(2) N-glycan. N -acetyl-D-glucosamine removal was inhibited by two N -acetylglucosaminidase inhibitors. Terminal GlcNAc was not released from [Manalpha1,6(Manalpha1,3)Manalpha 1,6] (GlcNAcbeta1,2Manalpha1,3)Manbeta1,4GlcNAcbeta1,4GlcNAc-R nor from the GlcNAcbeta1,2Manalpha1,6Manbeta- arm. These findings indicate that GLY-13 plays an important role in the synthesis of N-glycans by C. elegans and that therefore the worm should prove to be a suitable model for the study of the role of GnT I in nematode development. PMID:12603202

  1. Activin Enhances α- to β-Cell Transdifferentiation as a Source For β-Cells In Male FSTL3 Knockout Mice.

    PubMed

    Brown, Melissa L; Andrzejewski, Danielle; Burnside, Amy; Schneyer, Alan L

    2016-03-01

    Diabetes results from inadequate β-cell number and/or function to control serum glucose concentrations so that replacement of lost β-cells could become a viable therapy for diabetes. In addition to embryonic stem cell sources for new β-cells, evidence for transdifferentiation/reprogramming of non-β-cells to functional β-cells is accumulating. In addition, de-differentiation of β-cells observed in diabetes and their subsequent conversion to α-cells raises the possibility that adult islet cell fate is malleable and controlled by local hormonal and/or environmental cues. We previously demonstrated that inactivation of the activin antagonist, follistatin-like 3 (FSTL3) resulted in β-cell expansion and improved glucose homeostasis in the absence of β-cell proliferation. We recently reported that activin directly suppressed expression of critical α-cell genes while increasing expression of β-cell genes, supporting the hypothesis that activin is one of the local hormones controlling islet cell fate and that increased activin signaling accelerates α- to β-cell transdifferentiation. We tested this hypothesis using Gluc-Cre/yellow fluorescent protein (YFP) α-cell lineage tracing technology combined with FSTL3 knockout (KO) mice to label α-cells with YFP. Flow cytometry was used to quantify unlabeled and labeled α- and β-cells. We found that Ins+/YFP+ cells were significantly increased in FSTL3 KO mice compared with wild type littermates. Labeled Ins+/YFP+ cells increased significantly with age in FSTL3 KO mice but not wild type littermates. Sorting results were substantiated by counting fluorescently labeled cells in pancreatic sections. Activin treatment of isolated islets significantly increased the number of YFP+/Ins+ cells. These results suggest that α- to β-cell transdifferentiation is influenced by activin signaling and may contribute substantially to β-cell mass.

  2. Atypical Activin A and IL-10 Production Impairs Human CD16+ Monocyte Differentiation into Anti-Inflammatory Macrophages.

    PubMed

    González-Domínguez, Érika; Domínguez-Soto, Ángeles; Nieto, Concha; Flores-Sevilla, José Luis; Pacheco-Blanco, Mariana; Campos-Peña, Victoria; Meraz-Ríos, Marco A; Vega, Miguel A; Corbí, Ángel L; Sánchez-Torres, Carmen

    2016-02-01

    Human CD14(++)CD16(-) and CD14(+/lo)CD16(+) monocyte subsets comprise 85 and 15% of blood monocytes, respectively, and are thought to represent distinct stages in the monocyte differentiation pathway. However, the differentiation fates of both monocyte subsets along the macrophage (Mϕ) lineage have not yet been elucidated. We have now evaluated the potential of CD14(++) CD16(-) and CD16(+) monocytes to differentiate and to be primed toward pro- or anti-inflammatory Mϕs upon culture with GM-CSF or M-CSF, respectively (subsequently referred to as GM14, M14, GM16, or M16). Whereas GM16 and GM14 were phenotypic and functionally analogous, M16 displayed a more proinflammatory profile than did M14. Transcriptomic analyses evidenced that genes associated with M-CSF-driven Mϕ differentiation (including FOLR2, IL10, IGF1, and SERPINB2) are underrepresented in M16 with respect to M14. The preferential proinflammatory skewing of M16 relative to M14 was found to be mediated by the secretion of activin A and the low levels of IL-10 produced by M16. In fact, activin A receptor blockade during the M-CSF-driven differentiation of CD16(+) monocytes, or addition of IL-10-containing M14-conditioned medium, significantly enhanced their expression of anti-inflammatory-associated molecules while impairing their acquisition of proinflammatory-related markers. Thus, we propose that M-CSF drives CD14(++)CD16- monocyte differentiation into bona fide anti-inflammatory Mϕs in a self-autonomous manner, whereas M-CSF-treated CD16(+) monocytes generate Mϕs with a skewed proinflammatory profile by virtue of their high activin A expression unless additional anti-inflammatory stimuli such as IL-10 are provided. PMID:26729812

  3. Inhibition of Activin Receptor Type IIB Increases Strength and Lifespan in Myotubularin-Deficient Mice

    PubMed Central

    Lawlor, Michael W.; Read, Benjamin P.; Edelstein, Rachel; Yang, Nicole; Pierson, Christopher R.; Stein, Matthew J.; Wermer-Colan, Ariana; Buj-Bello, Anna; Lachey, Jennifer L.; Seehra, Jasbir S.; Beggs, Alan H.

    2011-01-01

    X-linked myotubular myopathy (XLMTM) is a congenital disorder caused by deficiency of the lipid phosphatase, myotubularin. Patients with XLMTM often have severe perinatal weakness that requires mechanical ventilation to prevent death from respiratory failure. Muscle biopsy specimens from patients with XLMTM exhibit small myofibers with central nuclei and central aggregations of organelles in many cells. It was postulated that therapeutically increasing muscle fiber size would cause symptomatic improvement in myotubularin deficiency. Recent studies have elucidated an important role for the activin-receptor type IIB (ActRIIB) in regulation of muscle growth and have demonstrated that ActRIIB inhibition results in significant muscle hypertrophy. To evaluate whether promoting muscle hypertrophy can attenuate symptoms resulting from myotubularin deficiency, the effect of ActRIIB-mFC treatment was determined in myotubularin-deficient (Mtm1δ4) mice. Compared with wild-type mice, untreated Mtm1δ4 mice have decreased body weight, skeletal muscle hypotrophy, and reduced survival. Treatment of Mtm1δ4 mice with ActRIIB-mFC produced a 17% extension of lifespan, with transient increases in weight, forelimb grip strength, and myofiber size. Pathologic analysis of Mtm1δ4 mice during treatment revealed that ActRIIB-mFC produced marked hypertrophy restricted to type 2b myofibers, which suggests that oxidative fibers in Mtm1δ4 animals are incapable of a hypertrophic response in this setting. These results support ActRIIB-mFC as an effective treatment for the weakness observed in myotubularin deficiency. PMID:21281811

  4. The glycoprotein-hormones activin A and inhibin A interfere with dendritic cell maturation

    PubMed Central

    Segerer, Sabine E; Müller, Nora; Brandt, Jens van den; Kapp, Michaela; Dietl, Johannes; Reichardt, Holger M; Rieger, Lorenz; Kämmerer, Ulrike

    2008-01-01

    Background Pregnancy represents an exclusive situation in which the immune and the endocrine system cooperate to prevent rejection of the embryo by the maternal immune system. While immature dendritic cells (iDC) in the early pregnancy decidua presumably contribute to the establishment of peripheral tolerance, glycoprotein-hormones of the transforming growth factor beta (TGF-beta) family including activin A (ActA) and inhibin A (InA) are candidates that could direct the differentiation of DCs into a tolerance-inducing phenotype. Methods To test this hypothesis we generated iDCs from peripheral-blood-monocytes and exposed them to TGF-beta1, ActA, as well as InA and Dexamethasone (Dex) as controls. Results Both glycoprotein-hormones prevented up-regulation of HLA-DR during cytokine-induced DC maturation similar to Dex but did not influence the expression of CD 40, CD 83 and CD 86. Visualization of the F-actin cytoskeleton confirmed that the DCs retained a partially immature phenotype under these conditions. The T-cell stimulatory capacity of DCs was reduced after ActA and InA exposure while the secretion of cytokines and chemokines was unaffected. Conclusion These findings suggest that ActA and InA interfere with selected aspects of DC maturation and may thereby help preventing activation of allogenic T-cells by the embryo. Thus, we have identified two novel members of the TGF-beta superfamily that could promote the generation of tolerance-inducing DCs. PMID:18460206

  5. Inhibition of activin receptor type IIB increases strength and lifespan in myotubularin-deficient mice.

    PubMed

    Lawlor, Michael W; Read, Benjamin P; Edelstein, Rachel; Yang, Nicole; Pierson, Christopher R; Stein, Matthew J; Wermer-Colan, Ariana; Buj-Bello, Anna; Lachey, Jennifer L; Seehra, Jasbir S; Beggs, Alan H

    2011-02-01

    X-linked myotubular myopathy (XLMTM) is a congenital disorder caused by deficiency of the lipid phosphatase, myotubularin. Patients with XLMTM often have severe perinatal weakness that requires mechanical ventilation to prevent death from respiratory failure. Muscle biopsy specimens from patients with XLMTM exhibit small myofibers with central nuclei and central aggregations of organelles in many cells. It was postulated that therapeutically increasing muscle fiber size would cause symptomatic improvement in myotubularin deficiency. Recent studies have elucidated an important role for the activin-receptor type IIB (ActRIIB) in regulation of muscle growth and have demonstrated that ActRIIB inhibition results in significant muscle hypertrophy. To evaluate whether promoting muscle hypertrophy can attenuate symptoms resulting from myotubularin deficiency, the effect of ActRIIB-mFC treatment was determined in myotubularin-deficient (Mtm1δ4) mice. Compared with wild-type mice, untreated Mtm1δ4 mice have decreased body weight, skeletal muscle hypotrophy, and reduced survival. Treatment of Mtm1δ4 mice with ActRIIB-mFC produced a 17% extension of lifespan, with transient increases in weight, forelimb grip strength, and myofiber size. Pathologic analysis of Mtm1δ4 mice during treatment revealed that ActRIIB-mFC produced marked hypertrophy restricted to type 2b myofibers, which suggests that oxidative fibers in Mtm1δ4 animals are incapable of a hypertrophic response in this setting. These results support ActRIIB-mFC as an effective treatment for the weakness observed in myotubularin deficiency. PMID:21281811

  6. Activin-Like Kinase 2 Functions in Peri-implantation Uterine Signaling in Mice and Humans

    PubMed Central

    Clementi, Caterina; Tripurani, Swamy K.; Large, Michael J.; Edson, Mark A.; Creighton, Chad J.; Hawkins, Shannon M.; Kovanci, Ertug; Kaartinen, Vesa; Lydon, John P.; Pangas, Stephanie A.; DeMayo, Francesco J.; Matzuk, Martin M.

    2013-01-01

    Implantation of a blastocyst in the uterus is a multistep process tightly controlled by an intricate regulatory network of interconnected ovarian, uterine, and embryonic factors. Bone morphogenetic protein (BMP) ligands and receptors are expressed in the uterus of pregnant mice, and BMP2 has been shown to be a key regulator of implantation. In this study, we investigated the roles of the BMP type 1 receptor, activin-like kinase 2 (ALK2), during mouse pregnancy by producing mice carrying a conditional ablation of Alk2 in the uterus (Alk2 cKO mice). In the absence of ALK2, embryos demonstrate delayed invasion into the uterine epithelium and stroma, and upon implantation, stromal cells fail to undergo uterine decidualization, resulting in sterility. Mechanistically, microarray analysis revealed that CCAAT/enhancer-binding protein β (Cebpb) expression is suppressed during decidualization in Alk2 cKO females. These findings and the similar phenotypes of Cebpb cKO and Alk2 cKO mice lead to the hypothesis that BMPs act upstream of CEBPB in the stroma to regulate decidualization. To test this hypothesis, we knocked down ALK2 in human uterine stromal cells (hESC) and discovered that ablation of ALK2 alters hESC decidualization and suppresses CEBPB mRNA and protein levels. Chromatin immunoprecipitation (ChIP) analysis of decidualizing hESC confirmed that BMP signaling proteins, SMAD1/5, directly regulate expression of CEBPB by binding a distinct regulatory sequence in the 3′ UTR of this gene; CEBPB, in turn, regulates the expression of progesterone receptor (PGR). Our work clarifies the conserved mechanisms through which BMPs regulate peri-implantation in rodents and primates and, for the first time, uncovers a linear pathway of BMP signaling through ALK2 to regulate CEBPB and, subsequently, PGR during decidualization. PMID:24244176

  7. Uterine activin receptor-like kinase 5 is crucial for blastocyst implantation and placental development.

    PubMed

    Peng, Jia; Monsivais, Diana; You, Ran; Zhong, Hua; Pangas, Stephanie A; Matzuk, Martin M

    2015-09-01

    Members of the transforming growth factor β (TGF-β) superfamily are key regulators in most developmental and physiological processes. However, the in vivo roles of TGF-β signaling in female reproduction remain uncertain. Activin receptor-like kinase 5 (ALK5) is the major type 1 receptor for the TGF-β subfamily. Absence of ALK5 leads to early embryonic lethality because of severe defects in vascular development. In this study, we conditionally ablated uterine ALK5 using progesterone receptor-cre mice to define the physiological roles of ALK5 in female reproduction. Despite normal ovarian functions and artificial decidualization in conditional knockout (cKO) mice, absence of uterine ALK5 resulted in substantially reduced female reproduction due to abnormalities observed at different stages of pregnancy, including implantation defects, disorganization of trophoblast cells, fewer uterine natural killer (uNK) cells, and impairment of spiral artery remodeling. In our microarray analysis, genes encoding proteins involved in cytokine-cytokine receptor interactions and NK cell-mediated cytotoxicity were down-regulated in cKO decidua compared with control decidua. Flow cytometry confirmed a 10-fold decrease in uNK cells in cKO versus control decidua. According to these data, we hypothesize that TGF-β acts on decidual cells via ALK5 to induce expression of other growth factors and cytokines, which are key regulators in luminal epithelium proliferation, trophoblast development, and uNK maturation during pregnancy. Our findings not only generate a mouse model to study TGF-β signaling in female reproduction but also shed light on the pathogenesis of many pregnancy complications in human, such as recurrent spontaneous abortion, preeclampsia, and intrauterine growth restriction. PMID:26305969

  8. A high-molecular-weight complex of membrane proteins BAP29/BAP31 is involved in the retention of membrane-bound IgD in the endoplasmic reticulum.

    PubMed

    Schamel, Wolfgang W A; Kuppig, Stephan; Becker, Bernd; Gimborn, Kerstin; Hauri, Hans-Peter; Reth, Michael

    2003-08-19

    B cell antigen receptors (BCRs) are multimeric transmembrane protein complexes comprising membrane-bound immunoglobulins (mIgs) and Ig-alpha/Ig-beta heterodimers. In most cases, transport of mIgs from the endoplasmic reticulum (ER) to the cell surface requires assembly with the Ig-alpha/Ig-beta subunits. In addition to Ig-alpha/Ig-beta, mIg molecules also bind two ER-resident membrane proteins, BAP29 and BAP31, and the chaperone heavy chain binding protein (BiP). In this article, we show that neither Ig-alpha/Ig-beta nor BAP29/BAP31 nor BiP bind simultaneously to the same mIgD molecule. Blue native PAGE revealed that only a minor fraction of intracellular mIgD is associated with high-molecular-weight BAP29/BAP31 complexes. BAP-binding to mIgs was found to correlate with ER retention of chimeric mIgD molecules. On high-level expression in Drosophila melanogaster S2 cells, mIgD molecules were detected on the cell surface in the absence of Ig-alpha/Ig-beta. This aberrant transport was prevented by coexpression of BAP29 and BAP31. Thus, BAP complexes contribute to ER retention of mIg complexes that are not bound to Ig-alpha/Ig-beta. Furthermore, the mechanism of ER retention of both BAP31 and mIgD is not through retrieval from a post-ER compartment, but true ER retention. In conclusion, BAP29 and BAP31 might be the long sought after retention proteins and/or chaperones that act on transmembrane regions of various proteins.

  9. Fibroblast growth factor, but not activin, is a potent activator of mitogen-activated protein kinase in Xenopus explants.

    PubMed Central

    Graves, L M; Northrop, J L; Potts, B C; Krebs, E G; Kimelman, D

    1994-01-01

    Isolated explants from the animal hemisphere of Xenopus embryos were incubated with Xenopus basic fibroblast growth factor (XbFGF) or human activin A. XbFGF incubation resulted in the rapid activation of mitogen-activated protein kinase (MAPK) and ribosomal S6 protein kinase (pp90rsk) in a dose-dependent manner with the highest levels of activation occurring at 50 ng/ml. Maximal activation occurred within 6-10 min after the addition of growth factor, and the activity of both kinases declined to unstimulated levels after 30 min. Activin was unable to activate either MAPK or pp90rsk in the Xenopus explants to a substantial level, although it induced dorsal mesoderm better than XbFGF under the same experimental conditions. The regulatory protein Xwnt-8 did not activate MAPK, nor did it enhance the activation of MAPK by XbFGF. XbFGF was able to activate MAPK through at least the midgastrula stage, suggesting that this family of growth factors may have a role in gastrula-stage events. Images PMID:7510404

  10. FGF signaling via MAPK is required early and improves Activin A-induced definitive endoderm formation from human embryonic stem cells

    SciTech Connect

    Sui, Lina; Mfopou, Josue K.; Geens, Mieke; Sermon, Karen; Bouwens, Luc

    2012-09-28

    Highlights: Black-Right-Pointing-Pointer Deep study the FGF signaling role during DE specification in the context of hESCs. Black-Right-Pointing-Pointer DE differentiation from hESCs has an early dependence on FGF signaling. Black-Right-Pointing-Pointer A serum-free DE protocol is developed based on the findings. Black-Right-Pointing-Pointer The DE cells showed potential to differentiate into pancreatic progenitor cells. -- Abstract: Considering their unlimited proliferation and pluripotency properties, human embryonic stem cells (hESCs) constitute a promising resource applicable for cell replacement therapy. To facilitate this clinical translation, it is critical to study and understand the early stage of hESCs differentiation wherein germ layers are defined. In this study, we examined the role of FGF signaling in Activin A-induced definitive endoderm (DE) differentiation in the absence of supplemented animal serum. We found that activated FGF/MAPK signaling is required at the early time point of Activin A-induced DE formation. In addition, FGF activation increased the number of DE cells compared to Activin A alone. These DE cells could further differentiate into PDX1 and NKX6.1 positive pancreatic progenitors in vitro. We conclude that Activin A combined with FGF/MAPK signaling efficiently induce DE cells in the absence of serum. These findings improve our understanding of human endoderm formation, and constitute a step forward in the generation of clinical grade hESCs progenies for cell therapy.

  11. Exogenous supplementation of Activin A enhances germ cell differentiation of human embryonic stem cells.

    PubMed

    Duggal, Galbha; Heindryckx, Björn; Warrier, Sharat; Taelman, Jasin; Van der Jeught, Margot; Deforce, Dieter; Chuva de Sousa Lopes, Susana; De Sutter, Petra

    2015-05-01

    Human embryonic stem cells (hESCs) derived in the presence of Activin A (ActA) demonstrate an increased differentiation propensity toward the germ cell lineage. In addition, mouse epiblast stem cells and mouse epiblast-like cells are poised toward germ cell differentiation and are derived in the presence of ActA. We therefore investigated whether supplementation with ActA enhances in vitro hESC differentiation toward germ cell lineage. ActA up-regulated early primordial germ cell (PGC) genes STELLA/DPPA3 (developmental pluripotency associated 3) and tyrosine kinase receptor cKIT in both ActA-derived and standard-derived hESCs indicating its role in priming hESCs toward the PGC lineage. Indeed, ActA plus bone morphogenic protein 4 (BMP4) strongly increased germ cell differentiation potential of hESCs based on the high expression of late PGC markers DAZL (deleted in azoospermia-like) and VASA/DDX4 (DEAD-box polypeptide 4) at mRNA and protein level. Hence, the combination of ActA with BMP4 provides an additional boost for hESCs to develop into postmigratory germ cells. Together with increased VASA expression in the presence of ActA and BMP4, we also observed up-regulation of endoderm-specific genes GATA4 (GATA binding protein 4) and GATA6. Finally, we were able to further mature these in vitro-derived PGC-like cells (PGCLCs) by culturing them in in vitro maturation (IVM) medium, resulting in the formation of germ cell-like clusters and induction of meiotic gene expression. In conclusion, we demonstrate for the first time a synergism between ActA and BMP4 in facilitating germ cell-directed differentiation of hESCs, which is enhanced by extended culture in IVM medium, as shown by cytoplasmic VASA-expressing PGCLCs. We propose a novel relationship between the endoderm and germ cell lineage during hESC differentiation.

  12. Stimulation of activin A expression in rat aortic smooth muscle cells by thrombin and angiotensin II correlates with neointimal formation in vivo.

    PubMed Central

    Pawlowski, J E; Taylor, D S; Valentine, M; Hail, M E; Ferrer, P; Kowala, M C; Molloy, C J

    1997-01-01

    Vasoactive GTP-binding protein-coupled receptor agonists (e.g., angiotensin II [AII] and alpha-thrombin) stimulate the production of mitogenic factors from vascular smooth muscle cells. In experiments to identify mitogens secreted from AII- or alpha-thrombin-stimulated rat aortic smooth muscle (RASM) cells, neutralizing antibodies directed against several growth factors (e.g., PDGF and basic fibroblast growth factor [basic FGF]) failed to inhibit the mitogenic activity of conditioned media samples derived from the cells. In this report, we found that polyclonal neutralizing antibodies directed against purified human placental basic FGF reduced the mitogenic activity of AII-stimulated RASM cell-conditioned media and in immunoblot experiments identified a 26-kD protein (14 kD under reducing conditions) that was distinct from basic FGF. After purification from RASM cell-conditioned medium, amino acid sequence analysis identified the protein as activin A, a member of the TGF-beta superfamily. Increased activin A expression was observed after treatment of the RASM cells with AII, alpha-thrombin, and the protein kinase C agonist PMA. In contrast, PDGF-BB or serum caused only a minor induction of this protein. Although activin A alone only weakly stimulated RASM cell DNA synthesis, it demonstrated a potent comitogenic effect in combination with either EGF or heparin-binding EGF-like growth factor in the RASM cells, increasing DNA synthesis by up to fourfold. Furthermore, in a rat carotid injury model, activin A mRNA was upregulated within 6 h after injury followed by increases in immunoreactive protein detected in the expanding neointima 7 and 14 d later. Taken together, these results indicate that activin A is a vascular smooth muscle cell-derived factor induced by vasoactive agonists that may, either alone or in combination with other vascular derived growth factors, have a role in neointimal formation after arterial injury. PMID:9239411

  13. Testing and characterizing enzymes and membrane-bound carrier proteins acting on amphipathic ligands in the presence of bilayer membrane material and soluble binding protein. Application to the uptake of oleate into isolated cells.

    PubMed Central

    Heirwegh, K P; Meuwissen, J A

    1992-01-01

    1. A multiphasic modelling approach [Heirwegh, Meuwissen, Vermeir & De Smedt (1988) Biochem. J. 254, 101-108] is applied to systems containing poorly water-soluble amphipathic reactants, membrane material, soluble binding protein and acceptor protein (enzyme or membrane-bound carrier protein). 2. The field of application is constrained by the assumptions (i) that the amount of acceptor-bound substrate is small compared with the total amount and (ii) that all preceding chemical reactions and steps of mass transport are rapid compared with the chemical change monitored. 3. Initial-rate formulae for systems in which an acceptor interacts with unbound or protein-bound ligand are given. The saturation curves are near-hyperbolic or sigmoidal, depending both (i) on the form of ligand (unbound or protein-bound) acted upon by the acceptor and (ii) on whether the assays are performed at constant concentration of soluble binding protein Cp or at constant substrate/binding-site molar ratio RS. 4. Several diagnostic features permit unequivocal distinction between acceptor action on unbound or protein-bound substrate. In the former case, saturation curves, run at the same constant concentration of one of several binding proteins of increasing binding affinity, will show progressively increasing inhibition, the shape changing from near-hyperbolic at Km' less than K1' to sigmoidal at Km' greater than K1'.Km' is the effective Michaelis constant of the acceptor and K1' the effective dissociation constant of the binding sites of the soluble protein (for the sites with the higher binding affinity, if several classes of binding site are present on the protein). Alternatively, the maximum velocity obtained at constant RS less than or equal to 1 should increase hyperbolically with RS/(1-RS) for a binding protein with a single class of binding site. The formula that applies when the binding protein contains two classes of independent binding site is also available. When the acceptor acts

  14. Regulation of FSHbeta and GnRH receptor gene expression in activin receptor II knockout male mice.

    PubMed

    Kumar, T Rajendra; Agno, Julio; Janovick, Jo Ann; Conn, P Michael; Matzuk, Martin M

    2003-12-30

    To examine in vivo, the local effects of inhibins and activins within the anterior pituitary, independent of their endocrine effects exerted from the gonad, in mediating FSH homeostasis, we used castrated knockout mice lacking either inhibin alpha or activin receptor II (ACVR2) alone or in combination. Compared to castrated wild-type (WT) mice, FSHbeta mRNA levels in the pituitaries of Acvr2 null mice were significantly downregulated in the absence of gonadal feedback. FSHbeta mRNA levels were not significantly higher in the pituitaries of castrated inhibin alpha null mice compared to those in Acvr2 null mice and remained the same in the pituitaries of castrated double mutant mice lacking both inhibin and ACVR2. In contrast to FSHbeta mRNA expression changes, pituitary FSH content was significantly reduced in Acvr2 null mice whereas it was only slightly upregulated in inhibin alpha null mice. Combined absence of both ACVR2 signaling and inhibins caused a decrease in FSH content compared to that in the absence of inhibins alone. These changes in pituitary content were in parallel to those in serum FSH levels in these three groups of castrated mice, suggesting that the unopposed actions of locally produced inhibins are dominant over those effects mediated by ACVR2 signaling to regulate FSH biosynthesis and secretion. Thus, our in vivo results demonstrate that within the pituitary, locally produced activins and inhibins exert their actions at distinct phases of FSH homeostasis. In an independent set of experiments, we tested whether in vivo signaling via ACVR2 is necessary for hypothalamic GnRH biosynthesis and for GnRH receptor expression. Our results demonstrate that in contrast to previous in vitro studies, signaling through ACVR2 is neither required for hypothalamic synthesis of GnRH peptide nor for expression of GnRH receptors in the anterior pituitary. We conclude that within the hypothalamic-pituitary short loop, ACVR2 signaling is critical only for FSH

  15. Matrigel and Activin A promote cell-cell contact and anti-apoptotic activity in cultured human retinal pigment epithelium cells.

    PubMed

    Guo, Xiaoling; Zhu, Deliang; Lian, Ruiling; Han, Yuting; Guo, Yonglong; Li, Zhijie; Tang, Shibo; Chen, Jiansu

    2016-06-01

    Age-related macular degeneration (AMD) is a leading cause of blindness among the aging population. Currently, replacement of diseased retinal pigment epithelium (RPE) cells with transplanted healthy RPE cells could be a feasible approach for AMD therapy. However, maintaining cell-cell contact and good viability of RPE cells cultured in vitro is difficult and fundamentally determines the success of RPE cell transplantation. This study was conducted to examine the role of Matrigel and Activin A (MA) in regulating cell-cell contact and anti-apoptotic activity in human RPE (hRPE) cells, as assessed by atomic force microscopy (AFM), scanning electron microscope (SEM), immunofluorescence staining, quantitative polymerase chain reaction (qPCR) analysis, Annexin V/propidium iodide (PI) analysis, mitochondrial membrane potential (△Ψ m) assays, intracellular reactive oxygen species (ROS) assays and Western blotting. hRPE cells cultured in vitro could maintain their epithelioid morphology after MA treatment over at least 4 passages. The contact of N-cadherin to the lateral cell border was promoted in hRPE cells at P2 by MA. MA treatment also enhanced the expression of tight junction-associated genes and proteins, such as Claudin-1, Claudin-3, Occludin and ZO-1, as well as polarized ZO-1 protein distribution and barrier function, in cultured hRPE cells. Moreover, MA treatment decreased apoptotic cells, ROS and Bax and increased △Ψ m and Bcl2 in hRPE cells under serum withdrawal-induced apoptosis. In addition, MA treatment elevated the protein expression levels of β-catenin and its target proteins, including Cyclin D1, c-Myc and Survivin, as well as the gene expression levels of ZO-1, β-catenin, Survivin and TCF-4, all of which could be down-regulated by the Wnt/β-catenin pathway inhibitor XAV-939. Taken together, MA treatment could effectively promote cell-cell contact and anti-apoptotic activity in hRPE cells, partly involving the Wnt/β-catenin pathway. This study

  16. Mutant activin-like kinase 2 in fibrodysplasia ossificans progressiva are activated via T203 by BMP type II receptors.

