scholarly journals Leukocyte integrin Mac-1 regulates thrombosis via interaction with platelet GPIbα

2017 ◽  
Vol 8 (1) ◽  
Author(s):  
Yunmei Wang ◽  
Huiyun Gao ◽  
Can Shi ◽  
Paul W. Erhardt ◽  
Alexander Pavlovsky ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Carotid Artery ◽  
Binding Site ◽  
Small Molecule ◽  
Therapeutic Potential ◽  
Bleeding Risk ◽  
Disease Models ◽  
Wild Type ◽  
Microvascular Injury

Abstract Inflammation and thrombosis occur together in many diseases. The leukocyte integrin Mac-1 (also known as integrin αMβ2, or CD11b/CD18) is crucial for leukocyte recruitment to the endothelium, and Mac-1 engagement of platelet GPIbα is required for injury responses in diverse disease models. However, the role of Mac-1 in thrombosis is undefined. Here we report that mice with Mac-1 deficiency (Mac-1 −/− ) or mutation of the Mac-1-binding site for GPIbα have delayed thrombosis after carotid artery and cremaster microvascular injury without affecting parameters of haemostasis. Adoptive wild-type leukocyte transfer rescues the thrombosis defect in Mac-1 −/− mice, and Mac-1-dependent regulation of the transcription factor Foxp1 contributes to thrombosis as evidenced by delayed thrombosis in mice with monocyte-/macrophage-specific overexpression of Foxp1. Antibody and small-molecule targeting of Mac-1:GPIbα inhibits thrombosis. Our data identify a new pathway of thrombosis involving leukocyte Mac-1 and platelet GPIbα, and suggest that targeting this interaction has anti-thrombotic therapeutic potential with reduced bleeding risk.

scholarly journals The Two ADF-H Domains of Twinfilin Play Functionally Distinct Roles in Interactions with Actin Monomers

2002 ◽  
Vol 13 (11) ◽  
pp. 3811-3821 ◽  
Author(s):  
Pauli J. Ojala ◽  
Ville O. Paavilainen ◽  
Maria K. Vartiainen ◽  
Roman Tuma ◽  
Alan G. Weeds ◽  
...  
Keyword(s):  
Binding Site ◽  
Binding Activity ◽  
Size Exclusion ◽  
Actin Dynamics ◽  
Actin Binding ◽  
Actin Monomer ◽  
Wild Type ◽  
Native Page ◽  
Exclusion Chromatography

Twinfilin is a ubiquitous and abundant actin monomer–binding protein that is composed of two ADF-H domains. To elucidate the role of twinfilin in actin dynamics, we examined the interactions of mouse twinfilin and its isolated ADF-H domains with G-actin. Wild-type twinfilin binds ADP-G-actin with higher affinity (K D = 0.05 μM) than ATP-G-actin (K D = 0.47 μM) under physiological ionic conditions and forms a relatively stable (k off = 1.8 s−1) complex with ADP-G-actin. Data from native PAGE and size exclusion chromatography coupled with light scattering suggest that twinfilin competes with ADF/cofilin for the high-affinity binding site on actin monomers, although at higher concentrations, twinfilin, cofilin, and actin may also form a ternary complex. By systematic deletion analysis, we show that the actin-binding activity is located entirely in the two ADF-H domains of twinfilin. Individually, these domains compete for the same binding site on actin, but the C-terminal ADF-H domain, which has >10-fold higher affinity for ADP-G-actin, is almost entirely responsible for the ability of twinfilin to increase the amount of monomeric actin in cosedimentation assays. Isolated ADF-H domains associate with ADP-G-actin with rapid second-order kinetics, whereas the association of wild-type twinfilin with G-actin exhibits kinetics consistent with a two-step binding process. These data suggest that the association with an actin monomer induces a first-order conformational change within the twinfilin molecule. On the basis of these results, we propose a kinetic model for the role of twinfilin in actin dynamics and its possible function in cells.

