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Robert L Medcalf

Publications and source records attributed to Robert L Medcalf.

16 recordsLinked to original sources
PubMed2006

Oncostatin M is a neuroprotective cytokine that inhibits excitotoxic injury in vitro and in vivo.

Oncostatin M (OsM) is a member of the interleukin (IL)-6 family of cytokines and is well known for its role in inflammation, cell proliferation, and hematopoiesis. OsM, together with its glycoprotein 130 containing receptor complex, is expressed and regulated in most cells of the central nervous system (CNS), yet the function of OsM within this compartment is poorly understood. Here we have investigated the effect of OsM using in vitro and in vivo models of excitotoxic injury. Using primary cultures of mouse cortical neurons, OsM was shown to reduce N-methyl-D-aspartate (NMDA) -induced neuronal death by 50% when added simultaneously with NMDA while pretreatment of neurons with OsM fully prevented NMDA toxicity indicating a profound protective effect of this cytokine. OsM was also shown to inhibit NMDA-mediated increase in levels of free intracellular calcium and to selectively reduce neuronal expression of the NR2C subunit of the NMDA receptor. Finally, using an in vivo model of excitotoxic injury, OsM significantly reduced the NMDA-induced lesion volume when coinjected with NMDA into the mouse striatum. Taken together, these results identify OsM as a powerful neuroprotective cytokine and provide a rational foundation to explore the therapeutic potential for OsM in diseases of the CNS.

Animals↗
PubMed2006

SerpinB2 is an inducible host factor involved in enhancing HIV-1 transcription and replication.

The serine protease inhibitor SerpinB2 (plasminogen activator inhibitor-2) is a major product of activated monocytes and macrophages and is substantially induced during most inflammatory processes. Distinct from its widely described extracellular role as an inhibitor of urokinase plasminogen activator, SerpinB2 has recently been shown to have an intracellular role as a retinoblastoma protein (Rb)-binding protein that inhibits Rb degradation. Here we show that HIV-1 infection and gp120 treatment of human peripheral blood mononuclear cells resulted in induction of SerpinB2. Furthermore, SerpinB2 expression in THP-1 monocyte/macrophage, Jurkat T, and HeLa cell lines increased replication of HIV-1 and enhanced transcription from the HIV-1 long terminal repeat (LTR) promoter by 3-10-fold. Increased HIV-1 gene expression and transcription was also observed in activated macrophages from SerpinB2+/+ mice compared with macrophages from SerpinB2-/- mice. The SerpinB2-mediated elevation of Rb protein levels appeared to be responsible for enhancing transcription from the core promoter region of the LTR by relieving HDM2-mediated inhibition of Sp1 and/or by increasing the Sp1/Sp3 expression ratios. This is the first report associating HIV-1 replication with SerpinB2 expression and illustrates that SerpinB2 is a potentially important inducible host factor that significantly promotes HIV-1 replication.

Animals↗
PubMed2006

Tissue-type plasminogen activator: a multifaceted modulator of neurotransmission and synaptic plasticity.

For over a decade, tissue-type plasminogen activator (t-PA), a serine protease classically known for its profibrinolytic role in the vasculature, has been implicated in numerous aspects of the synaptic plasticity process. But despite being the most intensively studied protease of the CNS, the mechanisms and molecular mediators behind the action of t-PA on synaptic efficacy remain largely undefined. Rather than examine the role of t-PA in proteolytic remodeling of the synaptic extracellular matrix, this review will focus on the evidence that defines t-PA as a direct modulator of neurotransmission and synaptic plasticity by impacting on glutamatergic and dopaminergic pathways.

Animals↗
PubMed2006

Stage specific gene expression of serpins and their cognate proteases during myeloid differentiation.

Proteases and their serpin inhibitors are abundantly expressed in haemopoietic and peripheral blood cells. There is, however, relatively little information about the role played by serpins in the control of protease activity within these cells and in the pericellular region. The observation that mutations in the neutrophil elastase gene, which cause cyclic and severe congenital neutropenia, are associated with protease maldistribution gives some clue as to the potential importance of inhibitor proteins. To begin to address the role of protease/inhibitor balance in blood cells we used reverse transcription polymerase chain reaction to examine protease and serpin gene expression in mature peripheral blood cells, differentiating haemopoietic progenitors, leukaemic blasts and haemopoietic cell lines. The results demonstrate stage-specific expression of proteases together with widespread expression of intra- and extra-cellular serpins. The elastase inhibitors monocyte neutrophil elastase inhibitor (MNEI) and antitrypsin (AT) showed overlapping expression. MNEI is predominantly expressed in early haemopoietic progenitors while antitrypsin is mainly expressed in more mature myeloid precursors, peripheral blood granulocytes and mononuclear cells. Our results give an overall picture of serpin and protease gene expression and draws attention to the potential importance of elastase regulators at all stages of myelopoiesis.