    PubMed

    Fujimoto, Mai; Ohte, Satoshi; Osawa, Kenji; Miyamoto, Arei; Tsukamoto, Sho; Mizuta, Takato; Kokabu, Shoichiro; Suda, Naoto; Katagiri, Takenobu

    2015-01-01

    Fibrodysplasia ossificans progressiva (FOP) is a genetic disorder characterized by progressive heterotopic ossification in soft tissues, such as the skeletal muscles. FOP has been shown to be caused by gain-of-function mutations in activin receptor-like kinase (ALK)-2, which is a type I receptor for bone morphogenetic proteins (BMPs). In the present study, we examined the molecular mechanisms that underlie the activation of intracellular signaling by mutant ALK2. Mutant ALK2 from FOP patients enhanced the activation of intracellular signaling by type II BMP receptors, such as BMPR-II and activin receptor, type II B, whereas that from heart disease patients did not. This enhancement was dependent on the kinase activity of the type II receptors. Substitution mutations at all nine serine and threonine residues in the ALK2 glycine- and serine-rich domain simultaneously inhibited this enhancement by the type II receptors. Of the nine serine and threonine residues in ALK2, T203 was found to be critical for the enhancement by type II receptors. The T203 residue was conserved in all of the BMP type I receptors, and these residues were essential for intracellular signal transduction in response to ligand stimulation. The phosphorylation levels of the mutant ALK2 related to FOP were higher than those of wild-type ALK2 and were further increased by the presence of type II receptors. The phosphorylation levels of ALK2 were greatly reduced in mutants carrying a mutation at T203, even in the presence of type II receptors. These findings suggest that the mutant ALK2 related to FOP is enhanced by BMP type II receptors via the T203-regulated phosphorylation of ALK2.

  17. Stimulation of circus movement by activin, bFGF and TGF-beta 2 in isolated animal cap cells of Xenopus laevis.

    PubMed

    Minoura, I; Nakamura, H; Tashiro, K; Shiokawa, K

    1995-01-01

    Lobopodium is a hyaline cytoplasmic protrusion which rotates circumferencially around a cell. This movement is called circus movement, which is seen in dissociated cells of amphibian embryos. Relative abundance of the lobopodia-forming cells changes temporally and spatially within Xenopus embryos, reflecting stage-dependent difference of morphogenetic movements. The lobopodia-forming activity of dissociated animal cap cells was stimulated strongly by activin and bFGF, and weakly by TGF-beta 2. In addition, activin A was found to stimulate cellular attachment to the substratum when the cultivation lasted long. Thus, mesoderm-inducing growth factors stimulate lobopodia formation and cellular movements which may be necessary for gastrulation and neurulation in Xenopus early embryos.

  18. Exogenous and endogenous protease inhibitors in the gut of the fall armyworm larvae, Spodoptera frugiperda.

    PubMed

    Lwalaba, Digali; Weidlich, Sandy; Hoffmann, Klaus H; Woodring, Joseph

    2010-06-01

    A dose-dependent inhibition of endogenous trypsin and aminopeptidase occurs in the lumen of Spodoptera frugiperda after feeding L6 larvae exogenous inhibitors soybean trypsin inhibitor (SBTI), tosyl-L-lysine chloromethyl ketone-HCl (TLCK), or bestatin, respectively, for 3 days. TLCK inhibits trypsin in tissue extracts and in secretions more strongly than SBTI. The aminopeptidase released into the lumen (containing the peritrophic membrane) is strongly inhibited by bestatin, but the membrane-bound enzyme is not. A bound enzyme may be more resistant to an inhibitor than unbound. A cross-class elevation of aminopeptidase activity occurs in response to ingested trypsin inhibitor, but there was no cross-class effect of aminopeptidase inhibitor (bestatin) on trypsin activity. An endogenous trypsin and aminopeptidase inhibitor is present in the lumen and ventricular cells. The strength of the endogenous trypsin inhibition seems to be in the same range as that resulting from ingestion of the exogenous inhibitor SBTI. In some insect species, considerable trypsin secretion occurs in unfed as well as in fed animals, and endogenous protease inhibitors might function to protect the ventricular epithelium by inactivation of trypsin when less food is available. PMID:20513059

  19. Tiling of R7 Axons in the Drosophila Visual System is Mediated Both by Transduction of an Activin Signal to the Nucleus and by Mutual Repulsion

    PubMed Central

    Ting, Chun-Yuan; Herman, Tory; Yonekura, Shinichi; Gao, Shuying; Wang, Jian; Serpe, Mihaela; O’Connor, Michael B.; Zipursky, S. Lawrence; Lee, Chi-Hon

    2009-01-01

    Summary The organization of neuronal wiring into layers and columns is a common feature of both vertebrate and invertebrate brains. In the Drosophila visual system, each R7 photoreceptor axon projects within a single column to a specific layer of the optic lobe. We refer to the restriction of terminals to single columns as tiling. In a genetic screen based on an R7-dependent behavior, we identified the Activin receptor Baboon and the nuclear import adaptor Importin-α3 as being required to prevent R7 axon terminals from overlapping with the terminals of R7s in neighboring columns. This tiling function requires the Baboon ligand, dActivin, the transcription factor, dSmad2, and retrograde transport from the growth cone to the R7 nucleus. We propose that dActivin is an autocrine signal that restricts R7 growth cone motility, and we demonstrate that it acts in parallel with a paracrine signal that mediates repulsion between R7 terminals. PMID:18054857

  20. Proteasome inhibitors.

    PubMed

    Teicher, Beverly A; Tomaszewski, Joseph E

    2015-07-01

    Proteasome inhibitors have a 20 year history in cancer therapy. The first proteasome inhibitor, bortezomib (Velcade, PS-341), a break-through multiple myeloma treatment, moved rapidly through development from bench in 1994 to first approval in 2003. Bortezomib is a reversible boronic acid inhibitor of the chymotrypsin-like activity of the proteasome. Next generation proteasome inhibitors include carfilzomib and oprozomib which are irreversible epoxyketone proteasome inhibitors; and ixazomib and delanzomib which are reversible boronic acid proteasome inhibitors. Two proteasome inhibitors, bortezomib and carfilzomib are FDA approved drugs and ixazomib and oprozomib are in late stage clinical trials. All of the agents are potent cytotoxics. The disease focus for all the proteasome inhibitors is multiple myeloma. This focus arose from clinical observations made in bortezomib early clinical trials. Later preclinical studies confirmed that multiple myeloma cells were indeed more sensitive to proteasome inhibitors than other tumor cell types. The discovery and development of the proteasome inhibitor class of anticancer agents has progressed through a classic route of serendipity and scientific investigation. These agents are continuing to have a major impact in their treatment of hematologic malignancies and are beginning to be explored as potential treatment agent for non-cancer indications. PMID:25935605

  1. Dynamics of CYP51: implications for function and inhibitor design

    PubMed Central

    Yu, Xiaofeng; Cojocaru, Vlad; Mustafa, Ghulam; Salo-Ahen, Outi M. H.; Lepesheva, Galina I.; Wade, Rebecca C.

    2015-01-01

    Sterol 14α-demethylase (cytochrome P450 family 51 (CYP51)) is an essential enzyme occurring in all biological kingdoms. In eukaryotes, it is located in the membrane of the endoplasmic reticulum. Selective inhibitors of trypanosomal CYP51s that do not affect the human CYP51 have been discovered in vitro and found to cure acute and chronic mouse Chagas disease without severe side effects in vivo. Crystal structures indicate that CYP51 may be more rigid than most CYPs, and it has been proposed that this property may facilitate antiparasitic drug design. Therefore, to investigate the dynamics of trypanosomal CYP51, we built a model of membrane-bound Trypanosoma brucei CYP51 and then performed molecular dynamics simulations of T. brucei CYP51 in membrane-bound and soluble forms. We compared the dynamics of T. brucei CYP51 with those of human CYP51, CYP2C9, and CYP2E1. In the simulations, the CYP51s display low mobility in the buried active site although overall mobility is similar in all the CYPs studied. The simulations suggest that in CYP51, pathway 2f serves as the major ligand access tunnel, and both pathways 2f (leading to membrane) and S (leading to solvent) can serve as ligand egress tunnels. Compared with the other CYPs, the residues at the entrance of the ligand access tunnels in CYP51 have higher mobility that may be necessary to facilitate the passage of its large sterol ligands. The water (W) tunnel is accessible to solvent during most of the simulations of CYP51, but its width is affected by the conformations of the heme's two propionate groups. These differ from those observed in the other CYPs studied because of differences in their hydrogen-bonding network. Our simulations give insights into the dynamics of CYP51 that complement the available experimental data and have implications for drug design against CYP51 enzymes. PMID:25601796

  2. Activin A/BMP2 chimera AB235 drives efficient redifferentiation of long term cultured autologous chondrocytes

    PubMed Central

    Jiménez, G.; López-Ruiz, E.; Kwiatkowski, W.; Montañez, E.; Arrebola, F.; Carrillo, E.; Gray, P. C.; Belmonte, J. C. Izpisua; Choe, S.; Perán, M.; Marchal, J. A.

    2015-01-01

    Autologous chondrocyte implantation (ACI) depends on the quality and quantity of implanted cells and is hindered by the fact that chondrocytes cultured for long periods of time undergo dedifferentiation. Here we have developed a reproducible and efficient chondrogenic protocol to redifferentiate chondrocytes isolated from osteoarthritis (OA) patients. We used morphological, histological and immunological analysis together with a RT-PCR detection of collagen I and collagen II gene expression to show that chondrocytes isolated from articular cartilage biopsies of patients and subjected to long-term culture undergo dedifferentiation and that these cells can be redifferentiated following treatment with the chimeric Activin A/BMP2 ligand AB235. Examination of AB235-treated cell pellets in both in vitro and in vivo experiments revealed that redifferentiated chondrocytes synthesized a cartilage-specific extracellular matrix (ECM), primarily consisting of vertically-orientated collagen fibres and cartilage-specific proteoglycans. AB235-treated cell pellets also integrated into the surrounding subcutaneous tissue following transplantation in mice as demonstrated by their dramatic increase in size while non-treated control pellets disintegrated upon transplantation. Thus, our findings describe an effective protocol for the promotion of redifferentiation of autologous chondrocytes obtained from OA patients and the formation of a cartilage-like ECM that can integrate into the surrounding tissue in vivo. PMID:26563344

  3. Blockade of the activin receptor IIb activates functional brown adipogenesis and thermogenesis by inducing mitochondrial oxidative metabolism.

    PubMed

    Fournier, Brigitte; Murray, Ben; Gutzwiller, Sabine; Marcaletti, Stefan; Marcellin, David; Bergling, Sebastian; Brachat, Sophie; Persohn, Elke; Pierrel, Eliane; Bombard, Florian; Hatakeyama, Shinji; Trendelenburg, Anne-Ulrike; Morvan, Frederic; Richardson, Brian; Glass, David J; Lach-Trifilieff, Estelle; Feige, Jerome N

    2012-07-01

    Brown adipose tissue (BAT) is a key tissue for energy expenditure via fat and glucose oxidation for thermogenesis. In this study, we demonstrate that the myostatin/activin receptor IIB (ActRIIB) pathway, which serves as an important negative regulator of muscle growth, is also a negative regulator of brown adipocyte differentiation. In parallel to the anticipated hypertrophy of skeletal muscle, the pharmacological inhibition of ActRIIB in mice, using a neutralizing antibody, increases the amount of BAT without directly affecting white adipose tissue. Mechanistically, inhibition of ActRIIB inhibits Smad3 signaling and activates the expression of myoglobin and PGC-1 coregulators in brown adipocytes. Consequently, ActRIIB blockade in brown adipose tissue enhances mitochondrial function and uncoupled respiration, translating into beneficial functional consequences, including enhanced cold tolerance and increased energy expenditure. Importantly, ActRIIB inhibition enhanced energy expenditure only at ambient temperature or in the cold and not at thermoneutrality, where nonshivering thermogenesis is minimal, strongly suggesting that brown fat activation plays a prominent role in the metabolic actions of ActRIIB inhibition.

  4. Graded changes in dose of a Xenopus activin A homologue elicit stepwise transitions in embryonic cell fate.

    PubMed

    Green, J B; Smith, J C

    1990-09-27

    The protein XTC-MIF, a Xenopus homologue of activin A and a potent mesoderm-inducing factor, can induce responding animal pole explants to form several different cell types in a dose-dependent manner, higher doses eliciting more dorso-anterior tissues. This graded response, characteristic of classically postulated morphogens, may underlie pattern formation, but the response of intact animal caps to XTC-MIF provides only a crude indication of trends. Here we report the effects of XTC-MIF on dispersed blastomeres rather than intact animal caps. Under these conditions, responding cells distinguish sharply between doses of pure XTC-MIF differing by less than 1.5-fold. Two different response thresholds have been found, defining three cell states. This suggests that XTC-MIF has an instructive effect. Notochord and muscle are both induced in the same narrow dose-range. Mixing treated with untreated cells does not seem to shift the dose thresholds, showing that at least some cells can stably record the received dose of inducing factor.

  5. Derivation of mesenchymal stromal cells from canine induced pluripotent stem cells by inhibition of the TGFβ/activin signaling pathway.

    PubMed

    Whitworth, Deanne J; Frith, Jessica E; Frith, Thomas J R; Ovchinnikov, Dmitry A; Cooper-White, Justin J; Wolvetang, Ernst J

    2014-12-15

    In this study we have generated canine mesenchymal stromal cells (MSCs), also known as mesenchymal stem cells, from canine induced pluripotent stem cells (ciPSCs) by small-molecule inhibition of the transforming growth factor beta (TGFβ)/activin signaling pathway. These ciPSC-derived MSCs (ciPSC-MSCs) express the MSC markers CD73, CD90, CD105, STRO1, cPDGFRβ and cKDR, in addition to the pluripotency factors OCT4, NANOG and REX1. ciPSC-MSCs lack immunostaining for H3K27me3, suggesting that they possess two active X chromosomes. ciPSC-MSCs are highly proliferative and undergo robust differentiation along the osteo-, chondro- and adipogenic pathways, but do not form teratoma-like tissues in vitro. Of further significance for the translational potential of ciPSC-MSCs, we show that these cells can be encapsulated and maintained within injectable hydrogel matrices that, when functionalized with bound pentosan polysulfate, dramatically enhance chondrogenesis and inhibit osteogenesis. The ability to efficiently derive large numbers of highly proliferative canine MSCs from ciPSCs that can be incorporated into injectable, functionalized hydrogels that enhance their differentiation along a desired lineage constitutes an important milestone towards developing an effective MSC-based therapy for osteoarthritis in dogs, but equally provides a model system for assessing the efficacy and safety of analogous approaches for treating human degenerative joint diseases. PMID:25055193

  6. Derivation of Mesenchymal Stromal Cells from Canine Induced Pluripotent Stem Cells by Inhibition of the TGFβ/Activin Signaling Pathway

    PubMed Central

    Frith, Jessica E.; Frith, Thomas J.R.; Ovchinnikov, Dmitry A.; Cooper-White, Justin J.; Wolvetang, Ernst J.

    2014-01-01

    In this study we have generated canine mesenchymal stromal cells (MSCs), also known as mesenchymal stem cells, from canine induced pluripotent stem cells (ciPSCs) by small-molecule inhibition of the transforming growth factor beta (TGFβ)/activin signaling pathway. These ciPSC-derived MSCs (ciPSC-MSCs) express the MSC markers CD73, CD90, CD105, STRO1, cPDGFRβ and cKDR, in addition to the pluripotency factors OCT4, NANOG and REX1. ciPSC-MSCs lack immunostaining for H3K27me3, suggesting that they possess two active X chromosomes. ciPSC-MSCs are highly proliferative and undergo robust differentiation along the osteo-, chondro- and adipogenic pathways, but do not form teratoma-like tissues in vitro. Of further significance for the translational potential of ciPSC-MSCs, we show that these cells can be encapsulated and maintained within injectable hydrogel matrices that, when functionalized with bound pentosan polysulfate, dramatically enhance chondrogenesis and inhibit osteogenesis. The ability to efficiently derive large numbers of highly proliferative canine MSCs from ciPSCs that can be incorporated into injectable, functionalized hydrogels that enhance their differentiation along a desired lineage constitutes an important milestone towards developing an effective MSC-based therapy for osteoarthritis in dogs, but equally provides a model system for assessing the efficacy and safety of analogous approaches for treating human degenerative joint diseases. PMID:25055193

  7. High circulating activin A level is associated with tumor progression and predicts poor prognosis in lung adenocarcinoma

    PubMed Central

    Hoda, Mir Alireza; Rozsas, Anita; Lang, Elisabeth; Klikovits, Thomas; Lohinai, Zoltan; Torok, Szilvia; Berta, Judit; Bendek, Matyas; Berger, Walter; Hegedus, Balazs; Klepetko, Walter; Renyi-Vamos, Ferenc; Grusch, Michael; Dome, Balazs; Laszlo, Viktoria

    2016-01-01

    Activin A (ActA)/follistatin (FST) signaling has been shown to be deregulated in different tumor types including lung adenocarcinoma (LADC). Here, we report that serum ActA protein levels are significantly elevated in LADC patients (n=64) as compared to controls (n=46, p=0.015). ActA levels also correlated with more advanced disease stage (p<0.0001) and T (p=0.0035) and N (p=0.0002) factors. M1 patients had significantly higher ActA levels than M0 patients (p<0.001). High serum ActA level was associated with poor overall survival (p<0.0001) and was confirmed as an independent prognostic factor (p=0.004). Serum FST levels were increased only in female LADC patients (vs. female controls, p=0.031). Two out of five LADC cell lines secreted biologically active ActA, while FST was produced in all of them. Transcripts of both type I and II ActA receptors were detected in all five LADC cell lines. In conclusion, our study does not only suggest that measuring blood ActA levels in LADC patients might improve the prediction of prognosis, but also indicates that this parameter might be a novel non-invasive biomarker for identifying LADC patients with organ metastases. PMID:26950277

  8. P7170: A Novel Molecule with Unique Profile of mTORC1/C2 and Activin Receptor-like Kinase 1 Inhibition Leading to Antitumor and Antiangiogenic Activity.

    PubMed

    Jalota-Badhwar, Archana; Bhatia, Dimple R; Boreddy, Srinivas; Joshi, Asavari; Venkatraman, Magesh; Desai, Nikesh; Chaudhari, Sarika; Bose, Julie; Kolla, Lakshmi S; Deore, Vijaykumar; Yewalkar, Nilambari; Kumar, Sanjay; Sharma, Rajiv; Damre, Anagha; More, Avinash; Sharma, Somesh; Agarwal, Veena R

    2015-05-01

    The mTOR pathway is often upregulated in cancer and thus intensively pursued as a target to design novel anticancer therapies. Approved and emerging drugs targeting the mTOR pathway have positively affected the clinical landscape. Recently, activin receptor-like kinase 1 (ALK1), belonging to the TGFβ receptor family, has been reported as an emerging target for antiangiogenic cancer therapy. Here, we describe a novel orally efficacious compound, P7170, that inhibits mTORC1/mTORC2/ALK1 activity with a potent cell growth inhibition. In cell-based assays, P7170 strongly inhibited (IC50 < 10 nmol/L) the phosphorylation of p70S6K (T389) and pAKT (S473). In many cancer cell lines, such as prostate, ovarian, colon, and renal, P7170 treatment resulted in marked cell growth inhibition. Furthermore, it induced G1-S cell-cycle arrest and autophagy. In vitro HUVEC tube formation, in vivo Matrigel plug, and rat aorta ring assays demonstrated that P7170 exhibited significant antiangiogenic activity. In addition, ALK1 knockdown studies in HUVEC confirmed that the antiangiogenic activity of P7170 was primarily due to ALK1 inhibition. Strong inhibition of ALK1 in addition to mTORC1/mTORC2 differentiates P7170 in its mechanism of action in comparison with existing inhibitors. In vivo mouse xenograft studies revealed P7170 to exhibit a significant dose-dependent tumor growth inhibition in a broad range of human tumor types when administered orally at 10 to 20 mg/kg doses. The distinctive pharmacological profile with favorable pharmacokinetic parameters and in vivo efficacy makes P7170 an attractive candidate for clinical development. It is currently being tested in phase I clinical studies.

  9. Platelet Inhibitors.

    PubMed

    Shifrin, Megan M; Widmar, S Brian

    2016-03-01

    Antithrombotic medications have become standard of care for management of acute coronary syndrome. Platelet adhesion, activation, and aggregation are essential components of platelet function; platelet-inhibiting medications interfere with these components and reduce incidence of thrombosis. Active bleeding is a contraindication for administration of platelet inhibitors. There is currently no reversal agent for platelet inhibitors, although platelet transfusion may be used to correct active bleeding after administration of platelet inhibitors. PMID:26897422

  10. N-Substituted Glutamyl Sulfonamides As Inhibitors Of Glutamate Carboxypeptidase II (GCP2)

    PubMed Central

    Blank, Brian R.; Alayoglu, Pinar; Engen, William; Choi, Joseph K.; Berkman, Clifford E.; Anderson, Marc O.

    2011-01-01

    Glutamate carboxypeptidase II (GCP2) is a membrane-bound cell-surface peptidase which is implicated in several neurological disorders, and is also over-expressed in prostate tumor cells. There is significant interest in the inhibition of GCP2 as a means of neuroprotection, while GCP2 inhibition as a method to treat prostate cancer remains a topic of further investigation. The key zinc-binding functional group of the well characterized classes of GCP2 inhibitors (phophonates and phosphoramidates) is tetrahedral and negatively charged at neutral pH, while glutamyl urea class of inhibitors possess a planar and neutral zinc-binding group. This study introduces a new class of GCP2 inhibitors, N-substituted glutamyl sulfonamides, which possess a neutral tetrahedral zinc-binding motif. A library containing 15 secondary sulfonamides and 4 tertiary (N-methyl) sulfonamides was prepared and evaluated for inhibitory potency against purified GCP2 enzyme activity. While most inhibitors lacked potency at 100 μM, short alkyl sulfonamides exhibited promising low micromolar potency, with the optimal inhibitor in this series being glutamyl N-propylsulfonamide (2g). Lastly, molecular docking was used to develop a model to formulate an explanation for the relative inhibitory potencies employed for this class of inhibitors. PMID:21219587

  11. Corrosion inhibitor

    SciTech Connect

    Wisotsky, M.J.; Metro, S.J.

    1989-10-31

    A corrosion inhibitor for use in synthetic ester lubricating oils is disclosed. It comprises an effective amount of: at least one aromatic amide; and at least one hydroxy substituted aromatic compound. The corrosion inhibitor thus formed is particularly useful in synthetic ester turbo lubricating oils.

  12. Heterozygous disruption of activin receptor-like kinase 1 is associated with increased arterial pressure in mice

    PubMed Central

    González-Núñez, María; Riolobos, Adela S.; Castellano, Orlando; Fuentes-Calvo, Isabel; de los Ángeles Sevilla, María; Oujo, Bárbara; Pericacho, Miguel; Cruz-Gonzalez, Ignacio; Pérez-Barriocanal, Fernando; ten Dijke, Peter; López-Novoa, Jose M.

    2015-01-01

    ABSTRACT The activin receptor-like kinase 1 (ALK-1) is a type I cell-surface receptor for the transforming growth factor-β (TGF-β) family of proteins. Hypertension is related to TGF-β1, because increased TGF-β1 expression is correlated with an elevation in arterial pressure (AP) and TGF-β expression is upregulated by the renin-angiotensin-aldosterone system. The purpose of this study was to assess the role of ALK-1 in regulation of AP using Alk1 haploinsufficient mice (Alk1+/−). We observed that systolic and diastolic AP were significantly higher in Alk1+/− than in Alk1+/+ mice, and all functional and structural cardiac parameters (echocardiography and electrocardiography) were similar in both groups. Alk1+/− mice showed alterations in the circadian rhythm of AP, with higher AP than Alk1+/+ mice during most of the light period. Higher AP in Alk1+/− mice is not a result of a reduction in the NO-dependent vasodilator response or of overactivation of the peripheral renin-angiotensin system. However, intracerebroventricular administration of losartan had a hypotensive effect in Alk1+/− and not in Alk1+/+ mice. Alk1+/− mice showed a greater hypotensive response to the β-adrenergic antagonist atenolol and higher concentrations of epinephrine and norepinephrine in plasma than Alk1+/+ mice. The number of brain cholinergic neurons in the anterior basal forebrain was reduced in Alk1+/− mice. Thus, we concluded that the ALK-1 receptor is involved in the control of AP, and the high AP of Alk1+/− mice is explained mainly by the sympathetic overactivation shown by these animals, which is probably related to the decreased number of cholinergic neurons. PMID:26398936

  13. A Soluble Activin Receptor IIB Fails to Prevent Muscle Atrophy in a Mouse Model of Spinal Cord Injury.

    PubMed

    Graham, Zachary A; Collier, Lauren; Peng, Yuanzhen; Saéz, Juan C; Bauman, William A; Qin, Weiping; Cardozo, Christopher P

    2016-06-15

    Myostatin (MST) is a potent regulator of muscle growth and size. Spinal cord injury (SCI) results in marked atrophy of muscle below the level of injury. Currently, there is no effective pharmaceutical treatment available to prevent sublesional muscle atrophy post-SCI. To determine whether inhibition of MST with a soluble activin IIB receptor (RAP-031) prevents sublesional SCI-induced muscle atrophy, mice were randomly assigned to the following groups: Sham-SCI; SCI+Vehicle group (SCI-VEH); and SCI+RAP-031 (SCI-RAP-031). SCI was induced by complete transection at thoracic level 10. Animals were euthanized at 56 days post-surgery. RAP-031 reduced, but did not prevent, body weight loss post-SCI. RAP-031 increased total lean tissue mass compared to SCI-VEH (14.8%). RAP-031 increased forelimb muscle mass post-SCI by 38% and 19% for biceps and triceps, respectively (p < 0.001). There were no differences in hindlimb muscle weights between the RAP-031 and SCI-VEH groups. In the gastrocnemius, messenger RNA (mRNA) expression was elevated for interleukin (IL)-6 (8-fold), IL-1β (3-fold), and tumor necrosis factor alpha (8-fold) in the SCI-VEH, compared to the Sham group. Muscle RING finger protein 1 mRNA was 2-fold greater in the RAP-031 group, compared to Sham-SCI. RAP-031 did not influence cytokine expression. Bone mineral density of the distal femur and proximal tibia were decreased post-SCI (-26% and -28%, respectively) and were not altered by RAP-031. In conclusion, MST inhibition increased supralesional muscle mass, but did not prevent sublesional muscle or bone loss, or the inflammation in paralyzed muscle.

  14. Fibrodysplasia ossificans progressiva-related activated activin-like kinase signaling enhances osteoclast formation during heterotopic ossification in muscle tissues.

