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PubMed2004

Reduction of DNA fragmentation and hydroxyl radical production by hyaluronic acid and chondroitin-4-sulphate in iron plus ascorbate-induced oxidative stress in fibroblast cultures.

Glycosaminoglycans (GAGs), components of extracellular matrix, are thought to play important roles in cell proliferation and differentiation in the repair process of injured tissue. Oxidative stress is one of the most frequent causes of tissue and cell injury and the consequent lipid peroxidation is the main manifestation of free radical damage. It has been found to play an important role in the evolution of cell death. Since several reports have shown that hyaluronic acid (HYA) and chondroitin-4-sulphate (C4S) are able to inhibit lipid peroxidation during oxidative stress, We investigated the antioxidant capacity of these GAGs in reducing oxidative damage in fibroblast cultures. Free radicals production was induced by the oxidizing system employing iron (Fe2+) plus ascorbate. We evaluated cell death, membrane lipid peroxidation, DNA damage, protein oxidation, hydroxyl radical (OH*) generation and endogenous antioxidant depletion in human skin fibroblast cultures. The exposition of fibroblasts to FeSO4 and ascorbate caused inhibition of cell growth and cell death, increased OH* production determined by the aromatic trap method; furthermore it caused DNA strand breaks and protein oxidation as shown by the DNA fragments analysis and protein carbonyl content, respectively. Moreover, it enhanced lipid peroxidation evaluated by the analysis of conjugated dienes (CD) and decreased antioxidant defenses assayed by means of measurement of superoxide dismutase (SOD) and catalase (CAT) activities. When fibroblasts were treated with two different doses of HYA or C4S a protective effect, following oxidative stress induction, was shown. In fact these GAGs were able to limit cell death, reduced DNA fragmentation and protein oxidation, decreased OH* generation, inhibited lipid peroxidation and improved antioxidant defenses. Our results confirm the antioxidant activity of HYA and C4S and this could represent a useful step in the understanding of the exact role played by GAGs in living organisms.

Alkadienes↗

PubMed2004

Effects of iron and phytic acid on production of extracellular radicals by Enterococcus faecalis.

Enterococcus faecalis is a human intestinal commensal that produces extracellular superoxide, hydrogen peroxide, and hydroxyl radical while colonizing the intestinal tract. To determine whether dietary factors implicated in colorectal cancer affect oxidant production by E. faecalis, radicals were measured in rats colonized with this microorganism while on diets supplemented with iron or phytic acid. Hydroxyl radical activity was measured by assaying for aromatic hydroxylation products of D-phenylalanine using reverse-phase high-performance liquid chromatography and electrochemical detection. In vitro, as expected, iron enhanced, and phytic acid decreased, hydroxyl radical formation by E. faecalis. For rats colonized with E. faecalis given supplemental dietary iron (740 mg elemental iron as ferric phosphate per kg diet) or phytic acid (1.2% w/w), no differences were found in concentrations of urinary ortho- or meta- isomers of D-phenylalanine compared to rats on a basal diet. Aqueous radicals in colonic contents were further assessed ex vivo by electron spin resonance using 5,5-dimethyl-1-pyrroline-N-oxide as a spin trap. Mixtures of thiyl (sulfur-centered) and oxygen-centered radicals were detected across all diets. In vitro, similar spectra were observed when E. faecalis was incubated with hydrogen sulfide, air-oxidized cysteine, or an alkylsulfide, as typical sulfur-containing compounds that might occur in colonic contents. In conclusion, intestinal colonization with E. faecalis in a rat model generates both thiyl and oxygen-centered radicals in colonic contents. Radical formation, however, was not significantly altered by short-term dietary supplementation with iron or phytic acid.

Animals↗

PubMed2006

A deletion variant study of the functional role of the Salmonella flagellin hypervariable domain region in motility.

