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At least 109 records · Page 6Linked to original sources
PubMed2000

Regulation of endothelial nitric oxide synthase expression by albumin-derived advanced glycosylation end products.

We examined whether albumin-derived advanced glycosylation end products (AGEs) downregulate the expression of endothelial nitric oxide synthase (NOS). Significant reductions in NOS activity and cGMP levels in bovine aortic endothelial cells were observed when exposed to different concentrations of albumin-derived AGEs. Western and Northern blot analyses showed significant decreases at the protein and transcript levels. Both reductions became evident after 24 hours of exposure. Nuclear run-on assays showed that AGE-BSA did not modify the transcription rate of the NOS III gene; however, AGE-BSA treatment markedly reduced the half-life of NOS III mRNA. In addition, AGE-treated endothelial cells displayed significant reduction on their antiplatelet properties. These results indicate that NOS expression is reduced by AGEs by increasing the rate of mRNA degradation and may be relevant to the impairment of some endothelial functions observed in diabetes and aging. The full text of this article is available at http://www.circresaha.org.

Animals↗
PubMed2001

Increased bleeding tendency and decreased susceptibility to thromboembolism in mice lacking the prostaglandin E receptor subtype EP(3).

BACKGROUND: Among the prostanoids, thromboxane (TX) A(2) is a potent stimulator of platelets, whereas prostaglandin (PG) I(2) inhibits their activation. The roles of PGE(2) in the regulation of platelet function have not been established, however, and the contribution of PGE(2) in hemostasis and thromboembolism is poorly understood. The present study was intended to clarify these roles of PGE(2) by using mice lacking the PGE(2) receptor subtype 3 (EP(3)(-/-) mice). METHODS AND RESULTS: Expression of mRNAs for EP(3) in murine platelets was confirmed by quantitative reverse transcription-polymerase chain reaction. PGE(2) and AE-248, a selective EP(3) agonist, showed concentration-dependent potentiation of platelet aggregation induced by U46619, a TXA(2) receptor agonist, although PGE(2) alone could not induce aggregation. PGE(2) and AE-248 increased cytosolic calcium ion concentration ([Ca(2+)](i)), and AE-248 inhibited the forskolin-induced increase in cytosolic cAMP concentration ([cAMP](i)), suggesting G(i) coupling of EP(3). The potentiating effects of PGE(2) and AE-248 on platelet aggregation along with their effects on [Ca(2+)](i) and [cAMP](i) were absent in EP(3)(-/-) mice. In vivo, the bleeding time was significantly prolonged in EP(3)(-/-) mice. Moreover, when mice were challenged intravenously with arachidonic acid, mortality and thrombus formation in the lung were significantly reduced in EP(3)(-/-) mice. CONCLUSIONS: - PGE(2) potentiated platelet aggregation induced by U46619 via EP(3) by increasing [Ca(2+)](i), decreasing [cAMP](i), or both. This potentiating action of PGE(2) via EP(3) is essential in mediating both physiological and pathological effects of PGE(2) in vivo.

15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5↗
PubMed2004

Platelet-derived lysophosphatidic acid supports the progression of osteolytic bone metastases in breast cancer.

The role of lysophosphatidic acid (LPA) in cancer is poorly understood. Here we provide evidence for a role of LPA in the progression of breast cancer bone metastases. LPA receptors LPA(1), LPA(2), and LPA(3) were expressed in human primary breast tumors and a series of human breast cancer cell lines. The inducible overexpression of LPA(1) in MDA-BO2 breast cancer cells specifically sensitized these cells to the mitogenic action of LPA in vitro. In vivo, LPA(1) overexpression in MDA-BO2 cells enhanced the growth of subcutaneous tumor xenografts and promoted bone metastasis formation in mice by increasing both skeletal tumor growth and bone destruction. This suggested that endogenous LPA was produced in the tumor microenvironment. However, MDA-BO2 cells or transfectants did not produce LPA. Instead, they induced the release of LPA from activated platelets which, in turn, promoted tumor cell proliferation and the LPA(1)-dependent secretion of IL-6 and IL-8, 2 potent bone resorption stimulators. Moreover, platelet-derived LPA deprivation in mice, achieved by treatment with the platelet antagonist Integrilin, inhibited the progression of bone metastases caused by parental and LPA(1)-overexpressing MDA-BO2 cells and reduced the progression of osteolytic lesions in mice bearing CHO-beta3wt ovarian cancer cells. Overall, our data suggest that, at the bone metastatic site, tumor cells stimulate the production of LPA from activated platelets, which enhances both tumor growth and cytokine-mediated bone destruction.