    PubMed

    Yano, Masato; Kawao, Naoyuki; Okumoto, Katsumi; Tamura, Yukinori; Okada, Kiyotaka; Kaji, Hiroshi

    2014-06-13

    Fibrodysplasia ossificans progressiva is characterized by extensive ossification within muscle tissues, and its molecular pathogenesis is responsible for the constitutively activating mutation (R206H) of the bone morphogenetic protein type 1 receptor, activin-like kinase 2 (ALK2). In this study, we investigated the effects of implanting ALK2 (R206H)-transfected myoblastic C2C12 cells into nude mice on osteoclast formation during heterotopic ossification in muscle and subcutaneous tissues. The implantation of ALK2 (R206H)-transfected C2C12 cells with BMP-2 in nude mice induced robust heterotopic ossification with an increase in the formation of osteoclasts in muscle tissues but not in subcutaneous tissues. The implantation of ALK2 (R206H)-transfected C2C12 cells in muscle induced heterotopic ossification more effectively than that of empty vector-transfected cells. A co-culture of ALK2 (R206H)-transfected C2C12 cells as well as the conditioned medium from ALK2 (R206H)-transfected C2C12 cells enhanced osteoclast formation in Raw264.7 cells more effectively than those with empty vector-transfected cells. The transfection of ALK2 (R206H) into C2C12 cells elevated the expression of transforming growth factor (TGF)-β, whereas the inhibition of TGF-β signaling suppressed the enhanced formation of osteoclasts in the co-culture with ALK2 (R206H)-transfected C2C12 cells and their conditioned medium. In conclusion, this study demonstrated that the causal mutation transfection of fibrodysplasia ossificans progressiva in myoblasts enhanced the formation of osteoclasts from its precursor through TGF-β in muscle tissues.

  15. Polyoxometalates--potent and selective ecto-nucleotidase inhibitors.

    PubMed

    Lee, Sang-Yong; Fiene, Amelie; Li, Wenjin; Hanck, Theodor; Brylev, Konstantin A; Fedorov, Vladimir E; Lecka, Joanna; Haider, Ali; Pietzsch, Hans-Jürgen; Zimmermann, Herbert; Sévigny, Jean; Kortz, Ulrich; Stephan, Holger; Müller, Christa E

    2015-01-15

    Polyoxometalates (POMs) are inorganic cluster metal complexes that possess versatile biological activities, including antibacterial, anticancer, antidiabetic, and antiviral effects. Their mechanisms of action at the molecular level are largely unknown. However, it has been suggested that the inhibition of several enzyme families (e.g., phosphatases, protein kinases or ecto-nucleotidases) by POMs may contribute to their pharmacological properties. Ecto-nucleotidases are cell membrane-bound or secreted glycoproteins involved in the hydrolysis of extracellular nucleotides thereby regulating purinergic (and pyrimidinergic) signaling. They comprise four distinct families: ecto-nucleoside triphosphate diphosphohydrolases (NTPDases), ecto-nucleotide pyrophosphatases/phosphodiesterases (NPPs), alkaline phosphatases (APs) and ecto-5'-nucleotidase (eN). In the present study, we evaluated the inhibitory potency of a series of polyoxometalates as well as chalcogenide hexarhenium cluster complexes at a broad range of ecto-nucleotidases. [Co4(H2O)2(PW9O34)2](10-) (5, PSB-POM142) was discovered to be the most potent inhibitor of human NTPDase1 described so far (Ki: 3.88 nM). Other investigated POMs selectively inhibited human NPP1, [TiW11CoO40](8-) (4, PSB-POM141, Ki: 1.46 nM) and [NaSb9W21O86](18-) (6, PSB-POM143, Ki: 4.98 nM) representing the most potent and selective human NPP1 inhibitors described to date. [NaP5W30O110](14-) (8, PSB-POM144) strongly inhibited NTPDase1-3 and NPP1 and may therefore be used as a pan-inhibitor to block ATP hydrolysis. The polyoxoanionic compounds displayed a non-competitive mechanism of inhibition of NPPs and eN, but appeared to be competitive inhibitors of TNAP. Future in vivo studies with selected inhibitors identified in the current study are warranted. PMID:25449596

  16. A myostatin and activin decoy receptor enhances bone formation in mice.

    PubMed

    Bialek, P; Parkington, J; Li, X; Gavin, D; Wallace, C; Zhang, J; Root, A; Yan, G; Warner, L; Seeherman, H J; Yaworsky, P J

    2014-03-01

    Myostatin is a member of the bone morphogenetic protein/transforming growth factor-β (BMP/TGFβ) super-family of secreted differentiation factors. Myostatin is a negative regulator of muscle mass as shown by increased muscle mass in myostatin deficient mice. Interestingly, these mice also exhibit increased bone mass suggesting that myostatin may also play a role in regulating bone mass. To investigate the role of myostatin in bone, young adult mice were administered with either a myostatin neutralizing antibody (Mstn-mAb), a soluble myostatin decoy receptor (ActRIIB-Fc) or vehicle. While both myostatin inhibitors increased muscle mass, only ActRIIB-Fc increased bone mass. Bone volume fraction (BV/TV), as determined by microCT, was increased by 132% and 27% in the distal femur and lumbar vertebrae, respectively. Histological evaluation demonstrated that increased BV/TV in both locations was attributed to increased trabecular thickness, trabecular number and bone formation rate. Increased BV/TV resulted in enhanced vertebral maximum compressive force compared to untreated animals. The fact that ActRIIB-Fc, but not Mstn-mAb, increased bone volume suggested that this soluble decoy receptor may be binding a ligand other than myostatin, that plays a role in regulating bone mass. This was confirmed by the significant increase in BV/TV in myostatin deficient mice treated with ActRIIB-Fc. Of the other known ActRIIB-Fc ligands, BMP3 has been identified as a negative regulator of bone mass. However, BMP3 deficient mice treated with ActRIIB-Fc showed similar increases in BV/TV as wild type (WT) littermates treated with ActRIIB-Fc. This result suggests that BMP3 neutralization is not the mechanism responsible for increased bone mass. The results of this study demonstrate that ActRIIB-Fc increases both muscle and bone mass in mice. Therefore, a therapeutic that has this dual activity represents a potential approach for the treatment of frailty. PMID:24333131

  17. Exogenous GDF9 but not Activin A, BMP15 or TGFβ alters tight junction protein transcript abundance in zebrafish ovarian follicles.

    PubMed

    Clelland, Eric S; Kelly, Scott P

    2011-04-01

    The tight junction (TJ) complex plays an important role in regulating paracellular permeability and provides mechanical stability in vertebrate epithelia and endothelia. In zebrafish ovarian follicles, TJ complexes in the follicular envelope degenerate as the follicles develop towards maturation. In the current study, transcript abundance of claudins (cldn d, g, h, 1, and 12) and occludins (ocln, and ocln b) were assessed in mid-vitellogenic follicles in response to treatment with exogenous growth factors that are reported to be involved in zebrafish follicle development (i.e. Activin A, BMP15, GDF9 and TGFβ). Exogenous GDF9 reduced the transcript abundance of cldn g, ocln and ocln b in mid-vitellogenic follicles, whereas Activin A, BMP15, and TGFβ had no effect. Subsequent studies with GDF9 revealed that this factor did not alter TJ protein transcript abundance in pre-vitellogenic follicles but did increase the abundance of ocln b in fully grown (maturing) follicles. GDF9 was also seen to increase the abundance of StAR mRNA in all but primary stage follicles. These data suggest a role for GDF9 in the regulation of TJ integrity in zebrafish ovarian follicles, perhaps in the facilitation of ovulation, and support a previously postulated role for GDF9 in zebrafish ovarian follicle development. In addition, data also support the idea that endocrine factors play an important role in the regulation of TJ proteins during ovarian follicle development.

  18. Changes in intrafollicular concentrations of free IGF-1, activin A, inhibin A, VEGF, estradiol, and prolactin before ovulation in mares.

    PubMed

    Bashir, S T; Ishak, G M; Gastal, M O; Roser, J F; Gastal, E L

    2016-05-01

    Changes in intrafollicular growth factors and hormones were evaluated in vivo in postdeviation and impending ovulation follicles. Mares (n = 30) were randomly assigned to five experimental groups based on target diameters of 25, 30, 35, 40 mm, and impending signs of ovulation. Furthermore, data belonging to two or more proximal diameter groups that were not different were combined and regrouped for each factor separately. Follicular fluid-free insulin-like growth factor 1 was highest (P < 0.003) in 35-mm follicles, followed by the 40-mm and impending ovulation follicle group, and the 25- to 30-mm follicle group. However, concentrations of insulin-like growth factor binding protein 2 in follicular fluid did not differ (P > 0.05) among groups. Additionally, follicular fluid activin A tended (P < 0.06) to be higher in impending ovulation follicles when compared with the 25- to 40-mm follicle group. Concentrations of intrafollicular estradiol were higher (P < 0.0001) in 40-mm and impending ovulation follicles than in the other follicle groups. Follicular fluid concentrations of inhibin A and vascular endothelial growth factor were lower (P < 0.05) in the 40-mm and the impending ovulation follicle group when compared with the 25- to 35-mm follicle group. Systemic and intrafollicular prolactin levels were lower (P < 0.05) in the impending ovulation group when compared with the 25- to 40-mm follicle group. Prolactin concentrations were higher (P < 0.05) in the follicular fluid than in the plasma. The novel findings of this study, a decrease in intrafollicular-free insulin-like growth factor 1, inhibin A, vascular endothelial growth factor, and prolactin during the final stages of follicular growth, document for the first time the occurrence of dynamic changes among intrafollicular factors and hormones during the stages of follicle dominance and as ovulation approaches. PMID:26895618

  19. Antisense-oligonucleotide mediated exon skipping in activin-receptor-like kinase 2: inhibiting the receptor that is overactive in fibrodysplasia ossificans progressiva.

    PubMed

    Shi, Songting; Cai, Jie; de Gorter, David J J; Sanchez-Duffhues, Gonzalo; Kemaladewi, Dwi U; Hoogaars, Willem M H; Aartsma-Rus, Annemieke; 't Hoen, Peter A C; ten Dijke, Peter

    2013-01-01

    Fibrodysplasia ossificans progressiva (FOP) is a rare heritable disease characterized by progressive heterotopic ossification of connective tissues, for which there is presently no definite treatment. A recurrent activating mutation (c.617G→A; R206H) of activin receptor-like kinase 2 (ACVR1/ALK2), a BMP type I receptor, has been shown as the main cause of FOP. This mutation constitutively activates the BMP signaling pathway and initiates the formation of heterotopic bone. In this study, we have designed antisense oligonucleotides (AONs) to knockdown mouse ALK2 expression by means of exon skipping. The ALK2 AON could induce exon skipping in cells, which was accompanied by decreased ALK2 mRNA levels and impaired BMP signaling. In addition, the ALK2 AON potentiated muscle differentiation and repressed BMP6-induced osteoblast differentiation. Our results therefore provide a potential therapeutic approach for the treatment of FOP disease by reducing the excessive ALK2 activity in FOP patients.

  20. Hormonal and photoperiodic modulation of testicular mRNAs coding for inhibin/activin subunits and follistatin in Clethrionomys glareolus, Schreber.

    PubMed

    Tähkä, K M; Kaipia, A; Toppari, J; Tähkä, S; Tuuri, T; Tuohimaa, P

    1998-07-01

    Photoperiodic and hormonal modulation of mRNAs for testicular inhibin/activin subunits and follistatin were studied in a seasonally breeding rodent, the bank vole (Clethrionomys glareolus). Photoperiod-induced testicular regression had no effect on the relatively low steady-state levels of follistatin mRNA. Inhibin alpha (I alpha) and beta B (I beta B) mRNA levels were significantly higher in regressed than in active gonads, but inhibin beta A was undetectable. The effect of gonadotropin administration on testicular weight and mRNA concentrations differed between the sexually active and quiescent voles. Neither FSH (1.2 U/kg; s.c. for 5 days) nor hCG (600 IU/kg; s.c. for 5 days) affected testicular weight in sexually active voles, whereas both gonadotropins significantly increased testicular weight in photo-regressed individuals. FSH had no effect on I alpha or I beta B mRNA concentrations in the active testes, whereas excessive hCG challenge induced a decrease in the steady-state levels of these mRNAs. FSH induced an increase in I alpha mRNA concentrations in the regressed gonad, whereas both gonadotropins concomitantly down-regulated I beta B mRNA levels. In conclusion, the high expression of I alpha and I beta B mRNA in the regressed testis imply autocrine and paracrine roles for inhibin/activin in the quiescent gonad of seasonal breeders. Inhibin alpha-subunit expression is at least partly under the control of FSH in the bank vole testis.

  1. Aldo-keto reductases in retinoid metabolism: search for substrate specificity and inhibitor selectivity.

    PubMed

    Porté, Sergio; Xavier Ruiz, F; Giménez, Joan; Molist, Iago; Alvarez, Susana; Domínguez, Marta; Alvarez, Rosana; de Lera, Angel R; Parés, Xavier; Farrés, Jaume

    2013-02-25

    Biological activity of natural retinoids requires the oxidation of retinol to retinoic acid (RA) and its binding to specific nuclear receptors in target tissues. The first step of this pathway, the reversible oxidoreduction of retinol to retinaldehyde, is essential to control RA levels. The enzymes of retinol oxidation are NAD-dependent dehydrogenases of the cytosolic medium-chain (MDR) and the membrane-bound short-chain (SDR) dehydrogenases/reductases. Retinaldehyde reduction can be performed by SDR and aldo-keto reductases (AKR), while its oxidation to RA is carried out by aldehyde dehydrogenases (ALDH). In contrast to SDR, AKR and ALDH are cytosolic. A common property of these enzymes is that they only use free retinoid, but not retinoid bound to cellular retinol binding protein (CRBP). The relative contribution of each enzyme type in retinoid metabolism is discussed in terms of the different subcellular localization, topology of membrane-bound enzymes, kinetic constants, binding affinity of CRBP for retinol and retinaldehyde, and partition of retinoid pools between membranes and cytoplasm. The development of selective inhibitors for AKR enzymes 1B1 and 1B10, of clinical relevance in diabetes and cancer, granted the investigation of some structure-activity relationships. Kinetics with the 4-methyl derivatives of retinaldehyde isomers was performed to identify structural features for substrate specificity. Hydrophilic derivatives were better substrates than the more hydrophobic compounds. We also explored the inhibitory properties of some synthetic retinoids, known for binding to retinoic acid receptors (RAR) and retinoid X receptors (RXR). Consistent with its substrate specificity towards retinaldehyde, AKR1B10 was more effectively inhibited by synthetic retinoids than AKR1B1. A RARβ/γ agonist (UVI2008) inhibited AKR1B10 with the highest potency and selectivity, and docking simulations predicted that its carboxyl group binds to the anion-binding pocket. PMID

  2. Calmodulin inhibitors trigger the proteolytic processing of membrane type-1 matrix metalloproteinase, but not its shedding in glioblastoma cells.

    PubMed Central

    Annabi, B; Pilorget, A; Bousquet-Gagnon, N; Gingras, D; Béliveau, R

    2001-01-01

    Most transmembrane proteins are subjected to limited proteolysis by cellular proteases, and stimulation of cleavage of membrane proteins by calmodulin (CaM) inhibitors was recently shown. The present study investigated the ability of several CaM inhibitors to induce the proteolytic cleavage of the membrane type-1 matrix metalloproteinase (MT1-MMP) from the cell surface of highly invasive U-87 glioblastoma cells. Although no shedding of a soluble MT1-MMP form was induced by CaM inhibitors in the conditioned media, we showed that these inhibitors induced MT1-MMP proteolytic processing to the 43 kDa membrane-bound inactive form that was not correlated with an increase in proMMP-2 activation but rather with an increase in tissue inhibitor of MMPs (TIMP)-2 expression levels. Moreover, this proteolytic processing was sensitive to marimastat suggesting the involvement of MMPs. Interestingly, CaM inhibitors antagonized concanavalin A- and cytochalasin D-induced proMMP-2 activation, and affected the cytoskeletal actin organization resulting in the loss of migratory potential of U-87 glioblastoma cells. Cytoplasmic tail-truncated MT1-MMP constructs expressed in COS-7 cells were also affected by CaM inhibitors suggesting that these inhibitors stimulated MT1-MMP proteolytic processing by mechanisms independent of the CaM-substrate interaction. We also propose that TIMP-2 acts as a negative regulator of MT1-MMP-dependent activities promoted by the action of CaM inhibitors in U-87 glioblastoma cells. PMID:11583578

  3. Electrochemical assay of α-glucosidase activity and the inhibitor screening in cell medium.

    PubMed

    Zhang, Juan; Liu, Ying; Wang, Xiaonan; Chen, Yangyang; Li, Genxi

    2015-12-15

    An electrochemical method is established in this work for the assay of α-glucosidase activity and the inhibitor screening through one-step displacement reaction, which can be directly used in cell medium. The displacement reaction can be achieved via strong binding of 4-aminophenyl-α-D-glucopyranoside (pAPG)/magnetic nanoparticles (MNPs) to pyrene boric acid (PBA) immobilized on the surface of graphite electrode (GE), compared to that of dopamine (DA)/sliver nanoparticles (AgNPs). Since α-glucosidase can specifically catalyze MNPs/pAPG into MNPs/pAP which has no binding capacity with PBA, the activity of both isolated and membrane bound enzyme can be well evaluated by using this proposed method. Meanwhile, signal amplification can be accomplished via the immobilization of DA at the outer layer of AgNPs, and the accuracy can be strengthened through magnetic separation. Moreover, this method can also be utilized for inhibitor screening not only in the medium containing the enzyme but also in cell medium. With good precision and accuracy, it may be extended to other proteases and their inhibitors as well. PMID:26201984

  4. Characterization of non-nitrocatechol pan and isoform specific catechol-O-methyltransferase inhibitors and substrates.

    PubMed

    Robinson, Ronald G; Smith, Sean M; Wolkenberg, Scott E; Kandebo, Monika; Yao, Lihang; Gibson, Christopher R; Harrison, Scott T; Polsky-Fisher, Stacey; Barrow, James C; Manley, Peter J; Mulhearn, James J; Nanda, Kausik K; Schubert, Jeffrey W; Trotter, B Wesley; Zhao, Zhijian; Sanders, John M; Smith, Robert F; McLoughlin, Debra; Sharma, Sujata; Hall, Dawn L; Walker, Tiffany L; Kershner, Jennifer L; Bhandari, Neetesh; Hutson, Pete H; Sachs, Nancy A

    2012-02-15

    Reduced dopamine neurotransmission in the prefrontal cortex has been implicated as causal for the negative symptoms and cognitive deficit associated with schizophrenia; thus, a compound which selectively enhances dopamine neurotransmission in the prefrontal cortex may have therapeutic potential. Inhibition of catechol-O-methyltransferase (COMT, EC 2.1.1.6) offers a unique advantage, since this enzyme is the primary mechanism for the elimination of dopamine in cortical areas. Since membrane bound COMT (MB-COMT) is the predominant isoform in human brain, a high throughput screen (HTS) to identify novel MB-COMT specific inhibitors was completed. Subsequent optimization led to the identification of novel, non-nitrocatechol COMT inhibitors, some of which interact specifically with MB-COMT. Compounds were characterized for in vitro efficacy versus human and rat MB and soluble (S)-COMT. Select compounds were administered to male Wistar rats, and ex vivo COMT activity, compound levels in plasma and cerebrospinal fluid (CSF), and CSF dopamine metabolite levels were determined as measures of preclinical efficacy. Finally, novel non-nitrocatechol COMT inhibitors displayed less potent uncoupling of the mitochondrial membrane potential (MMP) compared to tolcapone as well as nonhepatotoxic entacapone, thus mitigating the risk of hepatotoxicity. PMID:22860182

  5. Are soluble and membrane-bound rat brain acetylcholinesterase different

    SciTech Connect

    Andres, C.; el Mourabit, M.; Stutz, C.; Mark, J.; Waksman, A. )

    1990-11-01

    Salt-soluble and detergent-soluble acetylcholinesterases (AChE) from adult rat brain were purified to homogeneity and studied with the aim to establish the differences existing between these two forms. It was found that the enzymatic activities of the purified salt-soluble AChE as well as the detergent-soluble AChE were dependent on the Triton X-100 concentration. Moreover, the interaction of salt-soluble AChE with liposomes suggests amphiphilic behaviour of this enzyme. Serum cholinesterase (ChE) did not bind to liposomes but its activity was also detergent-dependent. Detergent-soluble AChE remained in solution below critical micellar concentrations of Triton X-100. SDS polyacrylamide gel electrophoresis of purified, Biobeads-treated and iodinated detergent-soluble 11 S AChE showed, under non reducing conditions, bands of 69 kD, 130 kD and greater than 250 kD corresponding, respectively, to monomers, dimers and probably tetramers of the same polypeptide chain. Under reducing conditions, only a 69 kD band was detected. It is proposed that an amphiphilic environment stabilizes the salt-soluble forms of AChE in the brain in vivo and that detergent-soluble Biobeads-treated 11 S AChE possess hydrophobic domain(s) different from the 20 kD peptide already described.

  6. Calcium Modulation of Plant Plasma Membrane-Bound Atpase Activities

    NASA Technical Reports Server (NTRS)

    Caldwell, C.

    1983-01-01

    The kinetic properties of barley enzyme are discussed and compared with those of other plants. Possibilities for calcium transport in the plasma membrane by proton pump and ATPase-dependent calcium pumps are explored. Topics covered include the ph phase of the enzyme; high affinity of barley for calcium; temperature dependence, activation enthalpy, and the types of ATPase catalytic sites. Attention is given to lipids which are both screened and bound by calcium. Studies show that barley has a calmodulin activated ATPase that is found in the presence of magnesium and calcium.

  7. Transient domain formation in membrane-bound organelles undergoing maturation

    NASA Astrophysics Data System (ADS)

    Dmitrieff, Serge; Sens, Pierre

    2013-12-01

    The membrane components of cellular organelles have been shown to segregate into domains as the result of biochemical maturation. We propose that the dynamical competition between maturation and lateral segregation of membrane components regulates domain formation. We study a two-component fluid membrane in which enzymatic reaction irreversibly converts one component into another and phase separation triggers the formation of transient membrane domains. The maximum domain size is shown to depend on the maturation rate as a power law similar to the one observed for domain growth with time in the absence of maturation, despite this time dependence not being verified in the case of irreversible maturation. This control of domain size by enzymatic activity could play a critical role in regulating exchange between organelles or within compartmentalized organelles such as the Golgi apparatus.

  8. Retinol dehydrogenases: membrane-bound enzymes for the visual function.

    PubMed

    Lhor, Mustapha; Salesse, Christian

    2014-12-01

    Retinoid metabolism is important for many physiological functions, such as differenciation, growth, and vision. In the visual context, after the absorption of light in rod photoreceptors by the visual pigment rhodopsin, 11-cis retinal is isomerized to all-trans retinal. This retinoid subsequently undergoes a series of modifications during the visual cycle through a cascade of reactions occurring in photoreceptors and in the retinal pigment epithelium. Retinol dehydrogenases (RDHs) are enzymes responsible for crucial steps of this visual cycle. They belong to a large family of proteins designated as short-chain dehydrogenases/reductases. The structure of these RDHs has been predicted using modern bioinformatics tools, which allowed to propose models with similar structures including a common Rossman fold. These enzymes undergo oxidoreduction reactions, whose direction is dictated by the preference and concentration of their individual cofactor (NAD(H)/NADP(H)). This review presents the current state of knowledge on functional and structural features of RDHs involved in the visual cycle as well as knockout models. RDHs are described as integral or peripheral enzymes. A topology model of the membrane binding of these RDHs via their N- and (or) C-terminal domain has been proposed on the basis of their individual properties. Membrane binding is a crucial issue for these enzymes because of the high hydrophobicity of their retinoid substrates.

  9. [Molecular cloning of the DNA sequence of activin beta A subunit gene mature peptides from panda and related species and its application in the research of phylogeny and taxonomy].

    PubMed

    Wang, Xiao-Jing; Wang, Xiao-Xing; Wang, Ya-Jun; Wang, Xi-Zhong; He, Guang-Xin; Chen, Hong-Wei; Fei, Li-Song

    2002-09-01

    Activin, which is included in the transforming growth factor-beta (TGF beta) superfamily of proteins and receptors, is known to have broad-ranging effects in the creatures. The mature peptide of beta A subunit of this gene, one of the most highly conserved sequence, can elevate the basal secretion of follicle-stimulating hormone (FSH) in the pituitary and FSH is pivotal to organism's reproduction. Reproduction block is one of the main reasons which cause giant panda to extinct. The sequence of Activin beta A subunit gene mature peptides has been successfully amplified from giant panda, red panda and malayan sun bear's genomic DNA by using polymerase chain reaction (PCR) with a pair of degenerate primers. The PCR products were cloned into the vector pBlueScript+ of Esherichia coli. Sequence analysis of Activin beta A subunit gene mature peptides shows that the length of this gene segment is the same (359 bp) and there is no intron in all three species. The sequence encodes a peptide of 119 amino acid residues. The homology comparison demonstrates 93.9% DNA homology and 99% homology in amino acid among these three species. Both GenBank blast search result and restriction enzyme map reveal that the sequences of Activin beta A subunit gene mature peptides of different species are highly conserved during the evolution process. Phylogeny analysis is performed with PHYLIP software package. A consistent phylogeny tree has been drawn with three different methods. The software analysis outcome accords with the academic view that giant panda has a closer relationship to the malayan sun bear than the red panda. Giant panda should be grouped into the bear family (Uersidae) with the malayan sun bear. As to the red panda, it would be better that this animal be grouped into the unique family (red panda family) because of great difference between the red panda and the bears (Uersidae).

  10. Interplay among membrane-bound lytic transglycosylase D1, the CreBC two-component regulatory system, the AmpNG-AmpDI-NagZ-AmpR regulatory circuit, and L1/L2 β-lactamase expression in Stenotrophomonas maltophilia.

    PubMed

    Huang, Yi-Wei; Wu, Chao-Jung; Hu, Rouh-Mei; Lin, Yi-Tsung; Yang, Tsuey-Ching

    2015-11-01

    Lytic transglycosylases (LTs) are an important class of enzymes involved in peptidoglycan (PG) cleavage, with the concomitant formation of an intramolecular 1,6-anhydromuramoyl reaction product. There are six annotated LT genes in the Stenotrophomonas maltophilia genome, including genes for five membrane-bound LTs (mltA, mltB1, mltB2, mltD1, and mltD2) and a gene for soluble LT (slt). Six LTs of S. maltophilia KJ were systematically mutated, yielding the ΔmltA, ΔmltB1, ΔmltB2, ΔmltD1, ΔmltD2, and Δslt mutants. Inactivation of mltD1 conferred a phenotype of elevated uninduced β-lactamase activity. The underlying mechanism responsible for this phenotype was elucidated by the construction of several mutants and determination of β-lactamase activity. The expression of the genes assayed was assessed by quantitative reverse transcriptase PCR and a promoter transcription fusion assay. The results demonstrate that ΔmltD1 mutant-mediated L1/L2 β-lactamase expression involved the creBC two-component regulatory system (TCS) and the ampNG-ampDI-nagZ-ampR regulatory circuit. The inactivation of mltD1 resulted in mltB1 and mltD2 upexpression in a creBC- and ampNG-dependent manner. The overexpressed MltB1 and MltD2 activity contributed to the expression of the L1/L2 β-lactamase genes via the ampNG-ampDI-nagZ-ampR regulatory circuit. These findings reveal, for the first time, a linkage between LTs, the CreBC TCS, the ampNG-ampDI-nagZ-ampR regulatory circuit, and L1/L2 β-lactamase expression in S. maltophilia.