Essential role of ER-α-dependent NO production in resveratrol-mediated inhibition of restenosis

2010 ◽  
Vol 299 (5) ◽  
pp. H1451-H1458 ◽  
Author(s):  
Alok R. Khandelwal ◽  
Valeria Y. Hebert ◽  
Tammy R. Dugas
Keyword(s):  
Smooth Muscle ◽  
Carotid Artery ◽  
Vascular Smooth Muscle ◽  
Smooth Muscle Cell ◽  
High Fat Diet ◽  
No Production ◽  
Wild Type ◽  
High Fat ◽  
Mediated Effects

Resveratrol (Resv), a red wine polyphenol, is known to exhibit vascular protective effects and reduce vascular smooth muscle cell mitogenesis. Vascular smooth muscle cell proliferation is a critical factor in the pathogenesis of restenosis, the renarrowing of vessels that often occurs after angioplasty and/or stent implantation. Although Resv has been shown to be an estrogen receptor (ER) modulator, the role of the ER in Resv-mediated protection against restenosis has not yet been elucidated in vivo. Therefore, with the use of a mouse carotid artery injury model, our objective was to determine the role of ER in modulating Resv-mediated effects on neointimal hyperplasia. Female wild-type and ER-α−/− mice were administered a high-fat diet ± Resv for 2 wk. A carotid artery endothelial denudation procedure was conducted, and the mice were administered a high-fat diet ± Resv for an additional 2 wk. Resv-treated wild-type mice exhibited a dramatic decrease in restenosis, with an increased arterial nitric oxide (NO) synthase (NOS) activity and NO production. However, in the ER-α−/− mice, Resv failed to afford protection and failed to increase NO production, apparently because of a decreased availability of the NOS cofactor tetrahydrobiopterin. To verify the role of NO in Resv-mediated effects, mice were coadministered Resv plus a nonselective NOS inhibitor, NG-nitro-l-arginine methyl ester (l-NAME). Cotreatment with l-NAME significantly attenuated the antirestenotic properties of Resv. These data thus suggest that Resv inhibits vascular proliferative responses after injury, predominately through an ER-α-dependent increase in NO production.

scholarly journals Role of Cyclin D3 in the Biology of Herpes Simplex Virus 1 ICP0

2001 ◽  
Vol 75 (4) ◽  
pp. 1888-1898 ◽  
Author(s):  
Charles Van Sant ◽  
Pascal Lopez ◽  
Sunil J. Advani ◽  
Bernard Roizman
Keyword(s):  
Cyclin D1 ◽  
Binding Site ◽  
Mutant Virus ◽  
Cyclin D3 ◽  
Wild Type Virus ◽  
Cell Protein ◽  
Cyclin D ◽  
Wild Type ◽  
Human Embryonic Lung