The eubacterial flagellum is a complex structure with an elongated extracellular filament that is composed primarily of many subunits of a flagellin protein. The highly conserved N and C termini of flagellin are important in its export and self-assembly, whereas the middle sequence region varies greatly in size and composition in different species and is known to be deletion-tolerant. In Salmonella typhimurium phase 1 flagellin, this "hypervariable" region encodes two solvent-exposed domains, D2 and D3, that form a knob-like feature on flagella fibers. The functional role of this structural feature in motility remains unclear. We investigated the structural and physiological role of the hypervariable region in flagella assembly, stability and cellular motility. A library of random internal deletion variants of S. typhimurium flagellin was constructed and screened for functional variants using a swarming agar motility assay. The relative cellular motility and propulsive force of ten representative variants were determined in semi-solid and liquid medium using colony swarming motility assays, video microscopy and optical trapping of single cells. All ten variants exhibited diminished motility, with varying extents of motility observed for internal deletions less than 75 residues and nearly complete loss of motility for deletions greater than 100 residues. The mechanical stability of the variant flagella fibers also decreased with increasing size of deletion. Comparison of the variant sequences with the wild-type sequence and structure indicated that all deletions involved loss of hydrophobic core residues, and removal of both partial and complete segments of secondary structure in the D2 and D3 domains. Homology modeling predicted disruptions of secondary structures in each variant. The hypervariable region D2 and D3 domains appear to stabilize the folded conformation of the flagellin protein and contribute to the mechanical stability and propulsive force of the flagella fibers.

Agar↗

PubMed1985

Uridylate-trapping sugar analogs in combination with inhibitors of uridylate synthesis de novo and 5-fluorouridine.

The combination of 3 types of antipyrimidines was studied in AS-30D hepatoma cells in suspension culture and in the rat in vivo. Cellular UTP and CTP pools can be depleted most effectively by combining an inhibitor of de novo UMP synthesis with sugar analogs diverting UMP to UDP-sugar analogs. The following UMP-trapping sugar analogs were employed: D-galactosamine, D-galactosone, D-glucosone, and D-glucosamine. These D-galactose and D-glucose analogs intensified the depletion of UTP and CTP pools induced by the following inhibitors of de novo UMP synthesis: acivicin, PALA, lapachol, pyrazofurin, and 6-azauridine. The sugar analogs, in the absence of inhibitors of the de novo pathway, enhanced the rate of de novo UMP synthesis several-fold, as indicated by incorporation of 14CO2 into intermediates and products of the pathway and by the expansion of the acid-soluble uracil nucleotide pool. Reduction of UTP and CTP contents to less than 5 and 15% of control, respectively, by D-galactosamine and PALA resulted in a decrease of the rate of RNA synthesis to 19% of control as calculated from the changes in specific activities of [14C]CTP and of [14C]cytidine in RNA after labeling with [14C]uridine. Hepatoma cells released uridine and cytidine into the extracellular fluid. This release was reduced to one third in UTP-deficient cells, indicating that pyrimidine nucleoside excretion is regulated by pyrimidine nucleotide levels, possibly by UTP and CTP regulation of uridine kinase. Determination of the rates of de novo pyrimidine synthesis, of the formation of RNA pyrimidines, and of pyrimidine nucleoside excretion indicates that de novo synthesis provides only about 67% of the pyrimidines required for the consuming processes. The difference, as well as the dilution of labeled pyrimidine nucleotide pools under conditions of a blocked de novo pathway, suggests a considerable salvage of pyrimidine nucleosides derived from RNA. This salvage of pyrimidines may be intracellular and/or by an excretion and re-uptake process. Depletion of UTP and CTP pools, induced in hepatoma cells by D-galactosamine and 6-azauridine, leads to growth inhibition in suspension culture; this inhibition becomes irreversible in an increasing percentage of cells, killing all cells after 20 hr of UTP deficiency. The enhanced uptake of 5-fluorouridine by UTP-deficient cells was associated with an increase of FUMP incorporation into RNA up to 4-fold and with stronger inhibition of cell growth.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗

PubMed1996

New concepts in tissue repair: skin as an example.