  11. HDAC Inhibitors.

    PubMed

    Olzscha, Heidi; Bekheet, Mina E; Sheikh, Semira; La Thangue, Nicholas B

    2016-01-01

    Lysine acetylation in proteins is one of the most abundant posttranslational modifications in eukaryotic cells. The dynamic homeostasis of lysine acetylation and deacetylation is dictated by the action of histone acetyltransferases (HAT) and histone deacetylases (HDAC). Important substrates for HATs and HDACs are histones, where lysine acetylation generally leads to an open and transcriptionally active chromatin conformation. Histone deacetylation forces the compaction of the chromatin with subsequent inhibition of transcription and reduced gene expression. Unbalanced HAT and HDAC activity, and therefore aberrant histone acetylation, has been shown to be involved in tumorigenesis and progression of malignancy in different types of cancer. Therefore, the development of HDAC inhibitors (HDIs) as therapeutic agents against cancer is of great interest. However, treatment with HDIs can also affect the acetylation status of many other non-histone proteins which play a role in different pathways including angiogenesis, cell cycle progression, autophagy and apoptosis. These effects have led HDIs to become anticancer agents, which can initiate apoptosis in tumor cells. Hematological malignancies in particular are responsive to HDIs, and four HDIs have already been approved as anticancer agents. There is a strong interest in finding adequate biomarkers to predict the response to HDI treatment. This chapter provides information on how to assess HDAC activity in vitro and determine the potency of HDIs on different HDACs. It also gives information on how to analyze cellular markers following HDI treatment and to analyze tissue biopsies from HDI-treated patients. Finally, a protocol is provided on how to detect HDI sensitivity determinants in human cells, based on a pRetroSuper shRNA screen upon HDI treatment. PMID:27246222

  12. Dual Inhibition of Activin/Nodal/TGF-β and BMP Signaling Pathways by SB431542 and Dorsomorphin Induces Neuronal Differentiation of Human Adipose Derived Stem Cells

    PubMed Central

    Madhu, Vedavathi; Dighe, Abhijit S.; Cui, Quanjun; Deal, D. Nicole

    2016-01-01

    Damage to the nervous system can cause devastating diseases or musculoskeletal dysfunctions and transplantation of progenitor stem cells can be an excellent treatment option in this regard. Preclinical studies demonstrate that untreated stem cells, unlike stem cells activated to differentiate into neuronal lineage, do not survive in the neuronal tissues. Conventional methods of inducing neuronal differentiation of stem cells are complex and expensive. We therefore sought to determine if a simple, one-step, and cost effective method, previously reported to induce neuronal differentiation of embryonic stem cells and induced-pluripotent stem cells, can be applied to adult stem cells. Indeed, dual inhibition of activin/nodal/TGF-β and BMP pathways using SB431542 and dorsomorphin, respectively, induced neuronal differentiation of human adipose derived stem cells (hADSCs) as evidenced by formation of neurite extensions, protein expression of neuron-specific gamma enolase, and mRNA expression of neuron-specific transcription factors Sox1 and Pax6 and matured neuronal marker NF200. This process correlated with enhanced phosphorylation of p38, Erk1/2, PI3K, and Akt1/3. Additionally, in vitro subcutaneous implants of SB431542 and dorsomorphin treated hADSCs displayed significantly higher expression of active-axonal-growth-specific marker GAP43. Our data offers novel insights into cell-based therapies for the nervous system repair. PMID:26798350

  13. Expression and distribution of forkhead activin signal transducer 2 (FAST2) during follicle development in mouse ovaries and pre-implantation embryos.

    PubMed

    Wang, Guiping; Liu, Linlin; Guo, Shujuan; Zhang, Cong

    2016-07-01

    Xenopus forkhead activin signal transducer 1 (xFAST 1) was first characterized in Xenopus as the transcriptional partner for Smad proteins. FAST2, which is the xFAST 1 homologues in mouse, is expressed during early developmental stages of the organism. However, the function of FAST2 in mouse ovaries and pre-implantation embryos is unclear. Therefore, we investigated its expression during these processes. In postnatal mice, FAST2 was expressed in oocytes and thecal cells from postnatal day (PD) 1 to PD 21. In gonadotropin-induced immature mice, FAST2 was expressed in oocytes, thecal cells and newly formed corpora lutea (CLs), but was expressed at a lower level in degenerated CLs. Similar results were observed upon western blot analyses. In meloxicam-treated immature mice, ovulation was inhibited and FAST2 was expressed in thecal cells, luteinized granulosa cells and entrapped oocytes. Immunofluorescence results showed that FAST2 was expressed in the cytoplasm and nucleus but not the nucleolus from the zygote to 8-cell embryo stage, after which it was localized to the cytoplasm of the morulae and inner cell mass of the blastocysts. Taken together, these observations suggest that FAST2 is expressed in a cell-specific manner during ovarian follicle development, ovulation, luteinization and early embryonic development, and that FAST2 might play important roles in these physiological processes. PMID:27432806

  14. Th2 lymphocytes migrating to the bone marrow under high-altitude hypoxia promote erythropoiesis via activin A and interleukin-9.

    PubMed

    Li, Peng; Zheng, Shan-jun; Jiang, Chun-hua; Zhou, Si-min; Tian, Huai-jun; Zhang, Gang; Gao, Yu-qi

    2014-09-01

    The mechanism of accelerated erythropoiesis under the hypoxic conditions of high altitude (HA) remains largely obscure. Here, we investigated the potential role of bone marrow (BM) T cells in the increased production of erythrocytes at HA. We found that mice exposed to a simulated altitude of 6,000 m for 1-3 weeks exhibited a significant expansion of BM CD4+ cells, mainly caused by increasing T helper 2 (Th2) cells. Using a coculture model of BM T cells and hematopoietic stem/progenitor cells, we observed that BM CD4+ cells from hypoxic mice induced erythroid output more easily, in agreement with the erythroid-enhancing effect observed for Th2-condition-cultured BM CD4+ cells. It was further demonstrated that elevated secretion of activin A and interleukin-9 by BM Th2 cells of hypoxic mice promoted erythroid differentiation of hematopoietic stem/progenitor cells and the growth of erythroblasts, respectively. Our study also provided evidence that the CXCL12-CXCR4 interaction played an important role in Th2 cell trafficking to the BM under HA conditions. These results collectively suggest that Th2 cells migrating to the BM during HA exposure have a regulatory role in erythropoiesis, which provides new insight into the mechanism of high altitude polycythemia.

  15. Ringhalexin from Hemachatus haemachatus: A novel inhibitor of extrinsic tenase complex

    PubMed Central

    Barnwal, Bhaskar; Jobichen, Chacko; Girish, Vallerinteavide Mavelli; Foo, Chun Shin; Sivaraman, J.; Kini, R. Manjunatha

    2016-01-01

    Anticoagulant therapy is used for the prevention and treatment of thromboembolic disorders. Blood coagulation is initiated by the interaction of factor VIIa (FVIIa) with membrane-bound tissue factor (TF) to form the extrinsic tenase complex which activates FX to FXa. Thus, it is an important target for the development of novel anticoagulants. Here, we report the isolation and characterization of a novel anticoagulant ringhalexin from the venom of Hemachatus haemachatus (African Ringhals Cobra). Amino acid sequence of the protein indicates that it belongs to the three-finger toxin family and exhibits 94% identity to an uncharacterized Neurotoxin-like protein NTL2 from Naja atra. Ringhalexin inhibited FX activation by extrinsic tenase complex with an IC50 of 123.8 ± 9.54 nM. It is a mixed-type inhibitor with the kinetic constants, Ki and Ki’ of 84.25 ± 3.53 nM and 152.5 ± 11.32 nM, respectively. Ringhalexin also exhibits a weak, irreversible neurotoxicity on chick biventer cervicis muscle preparations. Subsequently, the three-dimensional structure of ringhalexin was determined at 2.95 Å resolution. This study for the first time reports the structure of an anticoagulant three-finger toxin. Thus, ringhalexin is a potent inhibitor of the FX activation by extrinsic tenase complex and a weak, irreversible neurotoxin. PMID:27173146

  16. Ringhalexin from Hemachatus haemachatus: A novel inhibitor of extrinsic tenase complex.

    PubMed

    Barnwal, Bhaskar; Jobichen, Chacko; Girish, Vallerinteavide Mavelli; Foo, Chun Shin; Sivaraman, J; Kini, R Manjunatha

    2016-05-13

    Anticoagulant therapy is used for the prevention and treatment of thromboembolic disorders. Blood coagulation is initiated by the interaction of factor VIIa (FVIIa) with membrane-bound tissue factor (TF) to form the extrinsic tenase complex which activates FX to FXa. Thus, it is an important target for the development of novel anticoagulants. Here, we report the isolation and characterization of a novel anticoagulant ringhalexin from the venom of Hemachatus haemachatus (African Ringhals Cobra). Amino acid sequence of the protein indicates that it belongs to the three-finger toxin family and exhibits 94% identity to an uncharacterized Neurotoxin-like protein NTL2 from Naja atra. Ringhalexin inhibited FX activation by extrinsic tenase complex with an IC50 of 123.8 ± 9.54 nM. It is a mixed-type inhibitor with the kinetic constants, Ki and Ki' of 84.25 ± 3.53 nM and 152.5 ± 11.32 nM, respectively. Ringhalexin also exhibits a weak, irreversible neurotoxicity on chick biventer cervicis muscle preparations. Subsequently, the three-dimensional structure of ringhalexin was determined at 2.95 Å resolution. This study for the first time reports the structure of an anticoagulant three-finger toxin. Thus, ringhalexin is a potent inhibitor of the FX activation by extrinsic tenase complex and a weak, irreversible neurotoxin.

  17. Activin Decoy Receptor ActRIIB:Fc Lowers FSH and Therapeutically Restores Oocyte Yield, Prevents Oocyte Chromosome Misalignments and Spindle Aberrations, and Increases Fertility in Midlife Female SAMP8 Mice.

    PubMed

    Bernstein, Lori R; Mackenzie, Amelia C L; Lee, Se-Jin; Chaffin, Charles L; Merchenthaler, István

    2016-03-01

    Women of advanced maternal age (AMA) (age ≥ 35) have increased rates of infertility, miscarriages, and trisomic pregnancies. Collectively these conditions are called "egg infertility." A root cause of egg infertility is increased rates of oocyte aneuploidy with age. AMA women often have elevated endogenous FSH. Female senescence-accelerated mouse-prone-8 (SAMP8) has increased rates of oocyte spindle aberrations, diminished fertility, and rising endogenous FSH with age. We hypothesize that elevated FSH during the oocyte's FSH-responsive growth period is a cause of abnormalities in the meiotic spindle. We report that eggs from SAMP8 mice treated with equine chorionic gonadotropin (eCG) for the period of oocyte growth have increased chromosome and spindle misalignments. Activin is a molecule that raises FSH, and ActRIIB:Fc is an activin decoy receptor that binds and sequesters activin. We report that ActRIIB:Fc treatment of midlife SAMP8 mice for the duration of oocyte growth lowers FSH, prevents egg chromosome and spindle misalignments, and increases litter sizes. AMA patients can also have poor responsiveness to FSH stimulation. We report that although eCG lowers yields of viable oocytes, ActRIIB:Fc increases yields of viable oocytes. ActRIIB:Fc and eCG cotreatment markedly reduces yields of viable oocytes. These data are consistent with the hypothesis that elevated FSH contributes to egg aneuploidy, declining fertility, and poor ovarian response and that ActRIIB:Fc can prevent egg aneuploidy, increase fertility, and improve ovarian response. Future studies will continue to examine whether ActRIIB:Fc works via FSH and/or other pathways and whether ActRIIB:Fc can prevent aneuploidy, increase fertility, and improve stimulation responsiveness in AMA women. PMID:26713784

  18. Activin Decoy Receptor ActRIIB:Fc Lowers FSH and Therapeutically Restores Oocyte Yield, Prevents Oocyte Chromosome Misalignments and Spindle Aberrations, and Increases Fertility in Midlife Female SAMP8 Mice.

    PubMed

    Bernstein, Lori R; Mackenzie, Amelia C L; Lee, Se-Jin; Chaffin, Charles L; Merchenthaler, István

    2016-03-01

    Women of advanced maternal age (AMA) (age ≥ 35) have increased rates of infertility, miscarriages, and trisomic pregnancies. Collectively these conditions are called "egg infertility." A root cause of egg infertility is increased rates of oocyte aneuploidy with age. AMA women often have elevated endogenous FSH. Female senescence-accelerated mouse-prone-8 (SAMP8) has increased rates of oocyte spindle aberrations, diminished fertility, and rising endogenous FSH with age. We hypothesize that elevated FSH during the oocyte's FSH-responsive growth period is a cause of abnormalities in the meiotic spindle. We report that eggs from SAMP8 mice treated with equine chorionic gonadotropin (eCG) for the period of oocyte growth have increased chromosome and spindle misalignments. Activin is a molecule that raises FSH, and ActRIIB:Fc is an activin decoy receptor that binds and sequesters activin. We report that ActRIIB:Fc treatment of midlife SAMP8 mice for the duration of oocyte growth lowers FSH, prevents egg chromosome and spindle misalignments, and increases litter sizes. AMA patients can also have poor responsiveness to FSH stimulation. We report that although eCG lowers yields of viable oocytes, ActRIIB:Fc increases yields of viable oocytes. ActRIIB:Fc and eCG cotreatment markedly reduces yields of viable oocytes. These data are consistent with the hypothesis that elevated FSH contributes to egg aneuploidy, declining fertility, and poor ovarian response and that ActRIIB:Fc can prevent egg aneuploidy, increase fertility, and improve ovarian response. Future studies will continue to examine whether ActRIIB:Fc works via FSH and/or other pathways and whether ActRIIB:Fc can prevent aneuploidy, increase fertility, and improve stimulation responsiveness in AMA women.

  19. 5-(1,3-Benzothiazol-6-yl)-4-(4-methyl-1,3-thiazol-2-yl)-1H-imidazole derivatives as potent and selective transforming growth factor-β type I receptor inhibitors.

    PubMed

    Amada, Hideaki; Sekiguchi, Yoshinori; Ono, Naoya; Koami, Takeshi; Takayama, Tetsuo; Yabuuchi, Tetsuya; Katakai, Hironori; Ikeda, Akiko; Aoki, Mari; Naruse, Takumi; Wada, Reiko; Nozoe, Akiko; Sato, Masakazu

    2012-12-15

    A series of 5-(1,3-benzothiazol-6-yl)-4-(4-methyl-1,3-thiazol-2-yl)-1H-imidazole derivatives was synthesized as transforming growth factor-β (TGF-β) type I receptor (also known as activin-like kinase 5 or ALK5) inhibitors. These compounds were evaluated for their ALK5 inhibitory activity in an enzyme assay and for their TGF-β-induced Smad2/3 phosphorylation inhibitory activity in a cell-based assay. As a representative compound, 16i was a potent and selective ALK5 inhibitor, exhibiting a good enzyme inhibitory activity (IC(50) = 5.5 nM) as well as inhibitory activity against TGF-β-induced Smad2/3 phosphorylation at a cellular level (IC(50) = 36 nM). Furthermore, the topical application of 3% 16i lotion significantly inhibited Smad2 phosphorylation in Mouse skin (90% inhibition compared with vehicle-treated animals).

  20. Transcutaneous auricular vagus nerve stimulation regulates expression of growth differentiation factor 11 and activin-like kinase 5 in cerebral ischemia/reperfusion rats.

    PubMed

    Ma, Jingxi; Zhang, Lina; He, Guoqian; Tan, Xiaodan; Jin, Xinhao; Li, Changqing

    2016-10-15

    Growth differentiation factor 11 (GDF11), as a rejuvenation factor in heterochronic parabiosis, can increase proliferation of primary brain capillary endothelial cells (ECs). However, the angiogenic role of GDF11 in ischemia-induced brain injury is still unclear. There are no previous reports on the spatiotemporal expression of GDF11 in cerebral ischemia/reperfusion (I/R) rats. Our recent work has strongly suggested that transcutaneous auricular vagus nerve stimulation (ta-VNS) reduces infarct size and induces angiogenesis in focal cerebral I/R rats. This study focused on expression of GDF11 and activin-like kinase 5 (ALK5) and the effects of ta-VNS in a rat cerebral I/R model. For ta-VNS, electrical stimulation of the left cavum concha (1h duration) using percutaneous needles was initiated 30min after induction of ischemia. Expression of GDF11 was analyzed by enzyme-linked immunosorbent assay, immunohistochemistry, real-time polymerase chain reaction, and western blot 24h, 3d, and 7d after reperfusion. In addition, neurobehavioral function, EC proliferation, and expression of ALK5 in ECs in the peri-infarct cortex were measured. Results showed that levels of GDF11 were significantly elevated after cerebral I/R, both in plasma and the peri-infarct cerebral cortex. Interestingly, splenic GDF11 levels decreased after ischemia. ALK5 was expressed in ECs in the peri-infarct cerebral cortex where active vessel remodeling was noted. ta-VNS improved neurobehavioral recovery, upregulated cerebral GDF11 and downregulated splenic GDF11, indicating a brain-spleen communication during stroke. ta-VNS also increased expression of ALK5 in ECs and stimulated proliferation of ECs. These results suggest that, after cerebral ischemia, GDF11 redistributes and participates in angiogenesis as an angiogenic factor that acts at least in part through ALK5. GDF11/ALK5 may represent a new potential therapy target for stroke. PMID:27653860

  1. Activin A and equine chorionic gonadotropin recover reproductive dysfunction induced by neonatal exposure to an estrogenic endocrine disruptor in adult male mice.

    PubMed

    Warita, Katsuhiko; Okamoto, Kazutaka; Mutoh, Ken-Ichiro; Hasegawa, Yoshihisa; Yue, Zhan-Peng; Yokoyama, Toshifumi; Matsumoto, Yoshiki; Miki, Takanori; Takeuchi, Yoshiki; Kitagawa, Hiroshi; Sugawara, Teruo; Hoshi, Nobuhiko

    2008-01-01

    We aimed to elucidate the mechanism of action of estrogenic endocrine disruptors and the rescue of reproductive function, particularly the responsiveness of testes to eCG and/or activin A (ACT) after establishing reproductive disorders. Newborn male mice (n = 29) were randomly divided into an untreated group and three treatment groups that received diethylstilbestrol (DES; 100 mug per animal) subcutaneously on Postnatal Day 3 to establish reproductive disorders and daily treatment with PBS (controls: DES + PBS), eCG (eCG group: DES + eCG), or eCG + ACT (eCG + ACT group: DES + eCG + ACT) at 6-8 wk of age prior to mating. After treatment, the controls showed diminished Leydig cells in the testes and thin germ cell layers containing pyknotic germ cells and multinucleated cells. In the eCG and eCG + ACT groups, spermatids and Leydig cells increased markedly. The immunoexpression of androgen receptors in the eCG group and steroidogenic acute regulatory (STAR) protein in the eCG and eCG + ACT groups recovered to approximately the levels in the untreated group; plasma LH and testosterone levels also increased relative to those in the controls. In addition, the cell proliferation index, which is estimated from 5-bromo-2'-deoxyuridine immunoexpression in spermatogonia, increased significantly under eCG treatment, and even more with eCG + ACT. However, the numbers of germ and Leydig cells decreased at 12 wk of age. Thus, ACT and eCG help the testes to recover from the dysfunction induced by neonatal DES administration. Furthermore, the permanent male reproductive disorder induced by neonatal exposure to estrogenic agents may be more likely to result from dysfunction of the hypothalamic-pituitary axis than from dysfunction of the lower reproductive organs.

  2. Proton pump inhibitors

    MedlinePlus

    Proton pump inhibitors (PPIs) are medicines that work by reducing the amount of stomach acid made by ... Proton pump inhibitors are used to: Relieve symptoms of acid reflux, or gastroesophageal reflux disease (GERD). This ...

  3. The Expression of Human Cytomegalovirus MicroRNA MiR-UL148D during Latent Infection in Primary Myeloid Cells Inhibits Activin A-triggered Secretion of IL-6.

    PubMed

    Lau, Betty; Poole, Emma; Krishna, Benjamin; Sellart, Immaculada; Wills, Mark R; Murphy, Eain; Sinclair, John

    2016-08-05

    The successful establishment and maintenance of human cytomegalovirus (HCMV) latency is dependent on the expression of a subset of viral genes. Whilst the exact spectrum and functions of these genes are far from clear, inroads have been made for protein-coding genes. In contrast, little is known about the expression of non-coding RNAs. Here we show that HCMV encoded miRNAs are expressed de novo during latent infection of primary myeloid cells. Furthermore, we demonstrate that miR-UL148D, one of the most highly expressed viral miRNAs during latent infection, directly targets the cellular receptor ACVR1B of the activin signalling axis. Consistent with this, we observed upregulation of ACVR1B expression during latent infection with a miR-UL148D deletion virus (ΔmiR-UL148D). Importantly, we observed that monocytes latently infected with ΔmiR-UL148D are more responsive to activin A stimulation, as demonstrated by their increased secretion of IL-6. Collectively, our data indicates miR-UL148D inhibits ACVR1B expression in latently infected cells to limit proinflammatory cytokine secretion, perhaps as an immune evasion strategy or to postpone cytokine-induced reactivation until conditions are more favourable. This is the first demonstration of an HCMV miRNA function during latency in primary myeloid cells, implicating that small RNA species may contribute significantly to latent infection.

  4. The Expression of Human Cytomegalovirus MicroRNA MiR-UL148D during Latent Infection in Primary Myeloid Cells Inhibits Activin A-triggered Secretion of IL-6

    PubMed Central

    Lau, Betty; Poole, Emma; Krishna, Benjamin; Sellart, Immaculada; Wills, Mark R.; Murphy, Eain; Sinclair, John

    2016-01-01

    The successful establishment and maintenance of human cytomegalovirus (HCMV) latency is dependent on the expression of a subset of viral genes. Whilst the exact spectrum and functions of these genes are far from clear, inroads have been made for protein-coding genes. In contrast, little is known about the expression of non-coding RNAs. Here we show that HCMV encoded miRNAs are expressed de novo during latent infection of primary myeloid cells. Furthermore, we demonstrate that miR-UL148D, one of the most highly expressed viral miRNAs during latent infection, directly targets the cellular receptor ACVR1B of the activin signalling axis. Consistent with this, we observed upregulation of ACVR1B expression during latent infection with a miR-UL148D deletion virus (ΔmiR-UL148D). Importantly, we observed that monocytes latently infected with ΔmiR-UL148D are more responsive to activin A stimulation, as demonstrated by their increased secretion of IL-6. Collectively, our data indicates miR-UL148D inhibits ACVR1B expression in latently infected cells to limit proinflammatory cytokine secretion, perhaps as an immune evasion strategy or to postpone cytokine-induced reactivation until conditions are more favourable. This is the first demonstration of an HCMV miRNA function during latency in primary myeloid cells, implicating that small RNA species may contribute significantly to latent infection. PMID:27491954

  5. Multiple roles of Activin/Nodal, bone morphogenetic protein, fibroblast growth factor and Wnt/β-catenin signalling in the anterior neural patterning of adherent human embryonic stem cell cultures

    PubMed Central

    Lupo, Giuseppe; Novorol, Claire; Smith, Joseph R.; Vallier, Ludovic; Miranda, Elena; Alexander, Morgan; Biagioni, Stefano; Pedersen, Roger A.; Harris, William A.

    2013-01-01

    Several studies have successfully produced a variety of neural cell types from human embryonic stem cells (hESCs), but there has been limited systematic analysis of how different regional identities are established using well-defined differentiation conditions. We have used adherent, chemically defined cultures to analyse the roles of Activin/Nodal, bone morphogenetic protein (BMP), fibroblast growth factor (FGF) and Wnt/β-catenin signalling in neural induction, anteroposterior patterning and eye field specification in hESCs. We show that either BMP inhibition or activation of FGF signalling is required for effective neural induction, but these two pathways have distinct outcomes on rostrocaudal patterning. While BMP inhibition leads to specification of forebrain/midbrain positional identities, FGF-dependent neural induction is associated with strong posteriorization towards hindbrain/spinal cord fates. We also demonstrate that Wnt/β-catenin signalling is activated during neural induction and promotes acquisition of neural fates posterior to forebrain. Therefore, inhibition of this pathway is needed for efficient forebrain specification. Finally, we provide evidence that the levels of Activin/Nodal and BMP signalling have a marked influence on further forebrain patterning and that constitutive inhibition of these pathways represses expression of eye field genes. These results show that the key mechanisms controlling neural patterning in model vertebrate species are preserved in adherent, chemically defined hESC cultures and reveal new insights into the signals regulating eye field specification. PMID:23576785

  6. Multiple roles of Activin/Nodal, bone morphogenetic protein, fibroblast growth factor and Wnt/β-catenin signalling in the anterior neural patterning of adherent human embryonic stem cell cultures.

    PubMed

    Lupo, Giuseppe; Novorol, Claire; Smith, Joseph R; Vallier, Ludovic; Miranda, Elena; Alexander, Morgan; Biagioni, Stefano; Pedersen, Roger A; Harris, William A

    2013-04-01

    Several studies have successfully produced a variety of neural cell types from human embryonic stem cells (hESCs), but there has been limited systematic analysis of how different regional identities are established using well-defined differentiation conditions. We have used adherent, chemically defined cultures to analyse the roles of Activin/Nodal, bone morphogenetic protein (BMP), fibroblast growth factor (FGF) and Wnt/β-catenin signalling in neural induction, anteroposterior patterning and eye field specification in hESCs. We show that either BMP inhibition or activation of FGF signalling is required for effective neural induction, but these two pathways have distinct outcomes on rostrocaudal patterning. While BMP inhibition leads to specification of forebrain/midbrain positional identities, FGF-dependent neural induction is associated with strong posteriorization towards hindbrain/spinal cord fates. We also demonstrate that Wnt/β-catenin signalling is activated during neural induction and promotes acquisition of neural fates posterior to forebrain. Therefore, inhibition of this pathway is needed for efficient forebrain specification. Finally, we provide evidence that the levels of Activin/Nodal and BMP signalling have a marked influence on further forebrain patterning and that constitutive inhibition of these pathways represses expression of eye field genes. These results show that the key mechanisms controlling neural patterning in model vertebrate species are preserved in adherent, chemically defined hESC cultures and reveal new insights into the signals regulating eye field specification. PMID:23576785

  7. The Expression of Human Cytomegalovirus MicroRNA MiR-UL148D during Latent Infection in Primary Myeloid Cells Inhibits Activin A-triggered Secretion of IL-6.

    PubMed

    Lau, Betty; Poole, Emma; Krishna, Benjamin; Sellart, Immaculada; Wills, Mark R; Murphy, Eain; Sinclair, John

    2016-01-01

    The successful establishment and maintenance of human cytomegalovirus (HCMV) latency is dependent on the expression of a subset of viral genes. Whilst the exact spectrum and functions of these genes are far from clear, inroads have been made for protein-coding genes. In contrast, little is known about the expression of non-coding RNAs. Here we show that HCMV encoded miRNAs are expressed de novo during latent infection of primary myeloid cells. Furthermore, we demonstrate that miR-UL148D, one of the most highly expressed viral miRNAs during latent infection, directly targets the cellular receptor ACVR1B of the activin signalling axis. Consistent with this, we observed upregulation of ACVR1B expression during latent infection with a miR-UL148D deletion virus (ΔmiR-UL148D). Importantly, we observed that monocytes latently infected with ΔmiR-UL148D are more responsive to activin A stimulation, as demonstrated by their increased secretion of IL-6. Collectively, our data indicates miR-UL148D inhibits ACVR1B expression in latently infected cells to limit proinflammatory cytokine secretion, perhaps as an immune evasion strategy or to postpone cytokine-induced reactivation until conditions are more favourable. This is the first demonstration of an HCMV miRNA function during latency in primary myeloid cells, implicating that small RNA species may contribute significantly to latent infection. PMID:27491954

  8. Associations between Thyroid Hormones, Calcification Inhibitor Levels and Vascular Calcification in End-Stage Renal Disease

    PubMed Central

    Meuwese, Christiaan Lucas; Olauson, Hannes; Qureshi, Abdul Rashid; Ripsweden, Jonaz; Barany, Peter; Vermeer, Cees; Drummen, Nadja; Stenvinkel, Peter

    2015-01-01

    Introduction Vascular calcification is a common, serious and elusive complication of end-stage renal disease (ESRD). As a pro-calcifying risk factor, non-thyroidal illness may promote vascular calcification through a systemic lowering of vascular calcification inhibitors such as matrix-gla protein (MGP) and Klotho. Methods and Material In 97 ESRD patients eligible for living donor kidney transplantation, blood levels of thyroid hormones (fT3, fT4 and TSH), total uncarboxylated MGP (t-ucMGP), desphospho-uncarboxylated MGP (dp-ucMGP), descarboxyprothrombin (PIVKA-II), and soluble Klotho (sKlotho) were measured. The degree of coronary calcification and arterial stiffness were assessed by means of cardiac CT-scans and applanation tonometry, respectively. Results fT3 levels were inversely associated with coronary artery calcification (CAC) scores and measures of arterial stiffness, and positively with dp-ucMGP and sKlotho concentrations. Subfractions of MGP, PIVKA-II and sKlotho did not associate with CAC scores and arterial stiffness. fT4 and TSH levels were both inversely associated with CAC scores, but not with arterial stiffness. Discussion The positive associations between fT3 and dp-ucMGP and sKlotho suggest that synthesis of MGP and Klotho is influenced by thyroid hormones, and supports a link between non-thyroidal illness and alterations in calcification inhibitor levels. However, the absence of an association between serum calcification inhibitor levels and coronary calcification/arterial stiffness and the fact that MGP and Klotho undergo post-translational modifications underscore the complexity of this association. Further studies, measuring total levels of MGP and membrane bound Klotho, should examine this proposed pathway in further detail. PMID:26147960

  9. Outside or inside: role of the subcellular localization of DP4-like enzymes for substrate conversion and inhibitor effects.