ABSTRACT Earlier reports from this laboratory have shown that the promiscuous transactivator infected-cell protein 0 (ICP0) binds and stabilizes cyclin D3, that the binding site maps to aspartic acid 199 (D199), and that replacement of D199 with alanine abolishes binding and reduces the capacity of the mutant virus to replicate in quiescent cells or to cause mortality in mice infected by a peripheral site. The objective of this report was to investigate the role of cyclin D3 in the biology of ICP0. We report the following results. (i) Wild-type ICP0 activates cyclin D-dependent kinase 4 (cdk4) and stabilizes cyclin D1 although ICP0 does not interact with this cyclin. (ii) The D199A mutant virus (R7914) does not activate cdk4 or stabilize cyclin D1, and neither the wild-type nor the mutant virus activates cdk2. (iii) Early in infection of human embryonic lung (HEL) fibroblasts both wild-type and D199A mutant ICP0s colocalize with PML, and in these cells the ND10 nuclear structures are dispersed. Whereas wild-type ICP0 is transported to the cytoplasm between 3 and 9 h. after infection, ICPO containing the D199A substitution remains quantitatively in the nucleus. (iv) To examine the interaction of ICP0 with cyclin D3, we used a previously described mutant carrying a wild-type ICP0 but expressing cyclin D3 (R7801) and in addition constructed a virus (R7916) that was identical except that it carried the D199A-substituted ICP0. Early in infection with R7801, ICP0 colocalized with cyclin D3 in structures similar to those containing PML. At 3 h after infection, ICP0 was translocated to the cytoplasm whereas cyclin D3 remained in the nucleus. The translocation of ICP0 to the cytoplasm was accelerated in cells expressing cyclin D3 compared with that of ICP0 expressed by wild-type virus. In contrast, ICP0 carrying the D199A substitution remained in the nucleus and did not colocalize with cyclin D3. These studies suggest the following conclusions. (i) ICP0 brings to the vicinity of ND10 cyclin D3 and, in consequence, an activated cdk4. The metabolic events occurring at or near that structure and involving cyclin D3 cause the translocation of ICP0 to the cytoplasm. (ii) In the absence of the cyclin D3 binding site in ICP0, cyclin D3 is not brought to ND10, cyclin D is not stabilized, and the function responsible for the translocation of ICP0 is not expressed, and in quiescent HEL fibroblasts the yields of virus are reduced.

scholarly journals Modification Strategy of D-leucine Residue Addition on a Novel Peptide from Odorrana schmackeri, with Enhanced Bioactivity and In Vivo Efficacy

Toxins ◽  
2021 ◽  
Vol 13 (9) ◽  
pp. 611
Author(s):  
Aifang Yao ◽  
Yingxue Ma ◽  
Xiaoling Chen ◽  
Mei Zhou ◽  
Xinping Xi ◽  
...  
Keyword(s):  
Methicillin Resistant Staphylococcus Aureus ◽  
Therapeutic Potential ◽  
Wild Type ◽  
Anticancer Activities ◽  
Gram Positive Bacteria ◽  
Leucine Residue ◽  
Low Cytotoxicity ◽  
High Cytotoxicity

Brevinins are a well-characterised, frog-skin-derived, antimicrobial peptide (AMP) family, but their applications are limited by high cytotoxicity. In this study, a wild-type des-Leu2 brevinin peptide, named brevinin-1OS (B1OS), was identified from Odorrana schmackeri. To explore the significant role of the leucine residue at the second position, two variants, B1OS-L and B1OS-D-L, were designed by adding L-leucine and D-leucine residues at this site, respectively. The antibacterial and anticancer activities of B1OS-L and B1OS-D-L were around ten times stronger than the parent peptide. The activity of B1OS against the growth of Gram-positive bacteria was markedly enhanced after modification. Moreover, the leucine-modified products exerted in vivo therapeutic potential in an methicillin-resistant Staphylococcus aureus (MRSA)-infected waxworm model. Notably, the single substitution of D-leucine significantly increased the killing speed on lung cancer cells, where no viable H838 cells survived after 2 h of treatment with B1OS-D-L at 10 μM with low cytotoxicity on normal cells. Overall, our study suggested that the conserved leucine residue at the second position from the N-terminus is vital for optimising the dual antibacterial and anticancer activities of B1OS and proposed B1OS-D-L as an appealing therapeutic candidate for development.

scholarly journals Regulation of the Later Stages of Nodulation Stimulated by IPD3/CYCLOPS Transcription Factor and Cytokinin in Pea Pisum sativum L.