Several heparin-binding growth factors (HBGFs) are thought to play a key role in natural processes of tissue regeneration or repair after release from inflammatory or circulating cells and extracellular matrix-associated heparan sulfate proteoglycosaminoglycans. To clarify how the bioavailability of these HBGFs can help regulate wound-healing processes, we studied the healing effect of various chemically substituted dextrans (RGTA) selected for their affinity for HBGFs. One member of the RGTA family, RGTA11, obtained by substitution of carboxymethyl (CM), benzylamide (B) and benzylamide sulfonate (S) groups in a proportion of 110% (CM), 2.6% (B) and 36.5% (S) respectively was used in these studies. RGTA11 may potentiate the biological activity of fibroblast growth factors 1 and 2 and protect them against heat or pH inactivation and proteolytic degradation. RGTA11 was tested in a rat punch-biopsy skin-healing model for its ability to enhance wound repair. Wounds were filled with collagen plaster alone or soaked with RGTA, and skin regeneration was studied by histological analysis. In collagen plaster, RGTA11 affected both the kinetics and quality of restored skin. It seems likely that endogenous growth factors naturally released during the regeneration process are trapped and protected against natural proteases, thereby preserving their ability to stimulate tissue repair. Since most known growth factors have a nearly ubiquitous distribution and blind to heparin, our hypothesis was verified by studying the ability of RGTA to induce repair in damaged tissue. We demonstrated the RGTA could stimulate wound repair in various models, including bone, muscle, nerve, cornea and colonic anastomosis. The data presented here concern wound-healing in a deep skin model and suggest that heparan-like biopolymers constitute a new family of tissue-repair agents with a wide variety of potential uses. The efficiency of this approach in cases in which impaired healing is associated with a pathology, as in diabetes, remains to be determined.

Animals↗

PubMed1998

Copper-binding amyloid precursor protein undergoes a site-specific fragmentation in the reduction of hydrogen peroxide.

The extracellular domain of transmembrane Abeta amyloid precursor protein (APP) has a Cu(II) reducing activity upon Cu(II) binding associated with the formation of a new disulfide bridge. The complete assignment of the disulfide bond revealed the involvement of cysteines 144 and 158 around copper-binding histidine residues. The vulnerability of APP-Cu(I) complexes to reactive oxygen species was elaborated as a site-specific and random fragmentation of APP in a time-dependent manner and at low concentrations of H2O2. Analysis of the specific reaction revealed the generation of C-terminal polypeptides, containing the Abeta domain. APP catalyzed the reduction of H2O2 and oxidation of Cu(I) to Cu(II) in a "peroxidative" reaction in vitro. The resulting bound copper-hydroxyl radical intermediate [APP-Cu(II)(.OH)] then likely participated in a Fenton type of reaction with radical formation as a prerequisite for protein degradation. Evidence from two observations suggests that the reaction takes place in two phases. Bathocuproine, a trapping agent for Cu(I), abolished the initial fragmentation, and chelation of Cu(II) by DTPA (diethylenetriaminepentaacetic acid) interrupted the reaction cascade induced by H2O2 at later stages. Consequently, the results suggest that a cytotoxic gain-of-function of APP-Cu(I) complexes might result in a perturbation of free radical homeostasis. What significance such a perturbation may have for the pathogenesis of Alzheimer's disease remains to be determined.

Amino Acid Sequence↗

PubMed2000

Bait region cleavage and complex formation of human alpha2M with a Porphyromonas gingivalis W50 protease is not accompanied by enzyme inhibition.