    PubMed

    Bank, Ute; Heimburg, Anke; Wohlfarth, Astrid; Koch, Gudrun; Nordhoff, Karsten; Julius, Heiko; Helmuth, Martin; Breyer, Doreen; Reinhold, Dirk; Täger, Michael; Ansorge, Siegfried

    2011-03-01

    The discovery of the DP4-related enzymes DP8 and DP9 raised controversial discussion regarding the physiological and pathophysiological function of distinct members of the DP4 family. Particularly with regard to their potential relevance in regulating immune functions, it is of interest to know which role the subcellular distribution of the enzymes play. Synthetic substrates as well as low molecular weight inhibitors are widely used as tools, but little is yet known regarding their features in cell experiments, such as their plasma membrane penetration capacity. The fluorogenic substrates Gly-Pro-AMC or (Ala-Pro)₂-R110 predominantly detect plasma membrane-bound activities of viable cells (less than 0.1% of fluorochromes R110 or AMC inside viable cells after 1 h incubation). Additionally, the selective and non-selective DP8/9 inhibitors allo-Ile-isoindoline and Lys[Z(NO₂)]-pyrrolidide were found to be incapable of passing the plasma membrane easily. This suggests that previously reported cellular effects are not due to inhibition of the cytosolic enzymes DP8 or DP9. Moreover, our enzymatic studies with viable cells provided evidence that DP8 and/or DP9 are also present on the surface of immune cells under certain circumstances and could gain relevance particularly in the absence of DP4 expression. In summary, in cells which do express DP4 on the surface, this archetypical member of the DP4 family is the most relevant peptidase in the regulation of cellular functions.

  10. Ceapins are a new class of unfolded protein response inhibitors, selectively targeting the ATF6α branch

    PubMed Central

    Gallagher, Ciara M; Garri, Carolina; Cain, Erica L; Ang, Kenny Kean-Hooi; Wilson, Christopher G; Chen, Steven; Hearn, Brian R; Jaishankar, Priyadarshini; Aranda-Diaz, Andres; Arkin, Michelle R; Renslo, Adam R; Walter, Peter

    2016-01-01

    The membrane-bound transcription factor ATF6α plays a cytoprotective role in the unfolded protein response (UPR), required for cells to survive ER stress. Activation of ATF6α promotes cell survival in cancer models. We used cell-based screens to discover and develop Ceapins, a class of pyrazole amides, that block ATF6α signaling in response to ER stress. Ceapins sensitize cells to ER stress without impacting viability of unstressed cells. Ceapins are highly specific inhibitors of ATF6α signaling, not affecting signaling through the other branches of the UPR, or proteolytic processing of its close homolog ATF6β or SREBP (a cholesterol-regulated transcription factor), both activated by the same proteases. Ceapins are first-in-class inhibitors that can be used to explore both the mechanism of activation of ATF6α and its role in pathological settings. The discovery of Ceapins now enables pharmacological modulation all three UPR branches either singly or in combination. DOI: http://dx.doi.org/10.7554/eLife.11878.001 PMID:27435960

  11. The P-glycoprotein inhibitor GF120918 modulates Ca2+-dependent processes and lipid metabolism in Toxoplasma gondii.

    PubMed

    Bottova, Iveta; Sauder, Ursula; Olivieri, Vesna; Hehl, Adrian B; Sonda, Sabrina

    2010-01-01

    Up-regulation of the membrane-bound efflux pump P-glycoprotein (P-gp) is associated with the phenomenon of multidrug-resistance in pathogenic organisms, including protozoan parasites. In addition, P-gp plays a role in normal physiological processes, however our understanding of these P-gp functions remains limited. In this study we investigated the effects of the P-gp inhibitor GF120918 in Toxoplasma gondii, a model apicomplexan parasite and an important human pathogen. We found that GF120918 treatment severely inhibited parasite invasion and replication. Further analyses of the molecular mechanisms involved revealed that the P-gp inhibitor modulated parasite motility, microneme secretion and egress from the host cell, all cellular processes known to depend on Ca2+ signaling in the parasite. In support of a potential role of P-gp in Ca2+-mediated processes, immunoelectron and fluorescence microscopy showed that T. gondii P-gp was localized in acidocalcisomes, the major Ca2+ storage in the parasite, at the plasma membrane, and in the intravacuolar tubular network. In addition, metabolic labeling of extracellular parasites revealed that inhibition or down-regulation of T. gondii P-gp resulted in aberrant lipid synthesis. These results suggest a crucial role of T. gondii P-gp in essential processes of the parasite biology and further validate the potential of P-gp activity as a target for drug development. PMID:20386707

  12. The P-glycoprotein Inhibitor GF120918 Modulates Ca2+-Dependent Processes and Lipid Metabolism in Toxoplasma Gondii

    PubMed Central

    Bottova, Iveta; Sauder, Ursula; Olivieri, Vesna; Hehl, Adrian B.; Sonda, Sabrina

    2010-01-01

    Up-regulation of the membrane-bound efflux pump P-glycoprotein (P-gp) is associated with the phenomenon of multidrug-resistance in pathogenic organisms, including protozoan parasites. In addition, P-gp plays a role in normal physiological processes, however our understanding of these P-gp functions remains limited. In this study we investigated the effects of the P-gp inhibitor GF120918 in Toxoplasma gondii, a model apicomplexan parasite and an important human pathogen. We found that GF120918 treatment severely inhibited parasite invasion and replication. Further analyses of the molecular mechanisms involved revealed that the P-gp inhibitor modulated parasite motility, microneme secretion and egress from the host cell, all cellular processes known to depend on Ca2+ signaling in the parasite. In support of a potential role of P-gp in Ca2+-mediated processes, immunoelectron and fluorescence microscopy showed that T. gondii P-gp was localized in acidocalcisomes, the major Ca2+ storage in the parasite, at the plasma membrane, and in the intravacuolar tubular network. In addition, metabolic labeling of extracellular parasites revealed that inhibition or down-regulation of T. gondii P-gp resulted in aberrant lipid synthesis. These results suggest a crucial role of T. gondii P-gp in essential processes of the parasite biology and further validate the potential of P-gp activity as a target for drug development. PMID:20386707

  13. Ceapins are a new class of unfolded protein response inhibitors, selectively targeting the ATF6α branch.

    PubMed

    Gallagher, Ciara M; Garri, Carolina; Cain, Erica L; Ang, Kenny Kean-Hooi; Wilson, Christopher G; Chen, Steven; Hearn, Brian R; Jaishankar, Priyadarshini; Aranda-Diaz, Andres; Arkin, Michelle R; Renslo, Adam R; Walter, Peter

    2016-01-01

    The membrane-bound transcription factor ATF6α plays a cytoprotective role in the unfolded protein response (UPR), required for cells to survive ER stress. Activation of ATF6α promotes cell survival in cancer models. We used cell-based screens to discover and develop Ceapins, a class of pyrazole amides, that block ATF6α signaling in response to ER stress. Ceapins sensitize cells to ER stress without impacting viability of unstressed cells. Ceapins are highly specific inhibitors of ATF6α signaling, not affecting signaling through the other branches of the UPR, or proteolytic processing of its close homolog ATF6β or SREBP (a cholesterol-regulated transcription factor), both activated by the same proteases. Ceapins are first-in-class inhibitors that can be used to explore both the mechanism of activation of ATF6α and its role in pathological settings. The discovery of Ceapins now enables pharmacological modulation all three UPR branches either singly or in combination. PMID:27435960

  14. Long-term effects of the trehalase inhibitor trehazolin on trehalase activity in locust flight muscle.

    PubMed

    Wegener, Gerhard; Macho, Claudia; Schlöder, Paul; Kamp, Günter; Ando, Osamu

    2010-11-15

    Trehalase (EC 3.2.1.28) hydrolyzes the main haemolymph sugar of insects, trehalose, into the essential cellular substrate glucose. Trehalase in locust flight muscle is bound to membranes that appear in the microsomal fraction upon tissue fractionation, but the exact location in vivo has remained elusive. Trehalase has been proposed to be regulated by a novel type of activity control that is based on the reversible transformation of a latent (inactive) form into an overt (active) form. Most trehalase activity from saline-injected controls was membrane-bound (95%) and comprised an overt form (∼25%) and a latent form (75%). Latent trehalase could be assayed only after the integrity of membranes had been destroyed. Trehazolin, a potent tight-binding inhibitor of trehalase, is confined to the extracellular space and has been used as a tool to gather information on the relationship between latent and overt trehalase. Trehazolin was injected into the haemolymph of locusts, and the trehalase activity of the flight muscle was determined at different times over a 30-day period. Total trehalase activity in locust flight muscle was markedly inhibited during the first half of the interval, but reappeared during the second half. Inhibition of the overt form preceded inhibition of the latent form, and the time course suggested a reversible precursor-product relation (cycling) between the two forms. The results support the working hypothesis that trehalase functions as an ectoenzyme, the activity of which is regulated by reversible transformation of latent into overt trehalase. PMID:21037064

  15. DPP IV inhibitor blocks mescaline-induced scratching and amphetamine-induced hyperactivity in mice.

    PubMed

    Lautar, Susan L; Rojas, Camilo; Slusher, Barbara S; Wozniak, Krystyna M; Wu, Ying; Thomas, Ajit G; Waldon, Daniel; Li, William; Ferraris, Dana; Belyakov, Sergei

    2005-06-28

    Dipeptidyl peptidase IV (DPP IV) is a ubiquitous membrane-bound enzyme that cleaves the two N-terminal amino acids from peptides with a proline or alanine residue in the second position from the amino end. Potential substrates for DPP IV include several neuropeptides, suggesting a role for DPP IV in neurological processes. We have developed a potent DPP IV inhibitor (IC50 = 30 nM), 1-(2-amino-3-methyl-butyryl)-azetidine-2-carbonitrile (AMAC), which has shown efficacy in two established models of psychosis: mescaline-induced scratching and amphetamine-induced hyperactivity. In the mescaline-induced scratching model, AMAC treatment before mescaline administration reduced the number of scratching paroxysms by 68% (P < 0.01). The compound showed a dose-dependent effect, inhibiting significantly at 6, 20 and 60 mg/kg (37%, 39% and 68%, respectively). In the amphetamine-induced hyperactivity model, 50 and 60 mg/kg AMAC, given before injection of amphetamine, significantly reduced hyper-locomotion by 65% and 76%, respectively. Additionally, AMAC showed no significant activity in binding assays for 20 receptors thought to be involved in the pathology of schizophrenia, including dopamine, serotonin and glutamate. A structurally similar analog, 1-(2-dimethylamino-3-methyl-butyryl)-azetidine-2-carbonitrile (DAMAC), that does not inhibit DPP IV, was inactive in both models. Taken together, these data suggest that the antipsychotic effects of AMAC are the result of DPP IV inhibition.

  16. QseC Inhibitors as an Antivirulence Approach for Gram-Negative Pathogens

    PubMed Central

    Curtis, Meredith M.; Russell, Regan; Moreira, Cristiano G.; Adebesin, Adeniyi M.; Wang, Changguang; Williams, Noelle S.; Taussig, Ron; Stewart, Don; Zimmern, Philippe; Lu, Biao; Prasad, Ravi N.; Zhu, Chen; Rasko, David A.; Huntley, Jason F.; Falck, John R.

    2014-01-01

    ABSTRACT Invasive pathogens interface with the host and its resident microbiota through interkingdom signaling. The bacterial receptor QseC, which is a membrane-bound histidine sensor kinase, responds to the host stress hormones epinephrine and norepinephrine and the bacterial signal AI-3, integrating interkingdom signaling at the biochemical level. Importantly, the QseC signaling cascade is exploited by many bacterial pathogens to promote virulence. Here, we translated this basic science information into development of a potent small molecule inhibitor of QseC, LED209. Extensive structure activity relationship (SAR) studies revealed that LED209 is a potent prodrug that is highly selective for QseC. Its warhead allosterically modifies lysines in QseC, impairing its function and preventing the activation of the virulence program of several Gram-negative pathogens both in vitro and during murine infection. LED209 does not interfere with pathogen growth, possibly leading to a milder evolutionary pressure toward drug resistance. LED209 has desirable pharmacokinetics and does not present toxicity in vitro and in rodents. This is a unique antivirulence approach, with a proven broad-spectrum activity against multiple Gram-negative pathogens that cause mammalian infections. PMID:25389178

  17. Alternative intronic polyadenylation generates the interleukin-6 trans-signaling inhibitor sgp130-E10.

    PubMed

    Sommer, Jan; Garbers, Christoph; Wolf, Janina; Trad, Ahmad; Moll, Jens M; Sack, Markus; Fischer, Rainer; Grötzinger, Joachim; Waetzig, Georg H; Floss, Doreen M; Scheller, Jürgen

    2014-08-01

    Interleukin (IL)-6 signals via a receptor complex composed of the signal-transducing β-receptor gp130 and the non-signaling membrane-bound or soluble IL-6 receptor α (IL-6R, sIL-6R), which is referred to as classic and trans-signaling, respectively. IL-6 trans-signaling is functionally associated with the development of chronic inflammatory diseases and cancer. Soluble gp130 (sgp130) variants are natural inhibitors of trans-signaling. Differential splicing yields sgp130 isoforms. Here, we describe that alternative intronic polyadenylation in intron 10 of the gp130 transcript results in a novel mRNA coding for an sgp130 protein isoform (sgp130-E10) of 70-80 kDa. The sgp130-E10 protein was expressed in vivo in human peripheral blood mononuclear cells. To assess the biological activity of sgp130-E10, we expressed this variant as Fc-tagged fusion protein (sgp130-E10Fc). Recombinant sgp130-E10Fc binds to a complex of IL-6 and sIL-6R, but not to IL-6 alone, and specifically inhibits IL-6 trans-signaling. Thus, it might play an important role in the regulation of trans-signaling in vivo.

  18. Inhibitors and Antibody Fragments as Potential Anti-Inflammatory Therapeutics Targeting Neutrophil Proteinase 3 in Human Disease.

    PubMed

    Korkmaz, Brice; Lesner, Adam; Guarino, Carla; Wysocka, Magdalena; Kellenberger, Christine; Watier, Hervé; Specks, Ulrich; Gauthier, Francis; Jenne, Dieter E

    2016-07-01

    Proteinase 3 (PR3) has received great scientific attention after its identification as the essential antigenic target of antineutrophil cytoplasm antibodies in Wegener's granulomatosis (now called granulomatosis with polyangiitis). Despite many structural and functional similarities between neutrophil elastase (NE) and PR3 during biosynthesis, storage, and extracellular release, unique properties and pathobiological functions have emerged from detailed studies in recent years. The development of highly sensitive substrates and inhibitors of human PR3 and the creation of PR3-selective single knockout mice led to the identification of nonredundant roles of PR3 in cell death induction via procaspase-3 activation in cell cultures and in mouse models. According to a study in knockout mice, PR3 shortens the lifespan of infiltrating neutrophils in tissues and accelerates the clearance of aged neutrophils in mice. Membrane exposure of active human PR3 on apoptotic neutrophils reprograms the response of macrophages to phagocytosed neutrophils, triggers secretion of proinflammatory cytokines, and undermines immune silencing and tissue regeneration. PR3-induced disruption of the anti-inflammatory effect of efferocytosis may be relevant for not only granulomatosis with polyangiitis but also for other autoimmune diseases with high neutrophil turnover. Inhibition of membrane-bound PR3 by endogenous inhibitors such as the α-1-protease inhibitor is comparatively weaker than that of NE, suggesting that the adverse effects of unopposed PR3 activity resurface earlier than those of NE in individuals with α-1-protease inhibitor deficiency. Effective coverage of PR3 by anti-inflammatory tools and simultaneous inhibition of both PR3 and NE should be most promising in the future. PMID:27329045

  19. Inhibitors and Antibody Fragments as Potential Anti-Inflammatory Therapeutics Targeting Neutrophil Proteinase 3 in Human Disease.

    PubMed

    Korkmaz, Brice; Lesner, Adam; Guarino, Carla; Wysocka, Magdalena; Kellenberger, Christine; Watier, Hervé; Specks, Ulrich; Gauthier, Francis; Jenne, Dieter E

    2016-07-01

    Proteinase 3 (PR3) has received great scientific attention after its identification as the essential antigenic target of antineutrophil cytoplasm antibodies in Wegener's granulomatosis (now called granulomatosis with polyangiitis). Despite many structural and functional similarities between neutrophil elastase (NE) and PR3 during biosynthesis, storage, and extracellular release, unique properties and pathobiological functions have emerged from detailed studies in recent years. The development of highly sensitive substrates and inhibitors of human PR3 and the creation of PR3-selective single knockout mice led to the identification of nonredundant roles of PR3 in cell death induction via procaspase-3 activation in cell cultures and in mouse models. According to a study in knockout mice, PR3 shortens the lifespan of infiltrating neutrophils in tissues and accelerates the clearance of aged neutrophils in mice. Membrane exposure of active human PR3 on apoptotic neutrophils reprograms the response of macrophages to phagocytosed neutrophils, triggers secretion of proinflammatory cytokines, and undermines immune silencing and tissue regeneration. PR3-induced disruption of the anti-inflammatory effect of efferocytosis may be relevant for not only granulomatosis with polyangiitis but also for other autoimmune diseases with high neutrophil turnover. Inhibition of membrane-bound PR3 by endogenous inhibitors such as the α-1-protease inhibitor is comparatively weaker than that of NE, suggesting that the adverse effects of unopposed PR3 activity resurface earlier than those of NE in individuals with α-1-protease inhibitor deficiency. Effective coverage of PR3 by anti-inflammatory tools and simultaneous inhibition of both PR3 and NE should be most promising in the future.

  20. Histone Deacetylase Inhibitor Valproic Acid Promotes the Differentiation of Human Induced Pluripotent Stem Cells into Hepatocyte-Like Cells

    PubMed Central

    Kondo, Yuki; Iwao, Takahiro; Yoshihashi, Sachimi; Mimori, Kayo; Ogihara, Ruri; Nagata, Kiyoshi; Kurose, Kouichi; Saito, Masayoshi; Niwa, Takuro; Suzuki, Takayoshi; Miyata, Naoki; Ohmori, Shigeru; Nakamura, Katsunori; Matsunaga, Tamihide

    2014-01-01

    In this study, we aimed to elucidate the effects and mechanism of action of valproic acid on hepatic differentiation from human induced pluripotent stem cell-derived hepatic progenitor cells. Human induced pluripotent stem cells were differentiated into endodermal cells in the presence of activin A and then into hepatic progenitor cells using dimethyl sulfoxide. Hepatic progenitor cells were matured in the presence of hepatocyte growth factor, oncostatin M, and dexamethasone with valproic acid that was added during the maturation process. After 25 days of differentiation, cells expressed hepatic marker genes and drug-metabolizing enzymes and exhibited drug-metabolizing enzyme activities. These expression levels and activities were increased by treatment with valproic acid, the timing and duration of which were important parameters to promote differentiation from human induced pluripotent stem cell-derived hepatic progenitor cells into hepatocytes. Valproic acid inhibited histone deacetylase activity during differentiation of human induced pluripotent stem cells, and other histone deacetylase inhibitors also enhanced differentiation into hepatocytes. In conclusion, histone deacetylase inhibitors such as valproic acid can be used to promote hepatic differentiation from human induced pluripotent stem cell-derived hepatic progenitor cells. PMID:25084468

  1. Novel corrosion inhibitor technology

    SciTech Connect

    Van de Ven, P.; Fritz, P.; Pellet, R.

    1999-11-01

    A novel, patented corrosion inhibitor technology has been identified for use in heat transfer applications such as automotive and heavy-duty coolant. The new technology is based on a low-toxic, virtually depletion-free carboxylic acid corrosion inhibitor package that performs equally well in mono ethylene glycol and in less toxic propylene glycol coolants. An aqueous inhibitor concentrate is available to provide corrosion protection where freezing protection is not an issue. In the present paper, this inhibitor package is evaluated in the different base fluids: mono ethylene glycol, mono propylene glycol and water. Results are obtained in both standardized and specific corrosion tests as well as in selected field trials. These results indicate that the inhibitor package remains effective and retains the benefits previously identified in automotive engine coolant applications: excellent corrosion protection under localized conditions, general corrosion conditions as well as at high temperature.

  2. Active ingredients in Chinese medicines promoting blood circulation as Na+/K+-ATPase inhibitors

    PubMed Central

    Chen, Ronald JY; Jinn, Tzyy-rong; Chen, Yi-ching; Chung, Tse-yu; Yang, Wei-hung; Tzen, Jason TC

    2011-01-01

    The positive inotropic effect of cardiac glycosides lies in their reversible inhibition on the membrane-bound Na+/K+-ATPase in human myocardium. Steroid-like compounds containing a core structure similar to cardiac glycosides are found in many Chinese medicines conventionally used for promoting blood circulation. Some of them are demonstrated to be Na+/K+-ATPase inhibitors and thus putatively responsible for their therapeutic effects via the same molecular mechanism as cardiac glycosides. On the other hand, magnesium lithospermate B of danshen is also proposed to exert its cardiac therapeutic effect by effectively inhibiting Na+/K+-ATPase. Theoretical modeling suggests that the number of hydrogen bonds and the strength of hydrophobic interaction between the effective ingredients of various medicines and residues around the binding pocket of Na+/K+-ATPase are crucial for the inhibitory potency of these active ingredients. Ginsenosides, the active ingredients in ginseng and sanqi, substantially inhibit Na+/K+-ATPase when sugar moieties are attached only to the C-3 position of their steroid-like structure, equivalent to the sugar position in cardiac glycosides. Their inhibitory potency is abolished, however, when sugar moieties are linked to C-6 or C-20 position of the steroid nucleus; presumably, these sugar attachments lead to steric hindrance for the entrance of ginsenosides into the binding pocket of Na+/K+-ATPase. Neuroprotective effects of cardiac glycosides, several steroid-like compounds, and magnesium lithospermate B against ischemic stroke have been accordingly observed in a cortical brain slice-based assay model, and cumulative data support that effective inhibitors of Na+/K+-ATPase in the brain could be potential drugs for the treatment of ischemic stroke. PMID:21293466

  3. Active ingredients in Chinese medicines promoting blood circulation as Na+/K+ -ATPase inhibitors.

    PubMed

    Chen, Ronald J Y; Jinn, Tzyy-rong; Chen, Yi-ching; Chung, Tse-yu; Yang, Wei-hung; Tzen, Jason T C

    2011-02-01

    The positive inotropic effect of cardiac glycosides lies in their reversible inhibition on the membrane-bound Na(+)/K(+)-ATPase in human myocardium. Steroid-like compounds containing a core structure similar to cardiac glycosides are found in many Chinese medicines conventionally used for promoting blood circulation. Some of them are demonstrated to be Na(+)/K(+)-ATPase inhibitors and thus putatively responsible for their therapeutic effects via the same molecular mechanism as cardiac glycosides. On the other hand, magnesium lithospermate B of danshen is also proposed to exert its cardiac therapeutic effect by effectively inhibiting Na(+)/K(+)-ATPase. Theoretical modeling suggests that the number of hydrogen bonds and the strength of hydrophobic interaction between the effective ingredients of various medicines and residues around the binding pocket of Na(+)/K(+)-ATPase are crucial for the inhibitory potency of these active ingredients. Ginsenosides, the active ingredients in ginseng and sanqi, substantially inhibit Na(+)/K(+)-ATPase when sugar moieties are attached only to the C-3 position of their steroid-like structure, equivalent to the sugar position in cardiac glycosides. Their inhibitory potency is abolished, however, when sugar moieties are linked to C-6 or C-20 position of the steroid nucleus; presumably, these sugar attachments lead to steric hindrance for the entrance of ginsenosides into the binding pocket of Na(+)/K(+)-ATPase. Neuroprotective effects of cardiac glycosides, several steroid-like compounds, and magnesium lithospermate B against ischemic stroke have been accordingly observed in a cortical brain slice-based assay model, and cumulative data support that effective inhibitors of Na(+)/K(+)-ATPase in the brain could be potential drugs for the treatment of ischemic stroke.

  4. Pathway modulators and inhibitors.

    PubMed

    Smith, John A

    2009-07-01

    Inhibitors of specific cellular pathways are useful for investigating the roles of proteins of unknown function, and for selectively inhibiting a protein in complex pathways to uncover its relationships to other proteins in this and other interacting pathways. This appendix provides links to Web sites that describe cellular processes and pathways along with the various classes of inhibitors, numerous references, downloadable diagrams, and technical tips.

  5. Update on TNF Inhibitors.

    PubMed

    Kerdel, Francisco A

    2016-06-01

    The introduction of tumor necrosis factor (TNF)-α inhibitors dramatically improved the management of psoriasis. Some newer or investigational biologics with different mechanisms of action have demonstrated noninferiority or superiority to etanercept, the first self-injectable anti-TNF-α agent to become available in the United States. Nonetheless, TNF-α inhibitors are likely to remain a mainstay of therapy for many years.