Animals↗
PubMed1999

Protease-activated receptors 1 and 4 mediate activation of human platelets by thrombin.

Because of the role of thrombin and platelets in myocardial infarction and other pathological processes, identifying and blocking the receptors by which thrombin activates platelets has been an important goal. Three protease-activated receptors (PARs) for thrombin -- PAR1, PAR3, and PAR4 -- are now known. PAR1 functions in human platelets, and the recent observation that a PAR4-activating peptide activates human platelets suggests that PAR4 also acts in these cells. Whether PAR1 and PAR4 account for activation of human platelets by thrombin, or whether PAR3 or still other receptors contribute, is unknown. We have examined the roles of PAR1, PAR3, and PAR4 in platelets. PAR1 and PAR4 mRNA and protein were detected in human platelets. Activation of either receptor was sufficient to trigger platelet secretion and aggregation. Inhibition of PAR1 alone by antagonist, blocking antibody, or desensitization blocked platelet activation by 1 nM thrombin but only modestly attenuated platelet activation by 30 nM thrombin. Inhibition of PAR4 alone using a blocking antibody had little effect at either thrombin concentration. Strikingly, simultaneous inhibition of both PAR1 and PAR4 virtually ablated platelet secretion and aggregation, even at 30 nM thrombin. These observations suggest that PAR1 and PAR4 account for most, if not all, thrombin signaling in platelets and that antagonists that block these receptors might be useful antithrombotic agents.

Adenosine Triphosphate↗
PubMed2003

[Effects of higenamine on the cardio-circulatory system].

Higenamine (HG) is a potent cardioactive benzylisoquinoline alkaloid isolated from Aconiti tuber which has long been used as a cardiotonic in traditional Chinese medicine. HG exerts various effects on the cardio-circulatory system inotropic and chronotropic in isolated rat atria. It also relaxes isolated rat aorta. It inhibits epinephrine, ADP or collagen-induced platelet aggregation in platelet rich plasma. HG inhibits LPS-induced nitrate accumulation and the expression of iNOS mRNA in RAW 264.7 cells. HG lowers blood pressure in rats and increases the recovery rates in acute thrombosis model of mice, and lower the weight of thrombus formed in the arterio-venous shunt model of rats. Higenamine also has ameliorative effects in the LPS-induced DIC model.

Aconitum↗
PubMed1988

Characterization of an antibody directed against a 128 kDa glycoprotein involved in the thrombogenicity of the elastin-associated microfibrils.

We have developed a monospecific antiserum directed against a major glycoprotein in the elastin-associated microfibrils with an apparent molecular mass of 128 kDa (GP 128). When immunoblotting or enzyme-linked immunosorbent microassay was used, its IgGs recognized thrombospondin in a platelet lysate, but did not react with several basement-membrane-derived macromolecules, nor with plasma fibronectin. Similar patterns of immunofluorescence and immunoperoxidase were found after incubation of endothelial cells with either anti-GP 128 or anti-(platelet thrombospondin) IgGs. Both antibodies inhibited the microfibrils- and thrombin-induced platelet aggregation, and were without effect on the aggregation by other inducers. These results confirm that there is an antigenic homology between GP 128 and thrombospondin.

Blood Coagulation↗
PubMed1993

Cathepsin G--induced release of PAI-1 in the culture medium of endothelial cells: a new thrombogenic role for polymorphonuclear leukocytes?