Three isoforms of extracellular Arg-specific proteases of P. gingivalis, W50, HRgpA, RgpAcat and mt-RgpAcat, which are all products of the same gene, show identical enzymatic properties toward small chromogenic substrates but have different subunit organisation and molecular size. In order to examine the potential inhibition of these proteases in vivo by host protease inhibitors, the interaction of HRgpA (approximately 110 kDa) and RgpAcat (approximately 55 kDa) with human (alpha2M and their cytotoxicity toward cultured fibroblasts were investigated. Both enzymes formed complexes with (alpha2M as shown by gel filtration chromatography and both cleaved the 'bait' region at Arg696-Leu697. However, whereas (alpha2M-RgpAcat) complex was unable to hydrolyse large substrates such as hide powder azure, (alpha2M-HRgpA) complex hydrolysed both small and large substrates. HRgpA was able to bind to alpha2M saturated with trypsin and also to methylamine-treated alpha2M. This suggested that HRgpA is able to bind to both 'slow' and 'fast' forms of alpha2M and formation of (alpha2M:HRgpA) complex does not trap HRgpA and cause inhibition of activity toward hide powder azure. However, the (alpha2M-HRgpA) complex is not able to cleave other alpha2M molecules, which suggests that the active site of HRgpA in the complex is constrained probably due to steric reasons. The (alpha2M-HRgpA) complex was cytotoxic to 3T3 cells, causing them to round up and detach from the surface with a reduction in metabolic activity.

3T3 Cells↗

PubMed1991

Actin is a noncompetitive plasmin inhibitor.

Actin, one of the most abundant cellular proteins, circulates at micromolar concentrations in peripheral blood. Because actin released from dying cells may be trapped in fibrin clots that form at sites of tissue injury, we examined the effects of actin upon lysis of fibrin clots in vitro. Incorporation of native rabbit skeletal muscle actin into fibrin clots slowed their rates of lysis for periods of up to 24 h, an effect not seen when comparable concentrations of human IgG or bovine serum albumin were added instead. Actins isolated from a variety of sources inhibited plasmin's hydrolysis of the synthetic substrate S-2251 in a noncompetitive manner, with a Ki of a 0.6-3.1 microM. Inhibition was rapid, but covalent actin-plasmin complexes were not formed. Both epsilon-aminocaproic acid and tranexamic acid prevented actin's inhibition of plasmin, suggesting that accessible lysine residues of actin interact with the kringle (lysine-binding) regions of plasmin. Neither of the high-affinity actin-binding proteins of plasma (plasma gelsolin and vitamin D-binding protein) prevented actin from inhibiting plasmin. These findings suggest that actin released into the extracellular space following cell death may modulate plasmin action, and hence a number of plasmin-dependent biological responses, at sites of inflammation and tissue injury.

Actins↗

PubMed2006

Cellular adhesion responses to the heparin-binding (HepII) domain of fibronectin require heparan sulfate with specific properties.

Cell surface heparan sulfate (HS) proteoglycans are required in development and postnatal repair. Important classes of ligands for HS include growth factors and extracellular matrix macromolecules. For example, the focal adhesion component syndecan-4 interacts with the III(12-14) region of fibronectin (HepII domain) through its HS chains. The fine structure of HS is critical to growth factor responses, and whether this extends to matrix ligands is unknown but is suggested from in vitro experiments. Cell attachment to HepII showed that heparin oligosaccharides of >or=14 sugar residues were required for optimal inhibition. The presence of N-sulfated glucosamine in the HS was essential, whereas 2-O-sulfation of uronic acid or 6-O-sulfation of glucosamine had marginal effects. In the more complex response of focal adhesion formation through syndecan-4, N-sulfates were again required and also glucosamine 6-O-sulfate. The significance of polymer N-sulfation and sulfated domains in HS was confirmed by studies with mutant Chinese hamster ovary cells where heparan sulfation was compromised. Finally, focal adhesion formation was absent in fibroblasts synthesizing short HS chains resulting from a gene trap mutation in one of the two major glucosaminoglycan polymerases (EXT1). Several separate, specific properties of cell surface HS are therefore required in cell adhesion responses to the fibronectin HepII domain.