  6. Synthetic inhibitors of elastase.

    PubMed

    Edwards, P D; Bernstein, P R

    1994-03-01

    For more than two decades investigators around the world, in both academic and industrial institutions, have been developing inhibitors of human neutrophil elastase. A number of very elegant and insightful strategies have been reported. In the case of reversible peptidic inhibitors, this has resulted in the identification of some extremely potent compounds with dissociation constants in the 10(-11) M range. This is quite an accomplishment considering that these low molecular-weight inhibitors are only tri- and tetrapeptides. In the case of the heterocyclic-based inhibitors, the challenge of balancing the heterocycle's inherent reactivity and aqueous stability with the stability of the enzyme-inhibitor adduct has been meet by either using a latent, reactive functionality which is only activated within the enzyme, or by incorporating features which selectively obstruct deacylation but have little effect on the enzyme acylation step. The underlying goal of this research has been the identification of agents to treat diseases associated with HNE. Several animal models have been developed for evaluating the in vivo activity of elastase inhibitors, and compounds have been shown to be effective in all of these models by the intravenous, intratrachael or oral routes of administration. However, only a very small percentage of compounds have possessed all the necessary properties, including lack of toxicity, for progression into the clinic. The peptidyl TFMK ICI 200,880 (25-12) has many of the desired characteristics of a drug to treat the diseases associated with HNE: chemical stability, in vitro and in vivo activity, a long duration of action, and adequate metabolic stability. Currently ICI 200,880 is the only low molecular-weight HNE inhibitor known to be undergoing clinical trials, and may be the compound which finally demonstrates the clinical utility of a synthetic HNE inhibitor. PMID:8189835

  7. Crystal Structure of G Protein-coupled Receptor Kinase 5 in Complex with a Rationally Designed Inhibitor.

    PubMed

    Homan, Kristoff T; Waldschmidt, Helen V; Glukhova, Alisa; Cannavo, Alessandro; Song, Jianliang; Cheung, Joseph Y; Koch, Walter J; Larsen, Scott D; Tesmer, John J G

    2015-08-21

    G protein-coupled receptor kinases (GRKs) regulate cell signaling by initiating the desensitization of active G protein-coupled receptors. The two most widely expressed GRKs (GRK2 and GRK5) play a role in cardiovascular disease and thus represent important targets for the development of novel therapeutic drugs. In the course of a GRK2 structure-based drug design campaign, one inhibitor (CCG215022) exhibited nanomolar IC50 values against both GRK2 and GRK5 and good selectivity against other closely related kinases such as GRK1 and PKA. Treatment of murine cardiomyocytes with CCG215022 resulted in significantly increased contractility at 20-fold lower concentrations than paroxetine, an inhibitor with more modest selectivity for GRK2. A 2.4 Å crystal structure of the GRK5·CCG215022 complex was determined and revealed that the inhibitor binds in the active site similarly to its parent compound GSK180736A. As designed, its 2-pyridylmethyl amide side chain occupies the hydrophobic subsite of the active site where it forms three additional hydrogen bonds, including one with the catalytic lysine. The overall conformation of the GRK5 kinase domain is similar to that of a previously determined structure of GRK6 in what is proposed to be its active state, but the C-terminal region of the enzyme adopts a distinct conformation. The kinetic properties of site-directed mutants in this region are consistent with the hypothesis that this novel C-terminal structure is representative of the membrane-bound conformation of the enzyme.

  8. At-GDI1 from Arabidopsis thaliana encodes a rab-specific GDP dissociation inhibitor that complements the sec19 mutation of Saccharomyces cerevisiae.

    PubMed

    Zárský, V; Cvrcková, F; Bischoff, F; Palme, K

    1997-02-24

    Rab GTPases play a central role in the control of vesicular membrane traffic. These proteins cycle between cytosolic and membrane-bound compartments in a guanine nucleotide-dependent manner, a process that is regulated by several accessory proteins. Of particular interest are the Rab guanosine nucleotide diphosphate dissociation inhibitor proteins (Rab-GDI) which bind to prenylated Rab GTPases, slow the rate of GDP dissociation and escort GDP bound Rab proteins to their target membranes and retrieve them after completion of their catalytic cycle. We have cloned from Arabidopsis thaliana a cDNA coding for the Rab guanosine diphosphate dissociation inhibitor (AtGDI1) by functional complementation of the Saccharomyces cerevisiae sec19-1 mutant. The Arabidopsis cDNA potentially encodes a 49850 Da protein which is homologous to yeast GDI (49%) and to other members of the Rab-GDI family (49-63%). Northern blot analysis indicates that the mRNA is expressed in all tissues examined. The existence of a plant homologue of the Rab-GDI family indicates that the basic vesicle traffic control machinery may be highly conserved in plants as it is in yeast and mammals.

  9. Forces and Dynamics of Glucose and Inhibitor Binding to Sodium Glucose Co-transporter SGLT1 Studied by Single Molecule Force Spectroscopy*

    PubMed Central

    Neundlinger, Isabel; Puntheeranurak, Theeraporn; Wildling, Linda; Rankl, Christian; Wang, Lai-Xi; Gruber, Hermann J.; Kinne, Rolf K. H.; Hinterdorfer, Peter

    2014-01-01

    Single molecule force spectroscopy was employed to investigate the dynamics of the sodium glucose co-transporter (SGLT1) upon substrate and inhibitor binding on the single molecule level. CHO cells stably expressing rbSGLT1 were probed by using atomic force microscopy tips carrying either thioglucose, 2′-aminoethyl β-d-glucopyranoside, or aminophlorizin. Poly(ethylene glycol) (PEG) chains of different length and varying end groups were used as tether. Experiments were performed at 10, 25 and 37 °C to address different conformational states of SGLT1. Unbinding forces between ligands and SGLT1 were recorded at different loading rates by changing the retraction velocity, yielding binding probability, width of energy barrier of the binding pocket, and the kinetic off rate constant of the binding reaction. With increasing temperature, width of energy barrier and average life time increased for the interaction of SGLT1 with thioglucose (coupled via acrylamide to a long PEG) but decreased for aminophlorizin binding. The former indicates that in the membrane-bound SGLT1 the pathway to sugar translocation involves several steps with different temperature sensitivity. The latter suggests that also the aglucon binding sites for transport inhibitors have specific, temperature-sensitive conformations. PMID:24962566

  10. Complement inhibitor C4b-binding protein-friend or foe in the innate immune system?

    PubMed

    Blom, Anna M; Villoutreix, Bruno O; Dahlbäck, Björn

    2004-04-01

    The complement system constitutes an important component of the defence against foreign organisms, functioning both in innate and adaptive immune systems. It is potentially harmful also to the own organism and is therefore tightly regulated by a number of membrane-bound and soluble factors. C4b-binding protein (C4BP) is a potent circulating soluble inhibitor of the classical and lectin pathways of complement. In recent years, the relationships between the structure of C4BP and its functions have been elucidated using a combination of computer-based molecular analysis and recombinant DNA technologies. Moreover, two novel functions have recently been ascribed to C4BP. One is the ability of C4BP to localize complement regulatory activity to the surface of apoptotic cells via its interaction with the membrane-binding vitamin K-dependent protein S. The other is the ability of C4BP to act as a survival factor for B cells due to an interaction with CD40. The complement regulatory activity of C4BP is not only beneficial because it is also explored by pathogens such as Neisseria gonorrhoeae, Bordetella pertussis, Streptococcus pyogenes, Escherichia coli K1, and Candida albicans, that bind C4BP to their surfaces. This contributes to the serum resistance and the pathogenicity of these bacteria. In this review, the structural requirements and functional importance of the interactions between C4BP and its various ligands are discussed.

  11. Role of the complement system in rheumatoid arthritis and psoriatic arthritis: relationship with anti-TNF inhibitors.

    PubMed

    Ballanti, Eleonora; Perricone, Carlo; di Muzio, Gioia; Kroegler, Barbara; Chimenti, Maria Sole; Graceffa, Dario; Perricone, Roberto

    2011-08-01

    The complement system is an essential component of innate immunity and also plays an important role in modulating adaptive immunity. It comprises more than 30 plasma and membrane-bound proteins and can be activated through three pathways: the classical, the alternative and the lectin pathways. Its activation contributes to the pathogenesis of several autoimmune and inflammatory conditions. The evidence of complement activation in synovial fluid of Rheumatoid Arthritis (RA) patients is abundant, while few data exist in Psoriatic Arthritis (PsA) patients. Levels of complement proteins are generally depressed in the synovial fluid of patients with RA, reflecting consumption of complement. On the other hand, elevated levels of several complement cleavage products have been observed in synovial fluid. Involvement of complement in the pathogenesis of RA was also confirmed in animal models of arthritis: mice deficient for complement proteins are protected against the development of collagen-induced arthritis and administration of the anti-C5 monoclonal antibody prevents the onset of this arthritis. In the last decade anti-tumor necrosis factor agents have shown to be effective for the treatment of both RA and PsA and some studies suggest that the interaction between TNFα and complement system may contribute to the pathogenesis of these diseases. Reduction of the complement activation could be one of the mechanism by which TNFα-inhibitors exert their effectiveness in inflammatory arthritides. Because of these findings, complement could be an attractive therapeutic target both in RA and in PsA.

  12. Small-molecule caspase inhibitors

    NASA Astrophysics Data System (ADS)

    Zhenodarova, S. M.

    2010-02-01

    The review considers low-molecular weight inhibitors of caspases, cysteine proteases being key contributors to apoptosis (programmed cell death). The inhibitors with aspartic acid residues or various heterocyclic systems (both synthetic and natural) are covered. Their possible mechanisms of action are discussed. Data on inhibitor structure-activity relationship studies are systematically surveyed. The interactions of the non-peptide fragments of an inhibitor with the enzymes are examined. Examples of the use of some inhibitors for apoptosis suppression are provided.

  13. The novel complement inhibitor human CUB and Sushi multiple domains 1 (CSMD1) protein promotes factor I-mediated degradation of C4b and C3b and inhibits the membrane attack complex assembly.

    PubMed

    Escudero-Esparza, Astrid; Kalchishkova, Nikolina; Kurbasic, Emila; Jiang, Wen G; Blom, Anna M

    2013-12-01

    CUB and Sushi multiple domains 1 (CSMD1) is a transmembrane protein containing 15 consecutive complement control protein (CCP) domains, which are characteristic for complement inhibitors. We expressed a membrane-bound fragment of human CSMD1 composed of the 15 C-terminal CCP domains and demonstrated that it inhibits deposition of C3b by the classical pathway on the surface of Chinese hamster ovary cells by 70% at 6% serum and of C9 (component of membrane attack complex) by 90% at 1.25% serum. Furthermore, this fragment of CSMD1 served as a cofactor to factor I-mediated degradation of C3b. In all functional assays performed, well-characterized complement inhibitors were used as positive controls, whereas Coxsackie adenovirus receptor, a protein with no effect on complement, was a negative control. Moreover, attenuation of expression in human T47 breast cancer cells that express endogenous CSMD1 significantly increased C3b deposition on these cells by 45% at 8% serum compared with that for the controls. Furthermore, by expressing a soluble 17-21 CCP fragment of CSMD1, we found that CSMD1 inhibits complement by promoting factor I-mediated C4b/C3b degradation and inhibition of MAC assembly at the level of C7. Our results revealed a novel complement inhibitor for the classical and lectin pathways.

  14. The novel complement inhibitor human CUB and Sushi multiple domains 1 (CSMD1) protein promotes factor I-mediated degradation of C4b and C3b and inhibits the membrane attack complex assembly.

    PubMed

    Escudero-Esparza, Astrid; Kalchishkova, Nikolina; Kurbasic, Emila; Jiang, Wen G; Blom, Anna M

    2013-12-01

    CUB and Sushi multiple domains 1 (CSMD1) is a transmembrane protein containing 15 consecutive complement control protein (CCP) domains, which are characteristic for complement inhibitors. We expressed a membrane-bound fragment of human CSMD1 composed of the 15 C-terminal CCP domains and demonstrated that it inhibits deposition of C3b by the classical pathway on the surface of Chinese hamster ovary cells by 70% at 6% serum and of C9 (component of membrane attack complex) by 90% at 1.25% serum. Furthermore, this fragment of CSMD1 served as a cofactor to factor I-mediated degradation of C3b. In all functional assays performed, well-characterized complement inhibitors were used as positive controls, whereas Coxsackie adenovirus receptor, a protein with no effect on complement, was a negative control. Moreover, attenuation of expression in human T47 breast cancer cells that express endogenous CSMD1 significantly increased C3b deposition on these cells by 45% at 8% serum compared with that for the controls. Furthermore, by expressing a soluble 17-21 CCP fragment of CSMD1, we found that CSMD1 inhibits complement by promoting factor I-mediated C4b/C3b degradation and inhibition of MAC assembly at the level of C7. Our results revealed a novel complement inhibitor for the classical and lectin pathways. PMID:23964079

  15. Natural inhibitors of thrombin.

    PubMed

    Huntington, James A

    2014-04-01

    The serine protease thrombin is the effector enzyme of blood coagulation. It has many activities critical for the formation of stable clots, including cleavage of fibrinogen to fibrin, activation of platelets and conversion of procofactors to active cofactors. Thrombin carries-out its multiple functions by utilising three special features: a deep active site cleft and two anion binding exosites (exosite I and II). Similarly, thrombin inhibitors have evolved to exploit the unique features of thrombin to achieve rapid and specific inactivation of thrombin. Exogenous thrombin inhibitors come from several different protein families and are generally found in the saliva of haematophagous animals (blood suckers) as part of an anticoagulant cocktail that allows them to feed. Crystal structures of several of these inhibitors reveal how peptides and proteins can be targeted to thrombin in different and interesting ways. Thrombin activity must also be regulated by endogenous inhibitors so that thrombi do not occlude blood flow and cause thrombosis. A single protein family, the serpins, provides all four of the endogenous thrombin inhibitors found in man. The crystal structures of these serpins bound to thrombin have been solved, revealing a similar exosite-dependence on complex formation. In addition to forming the recognition complex, serpins destroy the structure of thrombin, allowing them to be released from cofactors and substrates for clearance. This review examines how the special features of thrombin have been exploited by evolution to achieve inhibition of the ultimate coagulation protease.

  16. SGLT2 inhibitors.

    PubMed

    Dardi, I; Kouvatsos, T; Jabbour, S A

    2016-02-01

    Diabetes mellitus is a serious health issue and an economic burden, rising in epidemic proportions over the last few decades worldwide. Although several treatment options are available, only half of the global diabetic population achieves the recommended or individualized glycemic targets. Sodium-glucose cotransporter 2 (SGLT2) inhibitors are a new class of antidiabetic agents with a novel insulin-independent action. SGLT2 is a transporter found in the proximal renal tubules, responsible for the reabsorption of most of the glucose filtered by the kidney. Inhibition of SGLT2 lowers the blood glucose level by promoting the urinary excretion of excess glucose. Due to their insulin-independent action, SGLT2 inhibitors can be used with any degree of beta-cell dysfunction or insulin resistance, related to a very low risk of hypoglycemia. In addition to improving glycemic control, SGLT2 inhibitors have been associated with a reduction in weight and blood pressure when used as monotherapy or in combination with other antidiabetic agents in patients with type 2 diabetes mellitus (T2DM). Treatment with SGLT2 inhibitors is usually well tolerated; however, they have been associated with an increased incidence of urinary tract and genital infections, although these infections are usually mild and easy to treat. SGLT2 inhibitors are a promising new option in the armamentarium of drugs for patients with T2DM. PMID:26362302

  17. Cholinesterase inhibitors from botanicals

    PubMed Central

    Ahmed, Faiyaz; Ghalib, Raza Murad; Sasikala, P.; Ahmed, K. K. Mueen

    2013-01-01

    Alzheimer's disease (AD) is a progressive neurodegenerative disease, wherein a progressive loss of cholinergic synapses occurs in hippocampus and neocortex. Decreased concentration of the neurotransmitter, acetylcholine (ACh), appears to be critical element in the development of dementia, and the most appropriate therapeutic approach to treat AD and other form of dementia is to restore acetylcholine levels by inhibiting both major form of cholinesterase: Acetylcholinesterase (AChE) and butyrylcholinesterase (BChE). Consequently, researches have focused their attention towards finding cholinesterase inhibitors from natural products. A large number of such inhibitors have been isolated from medicinal plants. This review presents a comprehensive account of the advances in field of cholinesterase inhibitor phytoconstituents. The structures of some important phytoconstituents (collected through www.Chemspider.com) are also presented and the scope for future research is discussed. PMID:24347920

  18. Cholinesterase inhibitors from botanicals.

    PubMed

    Ahmed, Faiyaz; Ghalib, Raza Murad; Sasikala, P; Ahmed, K K Mueen

    2013-07-01

    Alzheimer's disease (AD) is a progressive neurodegenerative disease, wherein a progressive loss of cholinergic synapses occurs in hippocampus and neocortex. Decreased concentration of the neurotransmitter, acetylcholine (ACh), appears to be critical element in the development of dementia, and the most appropriate therapeutic approach to treat AD and other form of dementia is to restore acetylcholine levels by inhibiting both major form of cholinesterase: Acetylcholinesterase (AChE) and butyrylcholinesterase (BChE). Consequently, researches have focused their attention towards finding cholinesterase inhibitors from natural products. A large number of such inhibitors have been isolated from medicinal plants. This review presents a comprehensive account of the advances in field of cholinesterase inhibitor phytoconstituents. The structures of some important phytoconstituents (collected through www.Chemspider.com) are also presented and the scope for future research is discussed. PMID:24347920

  19. Phosphodiesterase-5 inhibitors.

    PubMed

    Cockrill, Barbara A; Waxman, Aaron B

    2013-01-01

    Nitric oxide (NO) signaling plays a key role in modulating vascular tone and remodeling in the pulmonary circulation. The guanylate cyclase/cyclic guanylate monophosphate-signaling pathway primarily mediates nitric oxide signaling. This pathway is critical in normal regulation of the pulmonary vasculature, and is an important target for therapy in patients with pulmonary hypertension. In the pulmonary vasculature, degradation of cGMP is primarily regulated by PDE-5, and inhibition of this enzyme has important effects on pulmonary vasculature smooth muscle tone. Large randomized placebo-controlled trials of PDE-5 inhibitors demonstrated improved exercise capacity, hemodynamics and quality of life in adult patients with PAH. This chapter will discuss the mechanisms of NO signaling in the vasculature, characteristics of the PDE5-inhibitors approved for treatment of PH, and review available data on the use of phosphodiesterase inhibitors in PH. PMID:24092343

  20. TGF-β/BAMBI pathway dysfunction contributes to peripheral Th17/Treg imbalance in chronic obstructive pulmonary disease

    PubMed Central

    Zhang, Jian-Chu; Chen, Gang; Chen, Long; Meng, Zhao-Ji; Xiong, Xian-Zhi; Liu, Hong-Ju; Jin, Yang; Tao, Xiao-Nan; Wu, Jiang-Hua; Sun, Sheng-Wen

    2016-01-01

    BMP and activin membrane-bound inhibitor (BAMBI) is postulated to inhibit or modulate transforming growth factor β (TGF-β) signaling. Furthermore, strong upregulation of BAMBI expression following in vitro infection of chronic obstructive pulmonary disease (COPD) lung tissue has been demonstrated. In this study, we investigated whether TGF-β/BAMBI pathway is associated with COPD. Blood samples were obtained from 27 healthy controls (HC), 24 healthy smokers (HS) and 29 COPD patients. Elevated Th17/Treg ratios, and increased levels of BAMBI protein and mRNA (in plasma and CD4+ T cells respectively), were observed in COPD compared with HC and HS. BAMBI expression was first observed on human CD4+ T cells, with a typical membrane-bound pattern. The enhanced plasma BAMBI levels in COPD positively correlated with the increased plasma TGF-β1 levels and Th17/Treg ratio. Together, an impaired TGF-β/BAMBI pathway may promote the inflammation leading to Th17/Treg imbalance, which is a new mechanism in smokers who develop COPD. PMID:27549738

  1. The cytoskeletal inhibitors latrunculin A and blebbistatin exert antitumorigenic properties in human hepatocellular carcinoma cells by interfering with intracellular HuR trafficking.

    PubMed

    Doller, Anke; Badawi, Amel; Schmid, Tobias; Brauss, Thilo; Pleli, Thomas; zu Heringdorf, Dagmar Meyer; Piiper, Albrecht; Pfeilschifter, Josef; Eberhardt, Wolfgang

    2015-01-01

    The impact of the RNA-binding protein HuR for the post-transcriptional deregulation of tumor-relevant genes is well established. Despite of elevations in HuR expression levels, an increase in cytoplasmic HuR abundance in many cases correlates with a high grade of malignancy. Here, we demonstrated that administration of the actin-depolymerizing macrolide latrunculin A, or blebbistatin, an inhibitor of myosin II ATPase activity, caused a dose- and time-dependent reduction in the high cytoplasmic HuR content of HepG2 and Huh7 hepatocellular carcinoma (HCC) cells. Subcellular fractionation revealed that in addition, both inhibitors strongly attenuated cytoskeletal and membrane-bound HuR abundance and conversely increased the HuR amount in nuclear cell fractions. Concomitant with changes in intracellular HuR localization, both cytoskeletal inhibitors markedly decreased the half-lives of cyclooxygenase-2 (COX-2), cyclin A and cyclin D1 encoding mRNAs resulting in a significant reduction in their expression levels in HepG2 cells. Importantly, a similar reduction in the expression of these HuR targets was achieved by a RNA interference (RNAi)-mediated knockdown of either HuR or nonmuscle myoin IIA. Using polysomal fractionation, we further demonstrate that the decrease in cytoplasmic HuR by latrunculin A or blebbistatin is accompanied by a marked change in the allocation of HuR and its mRNA cargo from polysomes to ribonucleoprotein (RNP) particles. Functionally, the basal migration and prostaglandin E2 synthesis are similarly impaired in inhibitor-treated and stable HuR-knockdown HepG2 cells. Our data demonstrate that interfering with the actomyosin-dependent HuR trafficking may comprise a valid therapeutic option for antagonizing pathologic posttranscriptional gene expression by HuR and furthermore emphasize the potential benefit of HuR inhibitory strategies for treatment of HCC.

  2. Metabolic Targeting of Malignant Tumors: Small-Molecule Inhibitors of Bioenergetic Flux

    PubMed Central

    Mathupala, Saroj P.

    2012-01-01

    Metabolism in tumors deviates significantly from that of normal tissues. Increasingly, the underlying aberrant metabolic pathways are being considered as novel targets for cancer therapy. Denoted “metabolic targeting”, small molecule drugs are under investigation for focused inhibition of key metabolic steps that are utilized by tumors, since such inhibitors should harbor minimal toxicity towards surrounding normal tissues.This review will examine the primary biochemical pathways that tumors harness to enhance their bioenergetic capacity, which in turn, help their rapid proliferation and metastasis within the host. It is hoped that “metabolite-mimetic” drugs can be utilized to interfere with metabolic flux pathways active within the tumor, and across tumor-microenvironment boundary. In fact, the major pathways of mammalian metabolism, i.e., the carbohydrate, amino-acid, and fatty-acid metabolic pathways have been examined as putative targets for drug development, with some drug candidates advancing to phase II/III stages. In this regard, glucose metabolism, i.e., the glycolytic pathway - that predominates the bio-energetic flux in tumors, and the associated mitochondrial metabolism have received the most attention as suitable “druggable” targets, focused either at the pathway enzymes or at the plasma-membrane-bound metabolite transporters. Outlined in this review are pre-clinical studies that have led to the discovery of promising drug candidates to target tumor-metabolic flux, and ensuing patents, with descriptions of the biochemical rationale for the combinatorial strategy of a particular metabolic pathway-drug candidate pair. PMID:21110820

  3. Thiazole derivatives as inhibitors of cyclooxygenases in vitro and in vivo.

    PubMed

    El-Achkar, Ghewa A; Jouni, Mariam; Mrad, May F; Hirz, Taghreed; El Hachem, Nehme; Khalaf, Ali; Hammoud, Soukaina; Fayyad-Kazan, Hussein; Eid, Assaad A; Badran, Bassam; Merhi, Raghida Abou; Hachem, Ali; Hamade, Eva; Habib, Aïda

    2015-03-01

    Cyclooxygenases (COXs) are important membrane-bound heme containing enzymes important in platelet activation and inflammation. COX-1 is constitutively expressed in most cells whereas COX-2 is an inducible isoform highly expressed in inflammatory conditions. Studies have been carried out to evaluate thiazole derivatives as anti-inflammatory molecules. In this study, we investigated the in vitro and in vivo effects of two novel thiazole derivatives compound 1 (N-[4-(4-hydroxy-3-methoxyphenyl)-1,3-thiazol-2-yl] acetamide) and compound 2 (4-(2-amino-1,3-thiazol-4-yl)-2-methoxyphenol) on prostaglandin E2 (PGE2) production and COX activity in inflammatory settings. Our results reveal a potent inhibition of both compound 1 (IC50 9.01±0.01µM) and 2 (IC50 11.65±6.20µM) (Mean±S.E.M.) on COX-2-dependent PGE2 production. We also determined whether COX-1 activity was inhibited. Using cells stably over-expressing COX-1 and human blood platelets, we showed that compound 1 is a specific inhibitor of COX-1 with IC50 (5.56×10(-8)±2.26×10(-8)µM), whereas compound 2 did not affect COX-1. Both compounds exhibit anti-inflammatory effect in the dorsal air pouch model of inflammation as shows by inhibition of PGE2 secretion. Modeling analysis of docking in the catalytic site of COX-1 or COX-2 further confirmed the difference in the effect of these two compounds. In conclusion, this study contributes to the design of new anti-inflammatory agents and to the understanding of cyclooxygenase inhibition by thiazole. PMID:25617797

  4. Hemojuvelin Modulates Iron Stress During Acute Kidney Injury: Improved by Furin Inhibitor

    PubMed Central

    Young, Guang-Huar; Huang, Tao-Min; Wu, Che-Hsiung; Lai, Chun-Fu; Hou, Chun-Cheng; Peng, Kang-Yung; Liang, Chan-Jung; Lin, Shuei-Liong; Chang, Shih-Chung; Tsai, Pi-Ru; Wu, Kwan-Dun

    2014-01-01

    Abstract Aims: Free iron plays an important role in the pathogenesis of acute kidney injury (AKI) via the formation of hydroxyl radicals. Systemic iron homeostasis is controlled by the hemojuvelin-hepcidin-ferroportin axis in the liver, but less is known about this role in AKI. Results: By proteomics, we identified a 42 kDa soluble hemojuvelin (sHJV), processed by furin protease from membrane-bound hemojuvelin (mHJV), in the urine during AKI after cardiac surgery. Biopsies from human and mouse specimens with AKI confirm that HJV is extensively increased in renal tubules. Iron overload enhanced the expression of hemojuvelin-hepcidin signaling pathway. The furin inhibitor (FI) decreases furin-mediated proteolytic cleavage of mHJV into sHJV and augments the mHJV/sHJV ratio after iron overload with hypoxia condition. The FI could reduce renal tubule apoptosis, stabilize hypoxic induced factor-1, prevent the accumulation of iron in the kidney, and further ameliorate ischemic-reperfusion injury. mHJV is associated with decreasing total kidney iron, secreting hepcidin, and promoting the degradation of ferroportin at AKI, whereas sHJV does the opposite. Innovation: This study suggests the ratio of mHJV/sHJV affects the iron deposition during acute kidney injury and sHJV could be an early biomarker of AKI. Conclusion: Our findings link endogenous HJV inextricably with renal iron homeostasis for the first time, add new significance to early predict AKI, and identify novel therapeutic targets to reduce the severity of AKI using the FI. Antioxid. Redox Signal. 20, 1181–1194. PMID:23901875

  5. Pectin methylesterase inhibitor.

    PubMed

    Giovane, A; Servillo, L; Balestrieri, C; Raiola, A; D'Avino, R; Tamburrini, M; Ciardiello, M A; Camardella, L

    2004-02-12

    Pectin methylesterase (PME) is the first enzyme acting on pectin, a major component of plant cell wall. PME action produces pectin with different structural and functional properties, having an important role in plant physiology. Regulation of plant PME activity is obtained by the differential expression of several isoforms in different tissues and developmental stages and by subtle modifications of cell wall local pH. Inhibitory activities from various plant sources have also been reported. A proteinaceous inhibitor of PME (PMEI) has been purified from kiwi fruit. The kiwi PMEI is active against plant PMEs, forming a 1:1 non-covalent complex. The polypeptide chain comprises 152 amino acid residues and contains five Cys residues, four of which are connected by disulfide bridges, first to second and third to fourth. The sequence shows significant similarity with the N-terminal pro-peptides of plant PME, and with plant invertase inhibitors. In particular, the four Cys residues involved in disulfide bridges are conserved. On the basis of amino acid sequence similarity and Cys residues conservation, a large protein family including PMEI, invertase inhibitors and related proteins of unknown function has been identified. The presence of at least two sequences in the Arabidopsis genome having high similarity with kiwi PMEI suggests the ubiquitous presence of this inhibitor. PMEI has an interest in food industry as inhibitor of endogenous PME, responsible for phase separation and cloud loss in fruit juice manufacturing. Affinity chromatography on resin-bound PMEI can also be used to concentrate and detect residual PME activity in fruit and vegetable products.

  6. Acyclic peptide inhibitors of amylases.

    PubMed

    Pohl, Nicola

    2005-12-01

    In this issue of Chemistry and Biology, a library screening approach reveals a linear octapeptide inhibitor of alpha-amylases reached by de novo design . The selected molecule shares characteristics with naturally occurring protein inhibitors -- a result that suggests general rules for the design of peptide-based amylase inhibitors may be achievable.