Plants ◽  
2021 ◽  
Vol 11 (1) ◽  
pp. 56
Author(s):  
Elizaveta S. Rudaya ◽  
Polina Yu. Kozyulina ◽  
Olga A. Pavlova ◽  
Alexandra V. Dolgikh ◽  
Alexandra N. Ivanova ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Comparative Analysis ◽  
Pisum Sativum ◽  
Nodule Development ◽  
Rna Seq ◽  
Wild Type ◽  
Development Stages ◽  
Pisum Sativum L ◽  
Pea Mutant

The IPD3/CYCLOPS transcription factor was shown to be involved in the regulation of nodule primordia development and subsequent stages of nodule differentiation. In contrast to early stages, the stages related to nodule differentiation remain less studied. Recently, we have shown that the accumulation of cytokinin at later stages may significantly impact nodule development. This conclusion was based on a comparative analysis of cytokinin localization between pea wild type and ipd3/cyclops mutants. However, the role of cytokinin at these later stages of nodulation is still far from understood. To determine a set of genes involved in the regulation of later stages of nodule development connected with infection progress, intracellular accommodation, as well as plant tissue and bacteroid differentiation, the RNA-seq analysis of pea mutant SGEFix--2 (sym33) nodules impaired in these processes compared to wild type SGE nodules was performed. To verify cytokinin’s influence on late nodule development stages, the comparative RNA-seq analysis of SGEFix--2 (sym33) mutant plants treated with cytokinin was also conducted. Findings suggest a significant role of cytokinin in the regulation of later stages of nodule development.

Identification of a germ-cell-specific transcriptional repressor in the promoter of Tctex-1

Development ◽  
1995 ◽  
Vol 121 (2) ◽  
pp. 561-568 ◽  
Author(s):  
M.J. O'Neill ◽  
K. Artzt
Keyword(s):  
Germ Cell ◽  
Gene Family ◽  
Binding Site ◽  
Transcriptional Repressor ◽  
Chromosome 17 ◽  
Wild Type ◽  
Aberrant Expression ◽  
T Complex ◽  
T Haplotype

The Tctex-1 gene family maps to the t complex of the mouse and consists of four copies on chromosome 17 in both wild-type and t-haplotypes. Tctex-1 mRNA is eightfold overexpressed in male and female germ cells in t-haplotype compound heterozygotes (tx/ty). In order to determine the cause of this aberrant expression and the role of this gene family in spermatogenesis and oogenesis it was subjected to extensive molecular analysis. We find that Tctex-1 protein is present in sperm tails and oocytes and that it is present at equal levels in wild-type and t-haplotype testis. Surprisingly, the excess message in t-haplotypes is not translated. Sequence analysis of the gene family reveals that one copy in t-haplotypes has a mutated start codon. This same copy is deleted for a protein-binding motif in its promoter. This motif, GIM (Germ cell Inhibitory Motif) has strong homology to the Xenopus AP-2-binding site but does not appear to be a binding site for mammalian AP-2. A factor(s) present in testis and ovary, but absent in other mouse tissues binds specifically to this site. Transfection assays using Tctex-1 promoter constructs suggest that GIM functions as a transcriptional repressor. The possible role of Tctex-1 in t complex transmission ratio distortion and sterility is discussed.

PAX5/TEL Causes down Regulation of CD19 and TGFbeta Resistance in PreBI Cells.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 1416-1416
Author(s):  
Grazia Fazio ◽  
Chiara Palmi ◽  
Greta Giordano Attianese ◽  
Andrea Biondi ◽  
Antonius Rolink ◽  
...  
Keyword(s):  
Growth Rate ◽  
Cell Proliferation ◽  
Transcription Factor ◽  
Cell Differentiation ◽  
B Cell ◽  
Chimeric Protein ◽  
B Cell Differentiation ◽  
Wild Type ◽  
Down Regulation