Activated polymorphonuclear leukocytes (PMNs) may affect the integrity of blood vessels by endothelial cell injury. We investigated the effects of cathepsin G purified from human neutrophils on the fibrinolytic potential of cultured human umbilical vein endothelial cells (HUVECs). Cathepsin G (5 and 10 micrograms/ml) induced marked intercellular gap formation after 1 hour of treatment, whereas 1 microgram/ml did not, even after 6 hours incubation. In contrast, plasminogen activator inhibitor-1 (PAI-1) antigen levels, measured by a double antibody enzyme-linked immunosorbent assay, were significantly increased in culture media (CM) on cathepsin G (1 microgram/ml) treatment after 15 minutes (5.1 +/- 1.2 ng/ml vs 2.6 +/- 0.6 ng/ml for controls, p < 0.01) and 6 hours of incubation (69.6 +/- 17.5 ng/ml vs 40.0 +/- 9.0 ng/ml for controls, p < 0.01). Likewise, PAI activity, measured by reverse fibrin autography, increased on cell treatment with cathepsin G. Preincubation of cathepsin G with eglin C (10 micrograms/ml) almost completely abolished the increase in both PAI antigen and activity levels induced by cathepsin G. Cycloheximide, a protein synthesis inhibitor, did not block cathepsin G-induced PAI-1 release. PAI-1 mRNA levels were not affected by HUVEC treatment with cathepsin G (1 microgram/ml for 15 minutes), even after 24 hours. In the extracellular matrix (ECM) PAI-1 antigen levels decreased to 77% and 40% of controls, respectively, after 15 minutes and 6 hours of cathepsin G (1 micrograms/ml) treatment. Reverse fibrin autography also demonstrated a dose-dependent reduction of PAI activity in the ECM on 6 hours of cell treatment with 1 or 5 micrograms/ml cathepsin G. Moreover, ECM prepared from confluent HUVECs released PAI-1 in supernatants on 1 micrograms/ml cathepsin G incubation in a cell-free system. Tissue-type plasminogen activator (t-PA) activity was strongly depressed on cathepsin G treatment, both in CM from HUVECs or in a cell-free system. Finally, PAI-1 was also released from cathepsin G-stimulated platelets in a dose-dependent manner. In summary, our results support a potentially thrombogenic role of cathepsin G, which could impair the fibrinolytic potential of the endothelium. These data give a new insight into the mechanisms by which activated PMNs may promote thrombus formation. On the other hand, the decrease of PAI-1 in ECM could favor penetration and migration of inflammatory or tumor cells through the subendothelial layers.

Cathepsin G↗
PubMed1998

Granulocyte-macrophage colony-stimulating factor enhances EBV-induced synthesis of chemotactic factors in human neutrophils.

We have recently demonstrated that EBV binds to human neutrophils and stimulates a wide range of activities, including homeotypic aggregation, total RNA synthesis, and expression of the chemokines IL-8 and macrophage inflammatory protein-1alpha (MIP-1alpha). Neutrophil function is also known to be modulated by priming with granulocyte-macrophage colony-stimulating factor (GM-CSF). We have therefore investigated the modulation of EBV-induced activation of human neutrophils by GM-CSF. Treatment of neutrophils with GM-CSF before EBV activation enhanced the production of both MIP-1alpha and IL-8. The IL-8 produced under these conditions was biologically active as determined in the calcium mobilization assay. GM-CSF was also found to increase the ability of EBV to prime neutrophils for increased leukotriene B4 (LTB4) synthesis. Prior treatment of GM-CSF with neutralizing Abs inhibited these effects. GM-CSF also increased the specific binding of FITC-EBV to the neutrophil surface, as evaluated by fluorocytometry. Local production of GM-CSF in tissues invaded by EBV could therefore serve to potentiate a host defense mechanism directed toward the destruction of the infectious virus via increased production of chemotactic factors. Since both IL-8 and MIP-1alpha are reported to be chemoattractants in vitro for T cells and T and B cells, respectively, the ability of EBV to induce their production by neutrophils may enhance its ability to infect B and T lymphocytes via increased recruitment to sites of infection.

Adjuvants, Immunologic↗
PubMed1999

Sequence-dependent conformational differences of small RNAs revealed by native gel electrophoresis.