  7. [JAK2 inhibitors].

    PubMed

    Hernández Boluda, Juan Carlos; Gómez, Montse; Pérez, Ariadna

    2016-07-15

    Pharmacological inhibition of the kinase activity of JAK proteins can interfere with the signaling of immunomodulatory cytokines and block the constitutive activation of the JAK-STAT pathway that characterizes certain malignancies, including chronic myeloproliferative neoplasms. JAK inhibitors may, therefore, be useful to treat malignancies as well as inflammatory or immune disorders. Currently, the most significant advances have been made in the treatment of myelofibrosis, where these drugs may lead to a remarkable improvement in the control of hyperproliferative manifestations. However, available data suggest that this treatment is not curative of myelofibrosis. In general, JAK2 inhibition induces cytopaenias, with this being considered a class side-effect. By contrast, the extrahaematologic toxicity profile varies significantly among the different JAK inhibitors. At present, there are several clinical trials evaluating the combination of ruxolitinib with other drugs, in order to improve its therapeutic activity as well as reducing haematologic toxicity. PMID:27033437

  8. Coagulation inhibitors in inflammation.

    PubMed

    Esmon, C T

    2005-04-01

    Coagulation is triggered by inflammatory mediators in a number of ways. However, to prevent unwanted clot formation, several natural anticoagulant mechanisms exist, such as the antithrombin-heparin mechanism, the tissue factor pathway inhibitor mechanism and the protein C anticoagulant pathway. This review examines the ways in which these pathways are down-regulated by inflammation, thus limiting clot formation and decreasing the natural anti-inflammatory mechanisms that these pathways possess. PMID:15787615

  9. Neutrophil Elastase Inhibitors

    PubMed Central

    Groutas, William C.; Dou, Dengfeng; Alliston, Kevin R.

    2011-01-01

    Introduction Chronic obstructive pulmonary disease (COPD) constitutes a worldwide health problem. There is currently an urgent and unmet need for the development of small molecule therapeutics capable of blocking and/or reversing the progression of the disorder. Recent studies have greatly illuminated our understanding of the multiple pathogenic processes associated with COPD. Of paramount importance is the key role played by proteases, oxidative stress, apoptosis, and inflammation. Insights gained from these studies have made possible the exploration of new therapeutic approaches. Areas covered An overview of major developments in COPD research with emphasis on low molecular weight neutrophil elastase inhibitors is described in this review. Expert opinion Great strides have been made toward our understanding of the biochemical and cellular events associated with COPD. However, our knowledge regarding the inter-relationships among the multiple pathogenic mechanisms and their mediators involved is till limited. The problem is further compounded by the unavailability of suitable validated biomarkers for assessing the efficacy of potential therapeutic interventions. The complexity of COPD suggests that effective therapeutic interventions may require the administration of more than one agent such as, for instance, an HNE or MMP-12 inhibitor with an anti-inflammatory agent such as a phosphodiesterase-4 inhibitor, or a dual function agent capable of disrupting the cycle of proteolysis, apoptosis, inflammation and oxidative stress PMID:21235378

  10. mTOR kinase inhibitor pp242 causes mitophagy terminated by apoptotic cell death in E1A-Ras transformed cells

    PubMed Central

    Gordeev, Serguei A.; Bykova, Tatiana V.; Zubova, Svetlana G.; Bystrova, Olga A.; Martynova, Marina G.; Pospelov, Valery A.; Pospelova, Tatiana V.

    2015-01-01

    mTOR is a critical target for controlling cell cycle progression, senescence and cell death in mammalian cancer cells. Here we studied the role of mTOR-dependent autophagy in implementating the antiprolifrative effect of mTORC1-specific inhibitor rapamycin and ATP-competitive mTOR kinase inhibitor pp242. We carried out a comprehensive analysis of pp242- and rapamycin-induced autophagy in ERas tumor cells. Rapamycin exerts cytostatic effect on ERas tumor cells, thus causing a temporary and reversible cell cycle arrest, activation of non-selective autophagy not accompanied by cell death. The rapamycin-treated cells are able to continue proliferation after drug removal. The ATP-competitive mTORC1/mTORC2 kinase inhibitor pp242 is highly cytotoxic by suppressing the function of mTORC1-4EBP1 axis and mTORC1-dependent phosphorylation of mTORC1 target - ULK1-Ser757 (Atg1). In contrast to rapamycin, pp242 activates the selective autophagy targeting mitochondria (mitophagy). The pp242-induced mitophagy is accompanied by accumulation of LC3 and conversion of LC3-I form to LC3-II. However reduced degradation of p62/SQSTM indicates abnormal flux of autophagic process. According to transmission electron microscopy data, short-term pp242-treated ERas cells exhibit numerous heavily damaged mitochondria, which are included in single membrane-bound autophagic/autolysophagic vacuoles (mitophagy). Despite the lack of typical for apoptosis features, ERas-treated cells with induced mitophagy revealed the activation of caspase 3, 9 and nucleosomal DNA fragmentation. Thus, pp242 activates autophagy with suppressed later stages, leading to impaired recycling and accumulation of dysfunctional mitochondria and cell death. Better understanding of how autophagy determines the fate of a cell - survival or cell death, can help to development of new strategy for cancer therapy. PMID:26636543

  11. A sulfated polysaccharide from the sarcoplasmic reticulum of sea cucumber smooth muscle is an endogenous inhibitor of the Ca(2+)-ATPase.

    PubMed

    Landeira-Fernandez, A M; Aiello, K R; Aquino, R S; Silva, L C; Meis, L d; Mourão, P A

    2000-08-01

    Vesicles derived from the endoplasmic reticulum of sea cucumber smooth muscle retain a membrane bound Ca(2+)-ATPase that is able to transport Ca(2+) into the vesicles at the expense of ATP hydrolysis. In contrast with vesicles obtained from rabbit muscles, the activity of the Ca(2+)-dependent ATPase from sea cucumber is dependent on monovalent cations (K(+)>Na(+)>Li(+)). With the addition of highly sulfated polysaccharide to vesicle preparations from rabbit muscle, Ca(2+) uptake decreases sharply and becomes highly sensitive to monovalent cations, as observed with vesicles from sea cucumber muscle. These results led us to investigate the possible occurrence of a highly sulfated polysaccharide on vesicles from the endoplasmic reticulum of sea cucumber smooth muscle, acting as an "endogenous" Ca(2+)-ATPase inhibitor. In fact, vesicles derived from the invertebrate, but not from rabbit muscle, contain a highly sulfated polysaccharide. This compound inhibits Ca(2+) uptake in vesicles obtained from rabbit muscle and the inhibition is antagonized by monovalent cation. In addition, sea cucumber muscles contain high concentrations of another polysaccharide, which surrounds the muscle fibers, and was characterized as a fucosylated chondroitin sulfate. Possibly the occurrence of sulfated polysaccharides in the sea cucumber muscles is related with unique properties of the invertebrate body wall, which can rapidly and reversibly alter its mechanical properties, with change in length by more than 200%.

  12. Protein farnesyltransferase inhibitors.

    PubMed

    Ayral-Kaloustian, Semiramis; Salaski, Edward J

    2002-05-01

    Specific mutations in the ras gene impair the guanosine triphophatase (GTPase) activity of Ras proteins, which play a fundamental role in the signaling cascade, leading to uninterrupted growth signals and to the transformation of normal cells into malignant phenotypes. It has been shown that normal cells transfected with mutant ras gene become cancerous and that unfarnesylated, cytosolic mutant Ras protein does not anchor onto cell membranes and cannot induce this transformation. Posttranslational modification and plasma membrane association of mutant Ras is necessary for this transforming activity. Since its identification, the enzyme protein farnesyltransferase (FTase) that catalyzes the first and essential step of the three Ras-processing steps has emerged as the most promising target for therapeutic intervention. FTase has been implicated as a potential target in inhibiting the prenylation of a variety of proteins, thus in controlling varied disease states (e.g. cancer, neurofibromatosis, restenosis, viral hepatitis, bone resorption, parasitic infections, corneal inflammations, and diabetes) associated with prenyl modifications of Ras and other proteins. Furthermore, it has been suggested that FTase inhibitors indirectly help in inhibiting tumors via suppression of angiogenesis and induction of apoptosis. Major milestones have been achieved with small-molecule FTase inhibitors that show efficacy without toxicity in vitro, as well as in mouse models bearing ras-dependent tumors. With the determination of the crystal structure of mammalian FTase, existent leads have been fine-tuned and new potent molecules of diverse structural classes have been designed. A few of these molecules are currently in the clinic, with at least three drug candidates in Phase II studies and one in Phase III. This article will review the progress that has been reported with FTase inhibitors in drug discovery and in the clinic. PMID:12733981

  13. Physiology, biochemistry, and specific inhibitors of CH4, NH4+, and CO oxidation by methanotrophs and nitrifiers.

    PubMed Central

    Bédard, C; Knowles, R

    1989-01-01

    Ammonia oxidizers (family Nitrobacteraceae) and methanotrophs (family Methylococcaceae) oxidize CO and CH4 to CO2 and NH4+ to NO2-. However, the relative contributions of the two groups of organisms to the metabolism of CO, CH4, and NH4+ in various environments are not known. In the ammonia oxidizers, ammonia monooxygenase, the enzyme responsible for the conversion of NH4+ to NH2OH, also catalyzes the oxidation of CH4 to CH3OH. Ammonia monooxygenase also mediates the transformation of CH3OH to CO2 and cell carbon, but the pathway by which this is done is not known. At least one species of ammonia oxidizer, Nitrosococcus oceanus, exhibits a Km for CH4 oxidation similar to that of methanotrophs. However, the highest rate of CH4 oxidation recorded in an ammonia oxidizer is still five times lower than rates in methanotrophs, and ammonia oxidizers are apparently unable to grow on CH4. Methanotrophs oxidize NH4+ to NH2OH via methane monooxygenase and NH4+ to NH2OH via methane monooxygenase and NH2OH to NO2- via an NH2OH oxidase which may resemble the enzyme found in ammonia oxidizers. Maximum rates of NH4+ oxidation are considerably lower than in ammonia oxidizers, and the affinity for NH4+ is generally lower than in ammonia oxidizers. NH4+ does not apparently support growth in methanotrophs. Both ammonia monooxygenase and methane monooxygenase oxidize CO to CO2, but CO cannot support growth in either ammonia oxidizers or methanotrophs. These organisms have affinities for CO which are comparable to those for their growth substrates and often higher than those in carboxydobacteria. The methane monooxygenases of methanotrophs exist in two forms: a soluble form and a particulate form. The soluble form is well characterized and appears unrelated to the particulate. Ammonia monooxygenase and the particulate methane monooxygenase share a number of similarities. Both enzymes contain copper and are membrane bound. They oxidize a variety of inorganic and organic compounds, and

  14. Synthesis of Lysine Methyltransferase Inhibitors

    NASA Astrophysics Data System (ADS)

    Ye, Tao; Hui, Chunngai

    2015-07-01

    Lysine methyltransferase which catalyze methylation of histone and nonhistone proteins, play a crucial role in diverse biological processes and has emerged as a promising target for the development of various human diseases, including cancer, inflammation, and psychiatric disorders. However, inhibiting Lysine methyltransferases selectively has presented many challenges to medicinal chemists. During the past decade, lysine methyltransferase inhibitors covering many different structural classes have been designed and developed. In this review, we describe the development of selective, small-molecule inhibitors of lysine methyltransferases with an emphasis on their discovery and chemical synthesis. We highlight the current state of lysine methyltransferase inhibitors and discuss future directions and opportunities for lysine methyltransferase inhibitor discovery.

  15. High performance oilfield scale inhibitors

    SciTech Connect

    Duccini, Y.; Dufour, A.; Hann, W.M.; Sanders, T.W.; Weinstein, B.

    1997-08-01

    Sea water often reacts with the formation water in offshore fields to produce barium, calcium and strontium sulfate deposits that hinder oil production. Newer fields often have more difficult to control scale problems than older ones, and current technology scale inhibitors are not able to control the deposits as well as needed. In addition, ever more stringent regulations designed to minimize the impact of inhibitors on the environment are being enacted. Three new inhibitors are presented that overcome many of the problems of older technology scale inhibitors.

  16. The cytoskeletal inhibitors latrunculin A and blebbistatin exert antitumorigenic properties in human hepatocellular carcinoma cells by interfering with intracellular HuR trafficking

    SciTech Connect

    Doller, Anke; Badawi, Amel

    2015-01-01

    The impact of the RNA-binding protein HuR for the post-transcriptional deregulation of tumor-relevant genes is well established. Despite of elevations in HuR expression levels, an increase in cytoplasmic HuR abundance in many cases correlates with a high grade of malignancy. Here, we demonstrated that administration of the actin-depolymerizing macrolide latrunculin A, or blebbistatin, an inhibitor of myosin II ATPase activity, caused a dose- and time-dependent reduction in the high cytoplasmic HuR content of HepG2 and Huh7 hepatocellular carcinoma (HCC) cells. Subcellular fractionation revealed that in addition, both inhibitors strongly attenuated cytoskeletal and membrane-bound HuR abundance and conversely increased the HuR amount in nuclear cell fractions. Concomitant with changes in intracellular HuR localization, both cytoskeletal inhibitors markedly decreased the half-lives of cyclooxygenase-2 (COX-2), cyclin A and cyclin D{sub 1} encoding mRNAs resulting in a significant reduction in their expression levels in HepG2 cells. Importantly, a similar reduction in the expression of these HuR targets was achieved by a RNA interference (RNAi)-mediated knockdown of either HuR or nonmuscle myoin IIA. Using polysomal fractionation, we further demonstrate that the decrease in cytoplasmic HuR by latrunculin A or blebbistatin is accompanied by a marked change in the allocation of HuR and its mRNA cargo from polysomes to ribonucleoprotein (RNP) particles. Functionally, the basal migration and prostaglandin E{sub 2} synthesis are similarly impaired in inhibitor-treated and stable HuR-knockdown HepG2 cells. Our data demonstrate that interfering with the actomyosin-dependent HuR trafficking may comprise a valid therapeutic option for antagonizing pathologic posttranscriptional gene expression by HuR and furthermore emphasize the potential benefit of HuR inhibitory strategies for treatment of HCC. - Highlights: • We tested the effects of latrunculin A and blebbistatin on

  17. Osteocompatibility of Biofilm Inhibitors

    PubMed Central

    Rawson, Monica; Haggard, Warren; Jennings, Jessica A

    2014-01-01

    The demand for infection prevention therapies has led to the discovery of several biofilm inhibitors. These inhibiting signals are released by bacteria, fungi, or marine organisms to signal biofilm dispersal or disruption in Gram-positive, Gram-negative, and fungal microorganisms. The purpose of this study was to test the biocompatibility of five different naturally-produced biofilm chemical dispersal and inhibition signals with osteoblast-like cells: D-amino acids (D-AA), lysostaphin (LS), farnesol, cis-2-decenoic acid (C2DA), and desformyl flustrabromine (dFBr). In this preliminary study, compatibility of these anti-biofilm agents with differentiating osteoblasts was examined over a 21 days period at levels above and below concentrations active against bacterial biofilm. Anti-biofilm compounds listed above were serially diluted in osteogenic media and added to cultures of MC3T3 cells. Cell viability and cytotoxicity, after exposure to each anti-biofilm agent, were measured using a DNA assay. Differentiation characteristics of osteoblasts were determined qualitatively by observing staining of mineral deposits and quantitatively with an alkaline phosphatase assay. D-AA, LS, and C2DA were all biocompatible within the reported biofilm inhibitory concentration ranges and supported osteoblast differentiation. Farnesol and dFBr induced cytotoxic responses within the reported biofilm inhibitory concentration range and low doses of dFBr were found to inhibit osteoblast differentiation. At high concentrations, such as those that may be present after local delivery, many of these biofilm inhibitors can have effects on cellular viability and osteoblast function. Concentrations at which negative effects on osteoblasts occur should serve as upper limits for delivery to orthopaedic trauma sites and guide development of these potential therapeutics for orthopaedics. PMID:25505496

  18. Osteocompatibility of biofilm inhibitors.

    PubMed

    Rawson, Monica; Haggard, Warren; Jennings, Jessica A

    2014-01-01

    The demand for infection prevention therapies has led to the discovery of several biofilm inhibitors. These inhibiting signals are released by bacteria, fungi, or marine organisms to signal biofilm dispersal or disruption in Gram-positive, Gram-negative, and fungal microorganisms. The purpose of this study was to test the biocompatibility of five different naturally-produced biofilm chemical dispersal and inhibition signals with osteoblast-like cells: D-amino acids (D-AA), lysostaphin (LS), farnesol, cis-2-decenoic acid (C2DA), and desformyl flustrabromine (dFBr). In this preliminary study, compatibility of these anti-biofilm agents with differentiating osteoblasts was examined over a 21 days period at levels above and below concentrations active against bacterial biofilm. Anti-biofilm compounds listed above were serially diluted in osteogenic media and added to cultures of MC3T3 cells. Cell viability and cytotoxicity, after exposure to each anti-biofilm agent, were measured using a DNA assay. Differentiation characteristics of osteoblasts were determined qualitatively by observing staining of mineral deposits and quantitatively with an alkaline phosphatase assay. D-AA, LS, and C2DA were all biocompatible within the reported biofilm inhibitory concentration ranges and supported osteoblast differentiation. Farnesol and dFBr induced cytotoxic responses within the reported biofilm inhibitory concentration range and low doses of dFBr were found to inhibit osteoblast differentiation. At high concentrations, such as those that may be present after local delivery, many of these biofilm inhibitors can have effects on cellular viability and osteoblast function. Concentrations at which negative effects on osteoblasts occur should serve as upper limits for delivery to orthopaedic trauma sites and guide development of these potential therapeutics for orthopaedics. PMID:25505496

  19. Biological abatement of cellulase inhibitors

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Bio-abatement uses a fungus to metabolize and remove fermentation inhibitors. To determine whether bio-abatement could alleviate enzyme inhibitor effects observed in biomass liquors after pretreatment, corn stover at 10% (w/v) solids was pretreated with either dilute acid or liquid hot water. The ...

  20. Anthranilamide inhibitors of factor Xa.

    PubMed

    Mendel, David; Marquart, Angela L; Joseph, Sajan; Waid, Philip; Yee, Ying K; Tebbe, Anne Louise; Ratz, Andrew M; Herron, David K; Goodson, Theodore; Masters, John J; Franciskovich, Jeffry B; Tinsley, Jennifer M; Wiley, Michael R; Weir, Leonard C; Kyle, Jeffrey A; Klimkowski, Valentine J; Smith, Gerald F; Towner, Richard D; Froelich, Larry L; Buben, John; Craft, Trelia J

    2007-09-01

    SAR about the B-ring of a series of N(2)-aroyl anthranilamide factor Xa (fXa) inhibitors is described. B-ring o-aminoalkylether and B-ring p-amine probes of the S1' and S4 sites, respectively, afforded picomolar fXa inhibitors that performed well in in vitro anticoagulation assays.

  1. Tissue inhibitor of metalloproteinase-3 is up-regulated by transforming growth factor-beta1 in vitro and expressed in fibroblastic foci in vivo in idiopathic pulmonary fibrosis.

    PubMed

    García-Alvarez, Jorge; Ramirez, Remedios; Checa, Marco; Nuttall, Robert K; Sampieri, Clara L; Edwards, Dylan R; Selman, Moisés; Pardo, Annie

    2006-05-01

    Idiopathic pulmonary fibrosis (IPF) is characterized by fibroblast expansion and extracellular matrix accumulation. However, the mechanisms involved in matrix remodeling have not been elucidated. In this study, the authors aimed to evaluate the expression of the tissue inhibitors of matrix metalloproteinases (TIMPs) in human fibroblasts and whole tissues from IPF and normal lungs. They also determined the role of mitogen-activated protein kinase (MAPK) in TIMP3 expression. TIMP1, TIMP2, and TIMP3 were highly expressed in lung fibroblasts. Transforming growth factor (TGF)-beta1, a profibrotic mediator, induced strong up-regulation of TIMP3 at the mRNA and protein levels. The authors examined whether the MAPK pathway was involved in TGF-beta1-induced TIMP3 expression. TGF-beta1 induced the phosphorylation of p38 and extracellular signal-regulated kinase (ERK)1/2. Biochemical blockade of p38 by SB203580, but not of the ERK MAPK pathway, inhibited the effect of this factor. The effect was also blocked by the tyrosine kinase inhibitor genistein and by antagonizing TGF-beta1 receptor type I (activin-linked kinase [ALK5]). In IPF tissues TIMP3 gene expression was significantly increased and the protein was localized to fibroblastic foci and extracellular matrix. Our findings suggest that TGF-beta1-induced TIMP3 may be an important mediator in lung fibrogenesis.

  2. Proteinaceous alpha-amylase inhibitors.

    PubMed

    Svensson, Birte; Fukuda, Kenji; Nielsen, Peter K; Bønsager, Birgit C

    2004-02-12

    Proteins that inhibit alpha-amylases have been isolated from plants and microorganisms. These inhibitors can have natural roles in the control of endogenous alpha-amylase activity or in defence against pathogens and pests; certain inhibitors are reported to be antinutritional factors. The alpha-amylase inhibitors belong to seven different protein structural families, most of which also contain evolutionary related proteins without inhibitory activity. Two families include bifunctional inhibitors acting both on alpha-amylases and proteases. High-resolution structures are available of target alpha-amylases in complex with inhibitors from five families. These structures indicate major diversity but also some similarity in the structural basis of alpha-amylase inhibition. Mutational analysis of the mechanism of inhibition was performed in a few cases and various protein engineering and biotechnological approaches have been outlined for exploitation of the inhibitory function. PMID:14871655

  3. Authentic HIV-1 integrase inhibitors

    PubMed Central

    Liao, Chenzhong; Marchand, Christophe; Burke, Terrence R; Pommier, Yves; Nicklaus, Marc C

    2010-01-01

    HIV-1 integrase (IN) is indispensable for HIV-1 replication and has become a validated target for developing anti-AIDS agents. In two decades of development of IN inhibition-based anti-HIV therapeutics, a significant number of compounds were identified as IN inhibitors, but only some of them showed antiviral activity. This article reviews a number of patented HIV-1 IN inhibitors, especially those that possess high selectivity for the strand transfer reaction. These compounds generally have a polar coplanar moiety, which is assumed to chelate two magnesium ions in the binding site. Resistance to those compounds, when given to patients, can develop as a result of IN mutations. We refer to those compounds as authentic IN inhibitors. Continued drug development has so far delivered one authentic IN inhibitor to the market (raltegravir in 2007). Current and future attention will be focused on the development of novel authentic IN inhibitors with the goal of overcoming viral resistance. PMID:21426159

  4. Oxidized mucus proteinase inhibitor: a fairly potent neutrophil elastase inhibitor.

    PubMed Central

    Boudier, C; Bieth, J G

    1994-01-01

    N-chlorosuccinimide oxidizes one of the methionine residues of mucus proteinase inhibitor with a second-order rate constant of 1.5 M-1.s-1. Cyanogen bromide cleavage and NH2-terminal sequencing show that the modified residue is methionine-73, the P'1 component of the inhibitor's active centre. Oxidation of the inhibitor decreases its neutrophil elastase inhibitory capacity but does not fully abolish it. The kinetic parameters describing the elastase-oxidized inhibitor interaction are: association rate constant kass. = 2.6 x 10(5) M-1.s-1, dissociation rate constant kdiss. = 2.9 x 10(-3) s-1 and equilibrium dissociation constant Ki = 1.1 x 10(-8) M. Comparison with the native inhibitor indicates that oxidation decreases kass. by a factor of 18.8 and increases kdiss. by a factor of 6.4, and therefore leads to a 120-fold increase in Ki. Yet, the oxidized inhibitor may still act as a potent elastase inhibitor in the upper respiratory tract where its concentration is 500-fold higher than Ki, i.e. where the elastase inhibition is pseudo-irreversible. Experiments in vitro with fibrous human lung elastin, the most important natural substrate of elastase, support this view: 1.35 microM elastase is fully inhibited by 5-6 microM oxidized inhibitor whether the enzyme-inhibitor complex is formed in the presence or absence of elastin and whether elastase is pre-adsorbed on elastin or not. PMID:7945266

  5. Membrane-bound, pyridine nucleotide-independent L-lactate dehydrogenase of Rhodopseudomonas sphaeroides.

    PubMed Central

    Markwell, J P; Lascelles, J

    1978-01-01

    Rhodopseudomonas sphaeroides has a pyridine nucleotide-independent L-lactate dehydrogenase associated with the membrane fraction of cells grown either aerobically or phototrophically. The dehydrogenase is present in cells grown on a variety of carbon sources, but at levels less than 20% of that found in cells grown with DL-lactate. The dehydrogenase has been purified 45-fold from membranes of strain L-57, a non-photosynthetic mutant, by steps involving solubilization with lauryl dimethylamine oxide and three anion-exchange chromatography steps. The purified enzyme was specific for the L-isomer of lactate. The Km of the purified enzyme for L-lactate is 1.4 mM, whereas that of the membrane-associated enzyme is 0.5 mM. The enzyme activity was inhibited competitively by D-lactate and non-competitively by oxalate and oxamate. Quinacrine, a flavin analog, also inhibited the activity. The inducible enzyme may serve as a marker of membrane protein in studies of membrane development. PMID:304854

  6. Plant plasma membrane-bound staphylococcal-like DNases as a novel class of eukaryotic nucleases

    PubMed Central

    2012-01-01

    Background The activity of degradative nucleases responsible for genomic DNA digestion has been observed in all kingdoms of life. It is believed that the main function of DNA degradation occurring during plant programmed cell death is redistribution of nucleic acid derived products such as nitrogen, phosphorus and nucleotide bases. Plant degradative nucleases that have been studied so far belong mainly to the S1-type family and were identified in cellular compartments containing nucleic acids or in the organelles where they are stored before final application. However, the explanation of how degraded DNA components are exported from the dying cells for further reutilization remains open. Results Bioinformatic and experimental data presented in this paper indicate that two Arabidopsis staphylococcal-like nucleases, named CAN1 and CAN2, are anchored to the cell membrane via N-terminal myristoylation and palmitoylation modifications. Both proteins possess a unique hybrid structure in their catalytic domain consisting of staphylococcal nuclease-like and tRNA synthetase anticodon binding-like motifs. They are neutral, Ca2+-dependent nucleaces showing a different specificity toward the ssDNA, dsDNA and RNA substrates. A study of microarray experiments and endogenous nuclease activity revealed that expression of CAN1 gene correlates with different forms of programmed cell death, while the CAN2 gene is constitutively expressed. Conclusions In this paper we present evidence showing that two plant staphylococcal-like nucleases belong to a new, as yet unidentified class of eukaryotic nucleases, characterized by unique plasma membrane localization. The identification of this class of nucleases indicates that plant cells possess additional, so far uncharacterized, mechanisms responsible for DNA and RNA degradation. The potential functions of these nucleases in relation to their unique intracellular location are discussed. PMID:23102437

  7. Membrane-bound electron transport systems of an anammox bacterium: A complexome analysis.

    PubMed

    de Almeida, Naomi M; Wessels, Hans J C T; de Graaf, Rob M; Ferousi, Christina; Jetten, Mike S M; Keltjens, Jan T; Kartal, Boran

    2016-10-01

    Electron transport, or oxidative phosphorylation, is one of the hallmarks of life. To this end, prokaryotes evolved a vast variety of protein complexes, only a small part of which have been discovered and studied. These protein complexes allow them to occupy virtually every ecological niche on Earth. Here, we applied the method of proteomics-based complexome profiling to get a better understanding of the electron transport systems of the anaerobic ammonium-oxidizing (anammox) bacteria, the N2-producing key players of the global nitrogen cycle. By this method nearly all respiratory complexes that were previously predicted from genome analysis to be involved in energy and cell carbon fixation were validated. More importantly, new and unexpected ones were discovered. We believe that complexome profiling in concert with (meta)genomics offers great opportunities to expand our knowledge on bacterial respiratory processes at a rapid and massive pace, in particular in new and thus far poorly investigated non-model and environmentally-relevant species. PMID:27461995

  8. Divalent Counterions Tether Membrane-Bound Carbohydrates To Promote the Cohesion of Auditory Hair Bundles

    PubMed Central

    LeBoeuf, Adria C.; Ó Maoiléidigh, D.; Hudspeth, A.J.