Bone Marrow Cells↗
PubMed2005

Vampire bat salivary plasminogen activator (desmoteplase) inhibits tissue-type plasminogen activator-induced potentiation of excitotoxic injury.

BACKGROUND AND PURPOSE: In contrast to tissue-type plasminogen activator (tPA), vampire bat (Desmodus rotundus) salivary plasminogen activator (desmoteplase [DSPA]) does not promote excitotoxic injury when injected directly into the brain. We have compared the excitotoxic effects of intravenously delivered tPA and DSPA and determined whether DSPA can antagonize the neurotoxic and calcium enhancing effects of tPA. METHODS: The brain striatal region of wild-type c57 Black 6 mice was stereotaxically injected with N-methyl-d-Aspartate (NMDA); 24 hour later, mice received an intravenous injection of tPA or DSPA (10 mg/kg) and lesion size was assessed after 24 hours. Cell death and calcium mobilization studies were performed using cultures of primary murine cortical neurons. RESULTS: NMDA-mediated injury was increased after intravenous administration of tPA, whereas no additional toxicity was seen after administration of DSPA. Unlike DSPA, tPA enhanced NMDA-induced cell death and the NMDA-mediated increase in intracellular calcium levels in vitro. Moreover, the enhancing effects of tPA were blocked by DSPA. CONCLUSIONS: Intravenous administration of tPA promotes excitotoxic injury, raising the possibility that leakage of tPA from the vasculature into the parenchyma contributes to brain damage. The lack of such toxicity by DSPA further encourages its use as a thrombolytic agent in the treatment of ischemic stroke.

Animals↗
PubMed2005

The relationship between the prothrombin upstream sequence element and the G20210A polymorphism: the influence of a competitive environment for mRNA 3'-end formation.

The human prothrombin G20210A polymorphism located at the 3' cleavage site of the mRNA results in elevated plasma prothrombin levels and increased risk of venous thrombosis. This polymorphism has been shown to directly influence a variety of processes related to prothrombin mRNA metabolism. We have constructed plasmids that express the full-length prothrombin mRNA that is polyadenylated at its natural site. The A allele prothrombin variant was more efficient than the G allele at promoting cleavage at this site in the presence of a competing poly (A) sequence. In the absence of competition, both allelic variants give rise to a similar level of cleavage site heterogeneity. An upstream sequence element (USE) was also identified within the prothrombin 3'-UTR. When placed upstream of two competing poly (A) sites, the USE directed cleavage preferentially to the proximal poly (A) site. In the absence of competition, the USE had no effect on cleavage site selection. This study suggests that the basis for the increase in prothrombin expression in A allele carriers is not due to allelic changes in cleavage site selection per se. In addition, the functionality of USEs needs to be considered within the context of endogenous sequence architecture.

3' Untranslated Regions↗
PubMed2005

The undecided serpin. The ins and outs of plasminogen activator inhibitor type 2.

Plasminogen activator inhibitor type-2 (PAI-2) is a nonconventional serine protease inhibitor (serpin) with unique and tantalizing properties that is generally considered to be an authentic and physiological inhibitor of urokinase. However, the fact that only a small percentage of PAI-2 is secreted has been a long-standing argument for alternative roles for this serpin. Indeed, PAI-2 has been shown to have a number of intracellular roles: it can alter gene expression, influence the rate of cell proliferation and differentiation, and inhibit apoptosis in a manner independent of urokinase inhibition. Despite these recent advances in defining the intracellular function of PAI-2, it still remains one of the most mysterious and enigmatic members of the serpin superfamily.

Animals↗
PubMed2005

Meet the serpins.

Explore the source record for details and available documents.

Serine Proteinase Inhibitors↗
PubMed2005

Transcriptional and posttranscriptional regulation of the plasminogen activator system.