Abstract The PAX5/TEL chimeric gene was cloned from the translocation t(9;12)(q11;p13) in an ALL patient. Recent data indicate that the PAX5/TEL fusion defines the cytogenetic entity dic(9;12)(p13;p13), which accounts for about 1% of childhood ALL, almost exclusively B-progenitor ALL. PAX5/TEL is likely to be an aberrant transcription factor, resulting from joining the 5′ region of PAX5 (a transcription factor essential for B cell development) to the 3′ region of TEL/ETV6, containing the Ets-family DNA binding domain. We have cloned the FLAG-full length chimeric PAX5/TEL cDNA in the retroviral vector pMSCV-IRES-GFP (MigR1) to transduce target cells. We have demonstrated a specific nuclear localization of the chimeric protein in NIH3T3 by immunofluorescence analysis. Moreover, we observed a PAX5/TEL dependent decrease of the cellular growth rate in IL-3 dependent murine proB Ba/F3 cells. We further investigated the function of the PAX5/TEL chimeric protein as a potential oncoprotein in murine preBI cells, as a more physiological model. Murine PAX5 −/− preBI cells and wild type preBI cells were purified as B220+/c-KIT+ cells from mouse fetal liver and they were cultured on OP9 and DL1-OP9 stroma cells in presence of IL-7. The OP9 stroma supports B cell proliferation and survival; the DL1-OP9 stroma expresses Delta-like1, one of the Notch ligands, and it’s important to support T cell development. Both PAX5 −/− preBI cells and wild type preBI cells were transduced with the retroviral construct pMSCV-PAX5/TEL-IRES-GFP to analyze cell proliferation, differentiation and growth-dependence on IL-7. Wild type preBI cells expressing PAX5/TEL showed down modulation of CD19 when cultured on OP9 stroma in presence of IL-7; an inverse correlation was observed between the levels of expression of GFP and of CD19. The down modulation of CD19 can be involved in driving the preBI cell into differentiation block. A possible explanation of CD19 repression can rely on a potential competition between PAX5/TEL and endogenous PAX5 to bind PAX5 consensus region on DNA. On OP9 stroma, PAX5/TEL preBI cells are resistant to TGFbeta anti-proliferative and apoptotic effects, with a three-fold increased growth rate than control cells. Although the specific mechanism of PAX5/TEL disruption of TGFbeta signalling pathway remains to be investigated, we propose the TGFbeta resistance by PAX5/TEL as a way to evade the immunosurveillance. PAX5/TEL-preBI cells cultured on DL1-OP9 showed a different phenotype, with up-regulation of c-KIT and down-regulation of CD44. PAX5−/− preBI cells infected with PAX5TEL and grown on OP9 were CD19 negative even in the presence of PAX5TEL. On DL1-OP9 stroma, PAX5TEL cells were able to differentiate maintaining the developmental plasticity of PAX5 −/− preBI cells. These preliminary results indicate a role of PAX5/TEL as a transcription factor, potentially with a suppressor function, down regulating CD19 expression, thus suggesting a function on B cell differentiation. The chimera is able to interfere with TGFbeta pathway, inducing resistance and conferring an advantage in cell survival, evading the immunosurveillance. PAX5TEL do not replace PAX5 functions in PAX5−/− cells, it cannot activate PAX5 target genes as CD19, important for restoring B cell differentiation. Further analyeis are needed to better evaluate the role of PAX5/TEL protein, both in vivo and in vitro models.

Sp1 Inhibition Affects Cell Growth and Survival In Waldenstrom’s Macroglobulinemia Through a MYD88-Independent Pathway

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 3065-3065
Author(s):  
Mariateresa Fulciniti ◽  
Rajya Bandi ◽  
Nicola Amodio ◽  
Rao H. Prabhala ◽  
Mansa Munshi ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Cell Growth ◽  
Small Molecule ◽  
Combination Treatment ◽  
Low Grade ◽  
Growth And Survival ◽  
Equity Ownership ◽  
Myd88 Pathway ◽  
Myd88 L265p