Animals↗

PubMed1987

Protective effect of transient calcium reduction against reperfusion-induced arrhythmias in rat hearts.

By using the isolated rat heart with regional ischemia and a perfusate Ca2+ of 2.4 mM, we have observed a bell-shaped relationship between the incidence of reperfusion-induced ventricular fibrillation (VF) and the duration of preceding ischemia. With 5, 10, 20, 30, and 40 min of ischemia, 8, 83, 58, 25, and 8% of the hearts (n = 12 per group) exhibited irreversible VF. In additional studies (10 min ischemia) perfusate Ca2+ was reduced (to 0.1, 0.4, 0.8, 1.2, 1.6, and 2.0 mM) for 1 min before coronary occlusion (to trap low Ca2+ in the ischemic zone) and for 1 min before and throughout reperfusion. VF fell in a dose-dependent manner from its control incidence of 83% (2.4 mM Ca2+) to 17 (P less than 0.01), 25 (P less than 0.01), 41, 58, 91, and 83%, respectively. In further studies Ca2+ was reduced to 0.4 mM just before ischemia and also just before and during reperfusion, and the ischemic time was varied (5, 10, 20, 30, or 40 min), VF was reduced from its time-matched control (2.4 mM Ca2+) values of 8, 83, 58, 25, and 8% to 0, 25 (P less than 0.01), 8 (P less than 0.05), 0, and 0%, respectively. In a final series of experiments, Ca2+ reduction (to 0.4 mM) was carried out only during reperfusion (1 min before and throughout reperfusion). When time-vulnerability curves were constructed some protection was observed after 20 min of ischemia (VF fell from its control incidence of 58 to 8%). We conclude that extracellular Ca2+ concentration can influence vulnerability to reperfusion-induced arrhythmias.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed2003

Role of MAPK phosphorylation in cytoprotection by pro-vitamin C against oxidative stress-induced injuries in cultured cardiomyoblasts and perfused rat heart.

The reactive oxygen species (ROS) are known to be generated upon post-ischemic reperfusion (I/R) of the heart, and to injure cardiac muscle cells. The hydrogen peroxide-induced mortality of rat cardiomyoblasts H2c9 was markedly inhibited by previous administration with auto-oxidation-resistant pro-vitamin C, the 2-O-phosphorylated derivative (Asc2P) of ascorbic acid (Asc). The cytoprotection was partially counteracted by an inhibitor of MAPK (mitogen-activated protein kinase) kinase (MEK) as shown by DNA strand cleavage assay and mitochondrial dehydrogenase assay. Immunostains indicated that phosphorylated MAPK increased in the hydrogen peroxide-treated cardiomyoblasts, and that this action was moderately inhibited by Asc2P and restored nearly to the initial, pretreatment level by combined administration of the MEK inhibitor and Asc2P. The I/R-induced cell injuries in perfused rat hearts as estimated by extracellular release of the cardiac enzyme CPK were inhibited by 2-O-alpha-glucosylascorbic acid (Asc2G) and Asc, whereas the observed cytoprotection for the cardiomyoblasts was partially counteracted by the MEK inhibitor. The increase in phosphorylated MAPK in I/R-operated hearts was moderately inhibited by pro-vitamin C, but restored nearly to the normal non-operated level by combined administration with the MEK inhibitor. This is in contrast to no alteration in levels of non-phosphorylated MAPK for all the cases examined as shown by Western blots, consistent with results of immunostains for the cardiomyoblasts. The inhibitory effect of the MEK inhibitor on MAPK phosphorylation was, therefore, suggested to counteract the cytoprotective effects of pro-vitamin C via a thorough interruption of the phosphorylated MAPK signaling pathway. This was not true of ROS-related events; the scavenging effects of Asc2G and Asc on hydroxyl radicals generated from I/R-operated heart were not affected by combined administration with the MEK inhibitor, as shown by the spin-trapping DMPO-based ESR method.