The core protein components of the plasminogen activator (PA) system are two plasminogen activators, two plasminogen activator inhibitors and a urokinase type plasminogen activator-specific cell surface receptor. Various types of biological regulation are exerted through the interplay of these components mutually and with extracellular matrix proteins and cell membrane proteins, with or without involving proteolytic activity. Reflecting these diverse biological roles, the level and activity of each component of the PA system is under the control of a variety of regulatory mechanisms. The expression level of a protein reflects the level of the corresponding mRNA, which is essentially the net result of de novo synthesis, i.e. transcription, and degradation. Many recent studies have shown that the regulation of mRNA stability is dynamic and cell specific. Accordingly, we are learning that the mRNAs of the PA system are also the subject of diverse regulatory mechanisms. In this short review, we summarize current understanding of the transcriptional and mRNA-stability regulation of the PA system.

Adipocytes↗
PubMed2004

The t-PA -7351C>T enhancer polymorphism decreases Sp1 and Sp3 protein binding affinity and transcriptional responsiveness to retinoic acid.

We have previously identified a common polymorphism at the tissue-type plasminogen activator (t-PA) locus (-7351C>T), located within a GC-box in the retinoic acid (RA) and steroid hormone responsive t-PA enhancer. The aim of the present study was to functionally characterize this t-PA variant. Electrophoretic mobility shift assays (EMSAs) using crude nuclear extracts from human endothelial, HeLa, and NT2 neuronal cells revealed a 10-fold greater protein binding affinity to the wild-type C allele compared with the mutant T allele variant. Sp1 and Sp3 were identified as the GC-box binding proteins. Luciferase reporter assays showed that the C allele generated higher transcriptional activity after induction by RA, compared with the T allele variant. Further EMSAs showed that RA treatment enhanced Sp1/Sp3 binding to the GC-box. Formation of the Sp1/Sp3 containing complex was inhibited by anti-RA receptor (RAR) antibodies, suggesting that Sp1/Sp3 and RAR interact. The t-PA -7351C>T polymorphism is therefore functional at the level of transcription. The reduced binding affinity of Sp1/Sp3 to the T allele could explain our earlier observations of a reduced t-PA release and an increased risk of myocardial infarction in individuals carrying this allele.

Cytosine↗
PubMed2003

The structure of the zinc finger domain from human splicing factor ZNF265 fold.

Identification of the protein domains that are responsible for RNA recognition has lagged behind the characterization of protein-DNA interactions. However, it is now becoming clear that a range of structural motifs bind to RNA and their structures and molecular mechanisms of action are beginning to be elucidated. In this report, we have expressed and purified one of the two putative RNA-binding domains from ZNF265, a protein that has been shown to bind to the spliceosomal components U1-70K and U2AF35 and to direct alternative splicing. We show that this domain, which contains four highly conserved cysteine residues, forms a stable, monomeric structure upon the addition of 1 molar eq of Zn(II). Determination of the solution structure of this domain reveals a conformation comprising two stacked beta-hairpins oriented at approximately 80 degrees to each other and sandwiching the zinc ion; the fold resembles the zinc ribbon class of zinc-binding domains, although with one less beta-strand than most members of the class. Analysis of the structure reveals a striking resemblance to known RNA-binding motifs in terms of the distribution of key surface residues responsible for making RNA contacts, despite a complete lack of structural homology. Furthermore, we have used an RNA gel shift assay to demonstrate that a single crossed finger domain from ZNF265 is capable of binding to an RNA message. Taken together, these results define a new RNA-binding motif and should provide insight into the functions of the >100 uncharacterized proteins in the sequence data bases that contain this domain.

Amino Acid Sequence↗
PubMed2003

Inherent instability of plasminogen activator inhibitor type 2 mRNA is regulated by tristetraprolin.

Plasminogen activator inhibitor type 2 (PAI-2) is a serine protease inhibitor that is subject to regulation at the post-transcriptional level. At least two mRNA instability elements reside within the PAI-2 transcript; one in the coding region and another within the 3'-untranslated region (UTR). For the latter, a functional AU-rich motif (ARE) has been identified that provides a binding site for a number of cellular proteins, including the mRNA stability protein, HuR. In this study, we used the yeast three-hybrid system to screen a human leukocyte cDNA library to identify other proteins that associate with the PAI-2 ARE. This screen identified tristetraprolin (TTP) as a PAI-2 mRNA ARE-binding protein. UV cross-linking and immunoprecipitation experiments showed that TTP expressed in HEK293 cells could associate with the PAI-2 ARE in vitro. Co-transfection of plasmids expressing TTP and PAI-2 in HEK293 cells resulted in an increase in the decay rate of PAI-2 mRNA and loss of PAI-2 protein in a process that was dependent upon the PAI-2 3'-UTR. The 29-nt PAI-2 AU-rich element alone was also capable of conferring TTP-dependent mRNA instability to a reporter transcript. The extent of PAI-2 mRNA stability was remarkably sensitive to TTP since TTP-dependent PAI-2 mRNA decay occurred at TTP levels that were below Western blot detection limits. This study identifies TTP as a functional PAI-2 ARE-binding protein that modulates the post-transcriptional regulation of the PAI-2 gene.