    2011-01-01

    The cell membranes in the hair bundle of an auditory hair cell confront a difficult task as the bundle oscillates in response to sound: for efficient mechanotransduction, all the component stereocilia of the hair bundle must move essentially in unison, shearing at their tips yet maintaining contact without membrane fusion. One mechanism by which this cohesion might occur is counterion-mediated attachment between glycan components of apposed stereociliary membranes. Using capillary electrophoresis, we showed that the stereociliary glycocalyx acts as a negatively charged polymer brush. We found by force-sensing photomicrometry that the stereocilia formed elastic connections with one another to various degrees depending on the surrounding ionic environment and the presence of N-linked sugars. Mg2+ was a more potent mediator of attachment than was Ca2+. The forces between stereocilia produced chaotic stick-slip behavior. These results indicate that counterion-mediated interactions in the glycocalyx contribute to the stereociliary coherence that is essential for hearing. PMID:21943412

  9. The characterization of plasma membrane-bound tubulin of cauliflower using Triton X-114 fractionation.

    PubMed

    Sonesson, A; Berglund, M; Staxén, I; Widell, S

    1997-11-01

    The cortical microtubules determine how cellulose microfibrils are deposited in the plant cell wall and are thus important for the control of cell expansion. To understand how microtubules can control microfibril deposition, the components that link the microtubules to the plasma membrane (PM) of plant cells must be isolated. To obtain information on the properties of the tubulin-membrane associations, cauliflower (Brassica oleracea) PM was subjected to Triton X-114 fractionation, and the distribution of alpha- and beta-tubulin was analyzed using immunoblotting. Approximately one-half of the PM-associated tubulin was solubilized by Triton X-114 and 10 to 15% of both alpha- and beta-tubulin was recovered in the detergent phase (indicative of hydrophobic properties) and 30 to 40% was recovered in the aqueous phase. The hydrophobic tubulin could be released from the membrane by high pH extraction with preserved hydrophobicity. A large part of the PM-associated tubulin was found in the Triton-insoluble fraction. When this insoluble material was extracted a second time, a substantial amount of hydrophobic tubulin was released if the salt concentration was increased, suggesting that the hydrophobic tubulin was linked to a high-salt-sensitive protein aggregate that probably includes other components of the cytoskeleton. The hydrophobicity of a fraction of PM-associated tubulin could reflect a direct or indirect interaction of this tubulin with the lipid bilayer or with an integral membrane protein and may represent the anchoring of the cortical microtubules to the PM, a key element in the regulation of cell expansion.

  10. Membrane Bound GSK-3 Activates Wnt Signaling through Disheveled and Arrow

    PubMed Central

    Mannava, Anirudh G.; Tolwinski, Nicholas S.

    2015-01-01

    Wnt ligands and their downstream pathway components coordinate many developmental and cellular processes. In adults, they regulate tissue homeostasis through regulation of stem cells. Mechanistically, signal transduction through this pathway is complicated by pathway components having both positive and negative roles in signal propagation. Here we examine the positive role of GSK-3/Zw3 in promoting signal transduction at the plasma membrane. We find that targeting GSK-3 to the plasma membrane activates signaling in Drosophila embryos. This activation requires the presence of the co-receptor Arrow-LRP5/6 and the pathway activating protein Disheveled. Our results provide genetic evidence for evolutionarily conserved, separable roles for GSK-3 at the membrane and in the cytosol, and are consistent with a model where the complex cycles from cytosol to membrane in order to promote signaling at the membrane and to prevent it in the cytosol. PMID:25848770

  11. Radiation inactivation method provides evidence that membrane-bound mitochondrial creatine kinase is an oligomer

    SciTech Connect

    Quemeneur, E.; Eichenberger, D.; Goldschmidt, D.; Vial, C.; Beauregard, G.; Potier, M.

    1988-06-30

    Lyophilized suspensions of rabbit heart mitochondria have been irradiated with varying doses of gamma rays. Mitochondrial creatine kinase activity was inactivated exponentially with a radiation inactivation size of 352 or 377 kDa depending upon the initial medium. These values are in good agreement with the molecular mass previously deduced from by permeation experiments: 357 kDa. This is the first direct evidence showing that the native form of mitochondrial creatine kinase is associated to the inner membrane as an oligomer, very likely an octamer.

  12. Membrane-bound complement regulatory activity is decreased on vaccinia virus-infected cells.

    PubMed Central

    Baranyi, L; Okada, N; Baranji, K; Takizawa, H; Okada, H

    1994-01-01

    Decay accelerating factor (DAF), membrane cofactor protein (MCP), complement receptor 1 and mouse Crry are cell surface-bound complement regulatory proteins capable of inhibiting C3 convertase activity on cell membranes, and therefore provide a substantial protection from attack by homologous complement activated either by the classical or by the alternative pathway. Decrease in complement regulatory activity might lead to spontaneous complement deposition and subsequent cell injury. MoAb 5I2 can inhibit the complement regulatory activity of molecules on rat cells, resulting in deposition of homologous complement. The antigen recognized by 5I2 MoAb in rats is homologous to mouse Crry. Fifteen to 20 h after infection with vaccinia virus, in vitro cultured KDH-8 rat hepatoma cells show a strong decrease in expression of Crry-like antigen, and proved to be sensitive to complement deposition when 1:5 diluted normal rat serum was added to the culture medium as a source of complement. Addition of complement to the cultured KDH-8 cells infected with a very low dose of vaccinia virus (1 plaque-forming unit (PFU)/1000 cells) substantially reduced spreading of virus infection in the cell culture, while inactivation of complement by heat or zymosan treatment abrogated the protective effect. PMID:7923872

  13. Thiolation of polycarbophil enhances its inhibition of intestinal brush border membrane bound aminopeptidase N.

    PubMed

    Bernkop-Schnürch, A; Zarti, H; Walker, G F

    2001-11-01

    The purpose of this study was to evaluate the potential of polycarbophil-cysteine conjugates (PCP-Cys) as an oral excipient to protect leucine enkephalin (leu-enkp) from enzymatic degradation by the intestinal mucosa. Cysteine was covalently linked to polycarbophil by 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDAC). Inhibitory activity was tested towards isolated aminopeptidase N and excised intact pig intestinal mucosa, with native mucus. Aminopeptidase N activity was assayed spectrophotometrically using L-leucine p-nitroanilide (leu-pNA) as a synthetic substrate and against the model peptide drug leu-enkp, by high-performance liquid chromatography (HPLC). Free cysteine at 6.3 and 63 microM (pH 6) significantly (p < 0.05) inhibited aminopeptidase N activity, and PCP-Cys (0.25% w/v, pH 6) had a significantly (p < 0.05) greater inhibitory effect than PCP on the aminopeptidase N activity towards both substrates. PCP-Cys completely protected leu-enkp against aminopeptidase N activity over a 2-h incubation period, whereas 83 +/- 4 and 60 +/- 7% remained stable in the presence of PCP and buffer only, respectively. Leu-enkp in the absence and presence of PCP (0.25% w/v) at pH 6 was completely digested by the intact intestinal mucosa at the 60- and 90-min incubation time points, respectively, whereas in the presence of PCP-Cys (0.25% w/v, pH 6) 11 +/- 3.5% of leu-enkp remained at the 120-min time point. Thiolation of PCP increased the stability of leu-enkp against the enzymatic degradation by aminopeptidase N and the intact intestinal mucosa, identifying a promising new excipient for peroral delivery of peptides.

  14. Trans and surface membrane bound zervamicin IIB: 13C-MAOSS-NMR at high spinning speed.

    PubMed

    Raap, J; Hollander, J; Ovchinnikova, T V; Swischeva, N V; Skladnev, D; Kiihne, S

    2006-08-01

    Interactions between (15)N-labelled peptides or proteins and lipids can be investigated using membranes aligned on a thin polymer film, which is rolled into a cylinder and inserted into the MAS-NMR rotor. This can be spun at high speed, which is often useful at high field strengths. Unfortuantely, substrate films like commercially available polycarbonate or PEEK produce severe overlap with peptide and protein signals in (13)C-MAOSS NMR spectra. We show that a simple house hold foil support allows clear observation of the carbonyl, aromatic and C(alpha) signals of peptides and proteins as well as the ester carbonyl and choline signals of phosphocholine lipids. The utility of the new substrate is validated in applications to the membrane active peptide zervamicin IIB. The stability and macroscopic ordering of thin PC10 bilayers was compared with that of thicker POPC bilayers, both supported on the household foil. Sidebands in the (31)P-spectra showed a high degree of alignment of both the supported POPC and PC10 lipid molecules. Compared with POPC, the PC10 lipids are slightly more disordered, most likely due to the increased mobilities of the shorter lipid molecules. This mobility prevents PC10 from forming stable vesicles for MAS studies. The (13)C-peptide peaks were selectively detected in a (13)C-detected (1)H-spin diffusion experiment. Qualitative analysis of build-up curves obtained for different mixing times allowed the transmembrane peptide in PC10 to be distinguished from the surface bound topology in POPC. The (13)C-MAOSS results thus independently confirms previous findings from (15)N spectroscopy [Bechinger, B., Skladnev, D.A., Ogrel, A., Li, X., Rogozhkina, E.V., Ovchinnikova, T.V., O'Neil, J.D.J. and Raap, J. (2001) Biochemistry, 40, 9428-9437]. In summary, application of house hold foil opens the possibility of measuring high resolution (13)C-NMR spectra of peptides and proteins in well ordered membranes, which are required to determine the secondary and supramolecular structures of membrane active peptides, proteins and aggregates. PMID:16937243

  15. Purification and characterization of membrane-bound peroxidase from date palm leaves (Phoenix dactylifera L.)

    PubMed Central

    Al-Senaidy, Abdurrahman M.; Ismael, Mohammad A.

    2011-01-01

    Peroxidase from date palm (Phoenix dactylifera L.) leaves was purified to homogeneity and characterized biochemically. The enzyme purification included homogenization, extraction of pigments followed by consecutive chromatographies on DEAE-Sepharose and Superdex 200. The purification factor for purified date palm peroxidase was 17 with 5.8% yield. The purity was checked by SDS and native PAGE, which showed a single prominent band. The molecular weight of the enzyme was approximately 55 kDa as estimated by SDS–PAGE. The enzyme was characterized for thermal and pH stability, and kinetic parameters were determined using guaiacol as substrate. The optimum activity was between pH 5–6. The enzyme showed maximum activity at 55 °C and was fairly stable up to 75 °C, with 42% loss of activity. Date palm leaves peroxidase showed Km values of 0.77 and 0.045 mM for guaiacol and H2O2, respectively. These properties suggest that this enzyme could be a promising tool for applications in different analytical determinations as well as for treatment of industrial effluents at low cost. PMID:23961138

  16. Subunit Movements in Single Membrane-bound H+-ATP Synthases from Chloroplasts during ATP Synthesis

    PubMed Central

    Bienert, Roland; Rombach-Riegraf, Verena; Diez, Manuel; Gräber, Peter

    2009-01-01

    Subunit movements within the H+-ATP synthase from chloroplasts (CF0F1) are investigated during ATP synthesis. The γ-subunit (γCys-322) is covalently labeled with a fluorescence donor (ATTO532). A fluorescence acceptor (adenosine 5′-(β,γ-imino)triphosphate (AMPPNP)-ATTO665) is noncovalently bound to a noncatalytic site at one α-subunit. The labeled CF0F1 is integrated into liposomes, and a transmembrane pH difference is generated by an acid base transition. Single-pair fluorescence resonance energy transfer is measured in freely diffusing proteoliposomes with a confocal two-channel microscope. The fluorescence time traces reveal a repetitive three-step rotation of the γ-subunit relative to the α-subunit during ATP synthesis. Some traces show splitting into sublevels with fluctuations between the sublevels. During catalysis the central stalk interacts, with equal probability, with each αβ-pair. Without catalysis the central stalk interacts with only one specific αβ-pair, and no stepping between FRET levels is observed. Two inactive states of the enzyme are identified: one in the presence of AMPPNP and one in the presence of ADP. PMID:19864418

  17. Subunit movements in single membrane-bound H+-ATP synthases from chloroplasts during ATP synthesis.

    PubMed

    Bienert, Roland; Rombach-Riegraf, Verena; Diez, Manuel; Gräber, Peter

    2009-12-25

    Subunit movements within the H(+)-ATP synthase from chloroplasts (CF(0)F(1)) are investigated during ATP synthesis. The gamma-subunit (gammaCys-322) is covalently labeled with a fluorescence donor (ATTO532). A fluorescence acceptor (adenosine 5'-(beta,gamma-imino)triphosphate (AMPPNP)-ATTO665) is noncovalently bound to a noncatalytic site at one alpha-subunit. The labeled CF(0)F(1) is integrated into liposomes, and a transmembrane pH difference is generated by an acid base transition. Single-pair fluorescence resonance energy transfer is measured in freely diffusing proteoliposomes with a confocal two-channel microscope. The fluorescence time traces reveal a repetitive three-step rotation of the gamma-subunit relative to the alpha-subunit during ATP synthesis. Some traces show splitting into sublevels with fluctuations between the sublevels. During catalysis the central stalk interacts, with equal probability, with each alphabeta-pair. Without catalysis the central stalk interacts with only one specific alphabeta-pair, and no stepping between FRET levels is observed. Two inactive states of the enzyme are identified: one in the presence of AMPPNP and one in the presence of ADP.

  18. Reaction of (3H)meproadifen mustard with membrane-bound Torpedo acetylcholine receptor

    SciTech Connect

    Dreyer, E.B.; Hasan, F.; Cohen, S.G.; Cohen, J.B.

    1986-10-15

    The Torpedo nicotinic acetylcholine receptor (AChR) contains a binding site for aromatic amine noncompetitive antagonists that is distinct from the binding site for agonists and competitive antagonists. To characterize the location and function of this allosteric antagonist site, an alkylating analog of meproadifen has been synthesized, 2-(chloroethylmethylamino)-ethyl-2, 2-diphenylpentanoate HCl (meproadifen mustard). Reaction of (/sup 3/H)meproadifen mustard with AChR-rich membrane suspensions resulted in specific incorporation of label predominantly into the AChR alpha-subunit with minor incorporation into the beta-subunit. Specific labeling required the presence of high concentration of agonist and was inhibited by reversible noncompetitive antagonists including proadifen, meproadifen, perhydrohistrionicotoxin (HTX), and tetracaine when present at concentrations consistent with the binding affinity of these compounds for the allosteric antagonist site. No specific alkylation of the AChR alpha-subunit was detected in the absence of agonist, or in the presence of the partial agonist phenyltrimethylammonium or the competitive antagonists, d-tubocurarine, gallamine triethiodide, or decamethonium. Reaction with 35 microM meproadifen mustard for 70 min in the presence of carbamylcholine produced no alteration in the concentration of (/sup 3/H)ACh-binding sites, but decreased by 38 +/- 4% the number of allosteric antagonist sites as measured by (/sup 3/H)HTX binding. This decrease was not observed when the alkylation reaction was blocked by the presence of HTX. These results lead us to conclude that meproadifen mustard alkylates the allosteric antagonist site in the Torpedo AChR and that part of that site is associated with the AChR alpha-subunit.

  19. USP32 is an active, membrane-bound ubiquitin protease overexpressed in breast cancers.

    PubMed

    Akhavantabasi, Shiva; Akman, Hesna B; Sapmaz, Aysegul; Keller, Jennifer; Petty, Elizabeth M; Erson, Ayse E

    2010-08-01

    USP32, on chromosomal band 17q23.1-17q23.2, is a highly conserved but uncharacterized gene that gave rise during evolution to a well-known hominoid-specific proto-oncogene, USP6. We investigated the expression profile of USP32 in human tissues and examined its functions to gain insight into this novel member of the well-conserved ubiquitination system. We detected ubiquitous USP32 expression across tissues and confirmed the predicted deubiquitination function owing to the presence of conserved peptidase signature aspargine, cysteine, histidine, and aspartic acid domains of ubiquitin-specific proteases. A Golgi localization of GFP-fused USP32 was detected by fluorescent protection assay and BODIPY-TR staining. In addition, stable silencing of USP32 caused a significant decrease in the proliferation and migration rate of cells. Based on these and the fact that USP32 maps to 17q23, which is commonly amplified in breast cancers, we analyzed USP32 expression in breast cancer cells. We detected high expression of USP32 in 50% (9 of 18) of breast cancer cell lines and 22% (9 of 41) of primary breast tumors compared to mammary epithelial cells. In summary, we report the preliminary characterization of this novel deubiquitinating enzyme on 17q23 and demonstrate its functional role in the ubiquitin system and its potential involvement in tumorigenesis.

  20. The membrane bound bacterial lipocalin Blc is a functional dimer with binding preference for lysophospholipids

    PubMed Central

    Campanacci, Valérie; Bishop, Russell E.; Blangy, Stéphanie; Tegoni, Mariella; Cambillau, Christian

    2016-01-01

    Lipocalins, a widespread multifunctional family of small proteins (15–25 kDa) have been first described in eukaryotes and more recently in Gram-negative bacteria. Bacterial lipocalins belonging to class I are outer membrane lipoproteins, among which Blc from E. coli is the better studied. Blc is expressed under conditions of starvation and high osmolarity, conditions known to exert stress on the cell envelope. The structure of Blc that we have previously solved (V. Campanacci, D. Nurizzo, S. Spinelli, C. Valencia, M. Tegoni, C. Cambillau, FEBS Lett. 562 (2004) 183–188.) suggested its possible role in binding fatty acids or phospholipids. Both physiological and structural data on Blc, therefore, point to a role in storage or transport of lipids necessary for membrane maintenance. In order to further document this hypothesis for Blc function, we have performed binding studies using fluorescence quenching experiments. Our results indicate that dimeric Blc binds fatty acids and phospholipids in a micromolar Kd range. The crystal structure of Blc with vaccenic acid, an unsaturated C18 fatty acid, reveals that the binding site spans across the Blc dimer, opposite to its membrane anchored face. An exposed unfilled pocket seemingly suited to bind a polar group attached to the fatty acid prompted us to investigate lyso-phospholipids, which were found to bind in a nanomolar Kd range. We discuss these findings in terms of a potential role for Blc in the metabolism of lysophospholipids generated in the bacterial outer membrane. PMID:16920109

  1. Transport of membrane-bound mineral particles in blood vessels during chicken embryonic bone development.

    PubMed

    Kerschnitzki, Michael; Akiva, Anat; Ben Shoham, Adi; Koifman, Naama; Shimoni, Eyal; Rechav, Katya; Arraf, Alaa A; Schultheiss, Thomas M; Talmon, Yeshayahu; Zelzer, Elazar; Weiner, Stephen; Addadi, Lia

    2016-02-01

    During bone formation in embryos, large amounts of calcium and phosphate are taken up and transported to the site where solid mineral is first deposited. The initial mineral forms in vesicles inside osteoblasts and is deposited as a highly disordered calcium phosphate phase. The mineral is then translocated to the extracellular space where it penetrates the collagen matrix and crystallizes. To date little is known about the transport mechanisms of calcium and phosphate in the vascular system, especially when high transport rates are needed and the concentrations of these ions in the blood serum may exceed the solubility product of the mineral phase. Here we used a rapidly growing biological model, the chick embryo, to study the bone mineralization pathway taking advantage of the fact that large amounts of bone mineral constituents are transported. Cryo scanning electron microscopy together with cryo energy dispersive X-ray spectroscopy and focused-ion beam imaging in the serial surface view mode surprisingly reveal the presence of abundant vesicles containing small mineral particles in the lumen of the blood vessels. Morphologically similar vesicles are also found in the cells associated with bone formation. This observation directly implicates the vascular system in solid mineral distribution, as opposed to the transport of ions in solution. Mineral particle transport inside vesicles implies that far larger amounts of the bone mineral constituents can be transported through the vasculature, without the danger of ectopic precipitation. This introduces a new stage into the bone mineral formation pathway, with the first mineral being formed far from the bone itself. PMID:26481471

  2. An amperometric cholesterol biosensor based on epoxy resin membrane bound cholesterol oxidase

    PubMed Central

    Pundir, C.S.; Narang, Jagriti; Chauhan, Nidhi; Sharma, Preety; Sharma, Renu

    2012-01-01

    Background & objectives: The use of epoxy resin membrane as a support for immobilization of enzyme has resulted into improved sensitivity and stability of biosensors for uric acid, ascorbic acid and polyphenols. The present work was aimed to prepare an improved amperometric biosensor for determination of serum cholesterol required in the diagnostics and management of certain pathological conditions. Methods: Epoxy resin membrane with immobilized cholesterol oxidase was mounted on the cleaned platinum (Pt) electrode with a parafilm to construct a working electrode. This working electrode along with Ag/AgCl as reference and Ag wire as an auxiliary electrode were connected through a three terminal electrometer to construct a cholesterol biosensor. Results: The sensor showed optimum response within 25 sec at pH 7.0 and 45°C. The linear working range of biosensor was 1.0 to 8.0 mM cholesterol. Km and Imax for cholesterol were 5.0 mM and 9.09 μA, respectively. The biosensor measured serum cholesterol. The minimum detection limit of the sensor was 1.0 mM. The mean analytical recoveries of added cholesterol in serum (2.84 and 4.13 mM) were 91.4±2.8 and 92.3±3.1 per cent (n=6), respectively. Within and between assay coefficient of variation (CV) were <2 and <4 per cent, respectively. Biosensor had a storage life of 6 months at 4°C. Interpretation & conclusions: The use of epoxy resin membrane as a support for immobilization of cholesterol oxidase has resulted into an improved amperometric cholesterol biosensor. The present biosensor had an advantage over the existing biosensors as it worked at comparatively lower potential. PMID:23168704

  3. Intravacuolar Membranes Regulate CD8 T Cell Recognition of Membrane-Bound Toxoplasma gondii Protective Antigen.

    PubMed

    Lopez, Jodie; Bittame, Amina; Massera, Céline; Vasseur, Virginie; Effantin, Grégory; Valat, Anne; Buaillon, Célia; Allart, Sophie; Fox, Barbara A; Rommereim, Leah M; Bzik, David J; Schoehn, Guy; Weissenhorn, Winfried; Dubremetz, Jean-François; Gagnon, Jean; Mercier, Corinne; Cesbron-Delauw, Marie-France; Blanchard, Nicolas

    2015-12-15

    Apicomplexa parasites such as Toxoplasma gondii target effectors to and across the boundary of their parasitophorous vacuole (PV), resulting in host cell subversion and potential presentation by MHC class I molecules for CD8 T cell recognition. The host-parasite interface comprises the PV limiting membrane and a highly curved, membranous intravacuolar network (IVN) of uncertain function. Here, using a cell-free minimal system, we dissect how membrane tubules are shaped by the parasite effectors GRA2 and GRA6. We show that membrane association regulates access of the GRA6 protective antigen to the MHC I pathway in infected cells. Although insertion of GRA6 in the PV membrane is key for immunogenicity, association of GRA6 with the IVN limits presentation and curtails GRA6-specific CD8 responses in mice. Thus, membrane deformations of the PV regulate access of antigens to the MHC class I pathway, and the IVN may play a role in immune modulation.

  4. pMD-Membrane: A Method for Ligand Binding Site Identification in Membrane-Bound Proteins

    PubMed Central

    Gorfe, Alemayehu A.

    2015-01-01

    Probe-based or mixed solvent molecular dynamics simulation is a useful approach for the identification and characterization of druggable sites in drug targets. However, thus far the method has been applied only to soluble proteins. A major reason for this is the potential effect of the probe molecules on membrane structure. We have developed a technique to overcome this limitation that entails modification of force field parameters to reduce a few pairwise non-bonded interactions between selected atoms of the probe molecules and bilayer lipids. We used the resulting technique, termed pMD-membrane, to identify allosteric ligand binding sites on the G12D and G13D oncogenic mutants of the K-Ras protein bound to a negatively charged lipid bilayer. In addition, we show that differences in probe occupancy can be used to quantify changes in the accessibility of druggable sites due to conformational changes induced by membrane binding or mutation. PMID:26506102

  5. Core Steps of Membrane-Bound Peptidoglycan Biosynthesis: Recent Advances, Insight and Opportunities

    PubMed Central

    Teo, Alvin C. K.; Roper, David I.

    2015-01-01

    We are entering an era where the efficacy of current antibiotics is declining, due to the development and widespread dispersion of antibiotic resistance mechanisms. These factors highlight the need for novel antimicrobial discovery. A large number of antimicrobial natural products elicit their effect by directly targeting discrete areas of peptidoglycan metabolism. Many such natural products bind directly to the essential cell wall precursor Lipid II and its metabolites, i.e., preventing the utlisation of vital substrates by direct binding rather than inhibiting the metabolising enzymes themselves. Concurrently, there has been an increase in the knowledge surrounding the proteins essential to the metabolism of Lipid II at and across the cytoplasmic membrane. In this review, we draw these elements together and look to future antimicrobial opportunities in this area. PMID:27025638

  6. Reconstitution of selective HIV-1 RNA packaging in vitro by membrane-bound Gag assemblies

    PubMed Central

    Carlson, Lars-Anders; Bai, Yun; Keane, Sarah C; Doudna, Jennifer A; Hurley, James H

    2016-01-01

    HIV-1 Gag selects and packages a dimeric, unspliced viral RNA in the context of a large excess of cytosolic human RNAs. As Gag assembles on the plasma membrane, the HIV-1 genome is enriched relative to cellular RNAs by an unknown mechanism. We used a minimal system consisting of purified RNAs, recombinant HIV-1 Gag and giant unilamellar vesicles to recapitulate the selective packaging of the 5’ untranslated region of the HIV-1 genome in the presence of excess competitor RNA. Mutations in the CA-CTD domain of Gag which subtly affect the self-assembly of Gag abrogated RNA selectivity. We further found that tRNA suppresses Gag membrane binding less when Gag has bound viral RNA. The ability of HIV-1 Gag to selectively package its RNA genome and its self-assembly on membranes are thus interdependent on one another. DOI: http://dx.doi.org/10.7554/eLife.14663.001 PMID:27343348

  7. Optical Waveguide Lightmode Spectroscopic Techniques for Investigating Membrane-Bound Ion Channel Activities

    PubMed Central

    Székács, Inna; Kaszás, Nóra; Gróf, Pál; Erdélyi, Katalin; Szendrő, István; Mihalik, Balázs; Pataki, Ágnes; Antoni, Ferenc A.; Madarász, Emilia

    2013-01-01

    Optical waveguide lightmode spectroscopic (OWLS) techniques were probed for monitoring ion permeation through channels incorporated into artificial lipid environment. A novel sensor set-up was developed by depositing liposomes or cell-derived membrane fragments onto hydrophilic polytetrafluoroethylene (PTFE) membrane. The fibrous material of PTFE membrane could entrap lipoid vesicles and the water-filled pores provided environment for the hydrophilic domains of lipid-embedded proteins. The sensor surface was kept clean from the lipid holder PTFE membrane by a water- and ion-permeable polyethylene terephthalate (PET) mesh. The sensor set-up was tested with egg yolk lecithin liposomes containing gramicidin ion channels and with cell-derived membrane fragments enriched in GABA-gated anion channels. The method allowed monitoring the move of Na+ and organic cations through gramicidin channels and detecting the Cl–-channel functions of the (α5β2γ2) GABAA receptor in the presence or absence of GABA and the competitive GABA-blocker bicuculline. PMID:24339925

  8. Prohibitins act as a membrane-bound chaperone for the stabilization of mitochondrial proteins

    PubMed Central