Abstract Gene expression and proteomics studies have advanced our understanding of Waldenstrom’s macroglobulinemia (WM) and identified potential therapeutic targets, however, WM remains incurable. Therefore there is an urgent need for the development of novel chemotherapeutic agents targeting deregulated signaling pathways specifically present in WM. Based on role of transcription factor Sp1 in myeloma, we evaluated its molecular and functional role in WM. Our loss of function and Gain of function studies have highlighted a potential oncogenic role of Sp1 in WM. Reduction in Sp1 protein level following transient transfection of WM cells with Sp1 siRNA led to decreased WM cell viability. Conversely, overexpression of Sp1 promoted cell growth and increased IgM production in BCWM1 cell line, associated with an increased level of Sp1 dependent genes. These results demonstrate the role of Sp1 in WM cell growth and survival and provide rationale to therapeutically target Sp1 in WM using small molecule inhibitors of Sp1. We therefore evaluated the activity of Terameprocol (TMP), a small molecule with ability to inhibit Sp1-mediated transactivation by competing for binding to specific Sp1-domains within gene promoter regions. Treatment with TMP caused inhibition of WM and IgM-secreting low-grade lymphoma cell lines, as well as purified primary patient WM cell growth in a dose and time dependent fashion. Sp1 physically interacts with other TFs, influencing their activity. To identify TFs whose activity is controlled by Sp1 in WM cells, we analyzed the activation of 47 transcription factors in nuclear extracts from BCWM1 and MWCL1 cells that were siRNA-depleted for Sp1 or treated with TMP using a transcription factor profiling assay. Both depletion of Sp1 and TMP treatment decreased the activity of TFs, including STAT1, STAT3, and NF-κB, whereas other factors, such as p53, were not affected. As NF-κB and STAT-3 pathways have been shown to be constitutively activated in WM and to play a pivotal role in regulating growth and survival of WM cells, we have focused our further analysis on these TFs in an attempt to understand the molecular mechanism underlying the activity of Sp1 and its inhibition in WM. Enforced expression of Sp1 significantly induced NF-κB p65 (RelA) activity, and TMP was able to overcome this effect. Inhibition of Sp1 activity impairs basal and TNFα-stimulated NF-κB transcriptional activity as well as IL-6-induced STA3 activation in WM cells. Recent studies have reported the high frequency of the MYD88 L265P somatic mutation in patients with WM. To investigate the impact of MYD88 on the sensitivity of WM cells to Sp1 inhibition, we first analyzed effect of TMP on MYD88-silenced cells. MYD88 knockdown significantly inhibits BCWM1 cell growth compared with scrambled cells and the antitumor effect was more pronounced upon treatment with TMP. These results provided the rationale to investigate the activity of combination treatment between TMP and inhibitors known to impede the MYD88 pathway signaling. BCWM1 and MWCL1 WM cells were cultured in the absence or presence of a direct kinase inhibitor of IRAK 1 and 4 or the BTK inhibitor PCI32765. The combination treatment resulted in significant and synergistic dose-dependent antiproliferative effect and inhibition of NFkB p65 activity in MYD88 L265P–expressing WM cells suggesting that MYD88 and Sp1 pathways are both functional in WM but independent from each other. In summary, these results demonstrate Sp1 as an important transcription factor that regulates proliferation and survival of WM cells as well as IgM secreting low-grade lymphoma cells and provides preclinical rationale for clinical development of TMP in WM alone or in combination with inhibitors of MYD88 pathway. Disclosures: Anderson: oncopep: Equity Ownership; celgene: Consultancy; onyx: Consultancy; gilead: Consultancy; sanofi aventis: Consultancy; acetylon: Equity Ownership. Treon:Millennium: Consultancy.

scholarly journals Dissecting the Role of the N-Terminal Region of the Escherichia coli Global Transcription Factor FNR

2008 ◽  
Vol 190 (24) ◽  
pp. 8230-8233 ◽  
Author(s):  
Aixin Yan ◽  
Patricia J. Kiley
Keyword(s):  
Escherichia Coli ◽  
Transcription Factor ◽  
Terminal Region ◽  
Wild Type ◽  
Fnr Protein

ABSTRACT The role of the N-terminal region of the transcription factor FNR, which immediately precedes the first ligand (Cys20) of the [4Fe-4S] cluster, was investigated. We found that truncation mutants that removed residues 2 to 16 and 2 to 17 had wild-type levels of FNR protein but surprisingly altered O2 regulation.

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