In this study, we use native polyacrylamide gel electrophoresis and one-dimensional NMR spectroscopy to analyze small RNA hairpins containing a UUCG tetraloop. The aggregation state of one RNA 16-mer (5'-CGGCUUCGGUCGACCA-3') in the presence of Mg(2+) was confirmed by laser light scattering. Although it is widely known in the RNA field that some RNAs tend to aggregate, especially when present at high concentrations, the sequence elements responsible for this effect are rarely identified. In this work, we show that Mg(2+)-induced aggregation of the 16-mer RNA hairpin is sensitive to the presence of the 3'-terminal base and a specific 2'-hydroxyl group. Our study highlights the fact that even small changes in a particular RNA sequence can increase its tendency to undergo Mg(2+)-dependent aggregation in an unpredictable manner. Our analysis also shows that native gel electrophoresis is a sensitive probe of RNA conformation with the capability to detect differences apparently caused by subtle base stacking effects at the ends of helices.

Base Sequence↗
PubMed2004

Genome-wide RNA interference screen identifies previously undescribed regulators of polyglutamine aggregation.

Protein misfolding and the formation of aggregates are increasingly recognized components of the pathology of human genetic disease and hallmarks of many neurodegenerative disorders. As exemplified by polyglutamine diseases, the propensity for protein misfolding is associated with the length of polyglutamine expansions and age-dependent changes in protein-folding homeostasis, suggesting a critical role for a protein homeostatic buffer. To identify the complement of protein factors that protects cells against the formation of protein aggregates, we tested transgenic Caenorhabditis elegans strains expressing polyglutamine expansion yellow fluorescent protein fusion proteins at the threshold length associated with the age-dependent appearance of protein aggregation. We used genome-wide RNA interference to identify genes that, when suppressed, resulted in the premature appearance of protein aggregates. Our screen identified 186 genes corresponding to five principal classes of polyglutamine regulators: genes involved in RNA metabolism, protein synthesis, protein folding, and protein degradation; and those involved in protein trafficking. We propose that each of these classes represents a molecular machine collectively comprising the protein homeostatic buffer that responds to the expression of damaged proteins to prevent their misfolding and aggregation.

Animals↗
PubMed1978

Virion RNA species of the arenaviruses Pichinde, Tacaribe, and Tamiami.

The principal RNA species isolated from labeled preparations of the arenavirus Pichinde usually include a large viral RNA species L (apparent molecular weight = 3.2 X 10(6)), and a smaller viral RNA species S (apparent molecular weight = 1.6 X 10(6)). In addition, either little or considerable quantities of 28S rRNA as well as 18S rRNA can also be obtained in virus extracts, depending on the virus stock and growth conditions used to generate virus preparations. Similar RNA species have been identified in RNA extracted from Tacaribe and Tamiami arenavirus preparations. Oligonucleotide fingerprint analyses have confirmed the host ribosomal origin of the 28S and 18S species. Such analyses have also indicated that the Pichinde viral L and S RNA species each contain unique nucleotide sequences. Viral RNA preparations isolated by conventional phenol-sodium dodecyl sulfate extraction often have much of their L and S RNA species in the form of aggregates as visualized by either electron microscopy or oligonucleotide fingerprinting of material recovered from the top of gels (run by using undenatured RNA preparations). Circular and linear RNA forms have also been seen in electron micrographs of undenatured RNA preparations, although denatured viral RNA preparations have yielded mostly linear RNA species with few RNA aggregates or circular forms.

Arenaviridae↗
PubMed2005

Measurement of dendritic mRNA transport using ribosomal markers.

mRNA is transported to the dendritic regions by forming RNA granules, an aggregate of mRNA, ribosomal proteins, rRNA, and RNA-binding proteins such as Staufen. In this study, the dendritic transport of RNA granules was measured using the individual antibodies to ribosome-specific markers such as ribosomal L4 or S6 protein, and Y10B, a monoclonal antibody specific to rRNA. All the markers showed significant immunoreactivity in the dendritic regions of the hippocampal neurons. In addition, a GFP-tagged Staufen, a marker protein of the RNA granules, was colocalized with the Y10B and S6 signals in the dendrites. The S6 signals were also colocalized with the Y10B signals in the dendrites. Consistent with previous studies, the depolarization induced by KCl stimulation increased the ribosomal level, revealed by the S6 or Y10B immunostaining in the distal dendrites. These results demonstrate the utility of ribosomal markers for detecting the RNA granules or mRNA transport in dendrites.