Aldehyde Dehydrogenase↗

PubMed2001

Alternative splicing in the ligand binding domain of mouse ApoE receptor-2 produces receptor variants binding reelin but not alpha 2-macroglobulin.

LR7/8B and ApoER2 are recently discovered members of the low density lipoprotein (LDL) receptor family. Although structurally different, these two proteins are derived from homologous genes in chicken and man by alternative splicing and contain 7 or 8 LDL receptor ligand-binding repeats. Here we present the cDNA for ApoER2 cloned from mouse brain and describe splice variants in the ligand binding domain of this protein, which are distinct from those present in man and chicken. The cloned cDNA is coding for a receptor with only five LDL receptor ligand-binding repeats, i.e. comprising repeats 1-3, 7, and 8. Reverse transcriptase-polymerase chain reaction analysis of mRNA from murine brain revealed the existence of two additional transcripts. One is lacking repeat 8, and in the other repeat 8 is substituted for by a 13-amino acid insertion with a consensus site for furin cleavage arising from an additional small exon present in the murine gene. None of the transcripts in the mouse, however, contain repeats 4-6. In murine placenta only the form containing repeats 1-3 and 7 and the furin cleavage site is detectable. Analysis of the corresponding region of the murine gene showed the existence of 6 exons coding for a total of 8 ligand binding repeats, with one exon encoding repeats 4-6. Exon trapping experiments demonstrated that this exon is constitutively spliced out in all murine transcripts. Thus, the murine ApoER2 gene codes for receptor variants harboring either 4 or 5 binding repeats only. Recombinant expression of the 5-repeat and 4-repeat variants showed that repeats 1-3, 7, and 8 are sufficient for binding of beta-very low density lipoprotein and reelin, but not for recognition of alpha(2)-macroglobulin, which binds to the avian homologue of ApoER2 harboring 8 ligand binding repeats.

Alternative Splicing↗

PubMed1993

Origin of fibronectin in epiretinal membranes of proliferative vitreoretinopathy and proliferative diabetic retinopathy.

Fibronectins, high molecular multifunctional glycoproteins of the extracellular matrix and plasma, have been a popular area of research in the pathogenesis of proliferative disorders of the retina. Several immunohistochemical studies have revealed that fibronectin is a major constituent of epiretinal membranes and that the cell types involved in proliferative intraocular disorders may synthesise it. However, owing to the fact that plasma and cellular fibronectin are similar in their overall structure, the origin of fibronectin in epiretinal membranes has not yet been clearly defined. In this study, we used two monoclonal antibodies: FN-3, which recognises an extra domain present in the cellular but not plasma form of fibronectin; and FN-4, which reacts with an antigenic site on both plasma and cellular fibronectin. In 37 epiretinal membranes obtained from eyes with proliferative vitreoretinopathy and proliferative diabetic retinopathy, we demonstrated the presence of cellular fibronectin, thus indicating local production. The significantly stronger and positive immunostain with FN-4 in the same specimens suggests the colocalisation of plasma fibronectin, that may be derived from the breakdown of the blood-retinal barrier and trapped in membranes during their formation. In pathological vitreous we demonstrated both types of fibronectin by western blot analysis.

Blotting, Western↗

PubMed1996

Induction of p21 mediated by reactive oxygen species formed during the metabolism of aziridinylbenzoquinones by HCT116 cells.