3' Untranslated Regions↗
PubMed2003

Vampire bat salivary plasminogen activator (desmoteplase): a unique fibrinolytic enzyme that does not promote neurodegeneration.

BACKGROUND AND PURPOSE: Tissue-type plasminogen activator (tPA) promotes excitotoxic and ischemic injury within the brain. These findings have implications for the use of tPA in the treatment of acute ischemic stroke. The plasminogen activator from vampire bat (Desmodus rotundus) saliva (D rotundus salivary plasminogen activator [DSPA]; desmoteplase) is an effective plasminogen activator but, in contrast to tPA, is nearly inactive in the absence of a fibrin cofactor. The purpose of this study was to compare the ability of DSPA and tPA to promote kainate- and N-methyl-D-aspartate (NMDA)-induced neurodegeneration in tPA-/- mice and wild-type mice, respectively. METHODS: tPA-/- mice were infused intracerebrally with either tPA or DSPA. The degree of neuronal survival after hippocampal injection of kainate was assessed histochemically. Wild-type mice were used to assess the extent of neuronal damage after intrastriatal injection of NMDA in the presence of tPA or DSPA. Immunohistochemistry and fibrin zymography were used to evaluate DSPA and tPA antigen or activity. RESULTS: Infusion of tPA into tPA-/- mice restored sensitivity to kainate-mediated neurotoxicity and activation of microglia. DSPA was incapable of conferring sensitivity to kainate treatment, even when infused at 10-fold higher molar concentration than tPA. The presence of tPA also increased the lesion volume induced by NMDA injection into the striatum of wild-type mice, whereas DSPA had no effect. CONCLUSIONS: DSPA does not promote kainate- or NMDA-mediated neurotoxicity in vivo. These results provide significant impetus to evaluate DSPA in patients with ischemic stroke.

Animals↗
PubMed2002

Plasminogen activator inhibitor type 2: a regulator of monocyte proliferation and differentiation.

We have explored the role of plasminogen activator inhibitor type 2 (PAI-2) in THP-1 monocyte-like cells. These cells possess a mutation in the PAI-2 gene and do not produce an active PAI-2 protein. Transfection of THP-1 cells with plasmids expressing active PAI-2 reduced the cells' inherent adhesive properties and decreased the rate of cell proliferation. THP-1 cells expressing active PAI-2 also displayed an altered phenotype in response to phorbol ester-induced differentiation that was concomitant with a reduction in CD14 expression. THP-1 cells transfected with a variant PAI-2 containing a mutation in the reactive center (PAI-2(Ala380)) displayed no noticeable change in any of these parameters, suggesting the involvement of a PAI-2-sensitive serine protease(s). The antiproliferative effect of PAI-2 was attenuated by treating the PAI-2-expressing THP-1 cells with recombinant urokinase (u-PA), suggesting that PAI-2 was disruptive of a u-PA/u-PA receptor signaling pathway initiated on the cell surface. Consistent with this, treatment of wild-type THP-1 cells with recombinant PAI-2 also caused a reduction in cellular proliferation. These results implicate endogenous PAI-2 as a modulator of monocyte adhesion, proliferation, and differentiation.

Amino Acid Substitution↗
PubMed2002

Prothrombin G20210A is a bifunctional gene polymorphism.

The G20210A polymorphism has been shown to alter the efficiency of prothrombin mRNA processing. Here we show that the G20210A mutation also alters prothrombin mRNA stability. Three-fold more prothrombin protein and mRNA were produced in NIH-3T3 cells transfected with the prothrombin cDNAs containing the 20210A variant compared to cells expressing the 20210G variant. mRNA stability assays using chimeric globin transcripts harboring the G or A variant of the 97 nt prothrombin 3'-UTR indicated that the 20210G variant conferred greater instability to the globin reporter transcript than the A variant in transfected HepG2 cells. Both variants of the prothrombin 3'-UTR were shown to provide binding sites for a number of cellular proteins including HuR, an RNA binding protein associated with mRNA stability. Our results indicate that the G20210A is a bifunctional polymorphism, as it not only alters the efficiency of mRNA processing, but also the decay rate of prothrombin mRNA.

3' Untranslated Regions↗