Animals↗
PubMed1977

Comparison of hydrophobic and strongly hydrophilic cleavable crosslinking reagents in intermolecular bond formation in aggregates of proteins or protein-RNA.

Most of the bifunctional reagents in protein chemistry possess a strongly hydrophobic backbone, derived from aliphatic or aromatic hydrocarbons. Even bifunctionals of more than 30 A in length of this sort form intramolecular bridges preferentially. In recent years, the intermolecular crosslinking of physiological protein aggregates has gained in importance. As shown in the crosslinking of hemoglobin with two sets of hydrophobic and strongly hydrophilic reagents, derived from azo dyes and tartaric acid, respectively, in this case it is not primarily the length of the bifunctional, but the hydrophilic structure that will enhance intermolecular crosslinking. Artificial dimers of native structure may be obtained. For the crosslinking of RNA to protein, we have synthesized a new reagent, 3-(2-bromo-3-oxobutane-1-sulphonyl)-propionic acid p-nitrophenyl ester. In a two step reaction, it is attached to adenine and cytosine moieties at pH 6 first, and to lysine side chains at pH 7,5. The reagent has been applied to the poly-A sequence of globin messenger RNA nucleoprotein.

Adenosine↗
PubMed1976

Defective interfering passages of Sindbis virus: nature of the defective virion RNA.

Defective interfering particles of Sindbis virus contain 20S RNA identical to that found in BHK cells co-infected with standard and defective virions. We have characterized these RNAs by their oligonucleotide fingerprints. Most of the oligonucleotides were identical to those found in the mRNA (26S RNA) that codes for the virion structural proteins. Three oligonucleotides found in 20S RNA were absent from the 26S RNA pattern and may represent sequences from the 5' end of the virion RNA. Previous difficulties in describing the nature of the defective virion RNA were due to the aggregated state of the RNA. Nucleocapsids obtained from standard and defective virions were essentially the same size and had about the same density, suggesting that defective particles contain more than a single molecule of 20S RNA.

Base Sequence↗
PubMed1968

Ribonucleic acid synthesis in cells infected with influenza virus.

Virus-specific ribonucleic acid (RNA), synthesized in influenza virus-infected cells from 3.5 to 7.5 hr after infection, was studied. After velocity centrifugation in sucrose, three peaks of virus-specific RNA could be identified: 34S, 18S, and 11S. These RNA species are predominantly single-stranded and consist of 90% viral (plus) and 10% complementary (minus) RNA strands. Most (75%) of the complementary RNA is single-stranded, i.e., not part of RNA duplexes or replicative intermediates. The 34S RNA species is an aggregate of 18S and 14S RNA species. Both 18S and 11S RNA species are relatively heterogenous compared to 18S ribosomal RNA, and these species probably contain different RNA molecules having closely related sedimentation coefficients.

Animals↗
PubMed2003

Neurofilament RNA causes neurodegeneration with accumulation of ubiquitinated aggregates in cultured motor neurons.

The mechanisms whereby mutant gene expression triggers neurodegeneration are poorly understood but have generally been attributed to translated gene products. We now demonstrate direct neuropathic effects of untranslated RNA on cultured motor neurons. We show that expression of untranslated light neurofilament (NF-L) RNA sequence in the 3'UTR of an EGFP transgene (pEGFP/NF-L RNA) or in a separate expression vector (pRc/NF-L RNA) causes dose-dependent, neuron-specific motor neuron degeneration. Neither unfused EGFP protein (pEGFP/wt) nor EGFP-tagged NF-L protein (pEGFP/NF-L protein) has similar neuropathic effects. The findings are the first demonstration of a direct RNA-mediated neurotoxic effect. Moreover, the resulting neuropathological changes show that untranslated RNA can lead to early degeneration of neuritic processes and accumulations of ubiquitinated aggregates in the perikarya and nuclei of degenerating motor neurons. The latter findings are hallmark neuropathological features of neurodegenerative diseases and their occurrence as a result of altered RNA expression raises the prospects of an RNA-mediated component in the pathogenesis of neurodegenerative states.

Animals↗