Aziridinylbenzoquinones are a group of antitumor agents that elicit cytotoxicity by generating either alkylating intermediates or reactive oxygen species. The mechanism of toxicity may not always, however, involve profound damage of cellular constituents, but may involve a cytostatic effect through interference with the cell cycle. In this context, we have examined the induction of the cell cycle inhibitor p21 (WAF1, CIP1, or sdi1), whose overexpression suppresses the growth of various tumor cells, in human tumor cells metabolizing 3,6-diaziridinyl-1,4-benzoquinone (DZQ) and its C2,C5-substituted derivatives: 2,5-bis-(carboethoxyamino) (AZQ) and 2, 5-bis-2(-hydroxyethylamino) (BZQ). Both DZQ and AZQ were effectively activated by HCT116 human colonic carcinoma cells; the activation of the former involved largely a dicoumarol-sensitive activity, whereas that of the latter appeared to be accomplished primarily by one-electron transfer reductases. BZQ was not a substrate for the dicoumarol-sensitive enzyme in HCT116 cells. Cellular activation of the first two quinones was associated with formation of oxygen-centered radicals as detected by EPR in conjunction with the spin trap 5,5'-dimethyl-1-pyrroline-N-oxide. The redox transitions of DZQ involved hydroxyl radical formation and were strongly inhibited by catalase, whereas those of AZQ showed a strong superoxide anion component sensitive to superoxide dismutase. These signals were suppressed by N-acetylcysteine with concomitant production of a thiyl radical adduct. This suggests an effective electron transfer between the thiol and free radicals formed during the activation of these quinones. DZQ and AZQ induced significantly the expression of p21 in HCT116 cells, but a 10-fold higher concentration of AZQ was required to achieve the level of induction elicited by DZQ. BZQ had little effect on p21 expression. p21 induction at both mRNA and protein levels correlated with the inhibition of either cyclin-dependent kinase activity or cell proliferation. p21 induction elicited by the above quinones was inhibited by N-acetylcysteine, whereas the non-sulfur analog, N-acetylalanine, was without effect. Catalase and superoxide dismutase did not effect p21 induction by aziridinylbenzoquinones in HCT116 cells, thus suggesting that extracellular sources of oxygen radicals generated by plasma membrane reductases have no influence in the expression of this gene. Hydrogen peroxide, a product of quinone redox cycling, elicited an increase of p21 mRNA levels in HCT116 and K562 human chronic myelogenous leukemia cells. The latter lacks p53, one of the activators of p21 transcription, thus suggesting that p21 expression can be accomplished in a p53-independent manner in these cells. This study suggests that p21 induction is mediated by an increase in the cellular steady-state concentration of oxygen radicals and that the greater effectiveness in p21 induction by DZQ may be related to its efficient metabolism by NAD(P)H:quinone oxidoreductase activity in HCT116 cells.

Acetylcysteine↗

PubMed2002

The efflux of purine nucleobases and nucleosides from the rat brain.

The efflux of purine nucleobases and their nucleosides from the rat brain was investigated using the brain efflux index (BEI) method. Calculated BEI values showed that purine nucleobases had very rapid initial efflux after the intracerebral injection, which was followed by the slower efflux due to the intracellular trapping of labelled molecules and confirmed by the capillary depletion technique. The efflux of ribonucleosides was much slower than the efflux of nucleobases and the structure of the sugar moiety seemed to be important, since a significant difference in the efflux velocity between ribo- and deoxyribonucleosides was observed. The results of self- and cross-inhibition studies suggested that the efflux of test molecules was saturable and that purines shared the same transport system on the abluminal side of the blood-brain barrier.

Adenosine↗

PubMed2004

Decoy receptor 3 (DcR3) induces osteoclast formation from monocyte/macrophage lineage precursor cells.

Recent evidence indicates that the decoy receptor 3 (DcR3) of the TNF receptor superfamily, which initially though prevents cytokine responses of FasL, LIGHT and TL1A by binding and neutralization, can modulate monocyte function through reverse signaling. We show in this work that DcR3 can induce osteoclast formation from human monocytes, murine RAW264.7 macrophages, and bone marrow cells. DcR3-differentiated cells exhibit characteristics unique for osteoclasts, including polynuclear giant morphology, bone resorption, TRAP, CD51/61, and MMP-9 expression. Consistent with the abrogation of osteoclastogenic effect of DcR3 by TNFR-Fc, DcR3 treatment can induce osteoclastogenic cytokine TNF-alpha release through ERK and p38 MAPK signaling pathways. We conclude that DcR3 via coupling reverse signaling of ERK and p38 MAPK and stimulating TNF-alpha synthesis is a critical regulator of osteoclast formation. This action of DcR3 might play an important role in significant osteoclastic activity in osteolytic bone metastases.

Acid Phosphatase↗

PubMed1999

Protective effect of histidine on hydroxyl radical generation induced by potassium-depolarization in rat myocardium.

We investigated the efficacy of histidine on potassium-depolarization induced hydroxyl radical (*OH) generation in the extracellular fluid of rat myocardium by a flexibly mounted microdialysis technique (O system). After the rat was anesthetized, a microdialysis probe was implanted in the left ventricular myocardium, and then sodium salicylate in Ringer's solution (0.5 nmol/microl per minute) was infused to detect the generation of *OH as reflected by the nonenzymatic formation of 2,3-dihydroxybenzoic acid (DHBA). Infusion of KCl (70 mM) clearly produced an increase in *OH formation. However, when KCl in the presence of a high concentration of histidine (25 mM) was infused through the microdialysis probe, KCl failed to increase the 2,3-DHBA formation. To examine the effect of histidine on ischemia-reperfusion of the myocardium, the heart was subjected to myocardial ischemia for 15 min by occlusion of the left anterior descending coronary artery (LAD). When the heart was reperfused, a marked elevation of the levels of 2,3-DHBA was observed in the heart dialysate. However, when corresponding experiments were performed with histidine (25 mM)-pretreated animals, histidine prevented the ischemia-reperfusion induced *OH formation trapped as 2,3-DHBA. These results indicate that histidine may protect against K+-depolarization-evoked *OH generation in rat myocardium.

Animals↗

PubMed2003

Discovery of potent peptide-mimetic antagonists for the human thrombin receptor, protease-activated receptor-1 (PAR-1).

Protease-activated receptors (PARs) represent a unique family of seven-transmembrane G-protein-coupled receptors, which are enzymatically cleaved to expose a new extracellular N-terminus that acts as a tethered activating ligand. PAR-1 is cleaved and activated by the serine protease alpha-thrombin, is expressed in various tissues (e.g. platelets and vascular cells), and is involved in cellular responses associated with hemostasis, proliferation, and tissue injury. By using a de novo design approach, we have discovered a series of potent heterocycle-based peptide-miimetic antagonists of PAR-1, exemplified by advanced leads RWJ-56110 (22) and RWJ-58259 (32). These compounds are potent, selective PAR-1 antagonists, devoid of PAR-1 agonist and thrombin inhibitory activity: they bind to PAR-1, interfere with calcium mobilization and cellular functions associated with PAR-1, and do not affect PAR-2, PAR-3, or PAR-4. RWJ-56110 was determined to be a direct inhibitor of PAR-1 activation and internalization, without affecting PAR-1 N-terminal cleavage. At high concentrations of alpha-thrombin, RWJ-56110 fully blocked activation responses in human vascular cells, but not in human platelets; whereas, at high concentrations of TRAP-6, RWJ-56110 blocked activation responses in both cell types. This result is consistent with the presence of another thrombin receptor on human platelets, namely PAR-4. RWJ-56110 and RWJ-58259 clearly interrupt the binding of a tethered ligand to its receptor. RWJ-58259 demonstrated antirestenotic activity in a rat balloon angioplasty model and antithrombotic activity in a cynomolgus monkey arterial injury model. Such PAR-1 antagonists should not only serve as useful tools to delineate the physiological and pathophysiological roles of PAR-1, but also may have therapeutic potential for treating thrombosis and restenosis in humans.

Animals↗