Inactivation of the Reconstituted Oxoglutarate Carrier from Bovine Heart Mitochondria by Pyridoxal 5′-Phosphate

The effect of pyridoxal 5-phosphate and some other lysine reagents on the purified,reconstituted mitochondrial oxoglutarate transport protein has been investigated. The inhibition ofoxoglutarate/oxoglutarate exchange by pyridoxal 5-phosphate can be reversed by passing theproteoliposomes through a Sephadex column but the reduction of the Schiff's base by sodiumborohydride yielded an irreversible inactivation of the oxoglutarate carrier protein. Pyridoxal5-phosphate, which caused a time- and concentration-dependent inactivation of oxoglutaratetransport with an IC₅₀ of 0.5 mM, competed with the substrate for binding to the oxoglutaratecarrier (K _i = 0.4 mM). Kinetic analysis of oxoglutarate transport inhibition by pyridoxal5-phosphate indicated that modification of a single amino acid residue/carrier molecule wassufficient for complete inhibition of oxoglutarate transport. After reduction with sodiumborohydride [³H]pyridoxal 5-phosphate bound covalently to the oxoglutarate carrier. Incubation ofthe proteoliposomes with oxoglutarate or L-malate protected the carrier against inactivationand no radioactivity was found associated with the carrier protein. In contrast, glutarate andsubstrates of other mitochondrial carrier proteins were unable to protect the carrier. Mersalyl,which is a known sulfhydryl reagent, also failed to protect the oxoglutarate carrier againstinhibition by pyridoxal 5-phosphate. These results indicate that pyridoxal 5-phosphateinteracts with the oxoglutarate carrier at a site(s) (i.e., a lysine residue(s) and/or the amino-terminalglycine residue) which is essential for substrate translocation and may be localized at or nearthe substrate-binding site.     

CD4<Superscript>+</Superscript> T cells kill Id<Superscript>+</Superscript> B-lymphoma cells: FasLigand-Fas interaction is dominant in vitro but is redundant in vivo

B-lymphoma cells express a highly tumor-specific antigen, monoclonal Ig, which is a promising target for immunotherapy. Previous work has demonstrated that B-lymphoma cells spontaneously process their endogenous monoclonal Ig and present variable (V) region peptides (Id-peptides) on their MHC class II molecules to CD4⁺ T cells. Id-specific CD4⁺ T cells protect mice against B-lymphoma cells in the absence of anti-idiotypic antibodies. The molecular mechanism by which Id-specific CD4⁺ T cells kill B-lymphoma cells is hitherto unknown. We here demonstrate in an Id-specific T-cell receptor (TCR)–transgenic mouse model that Id-specific CD4⁺ T cells induce apoptosis of Fas⁺ B-lymphoma cells in vitro by FasLigand (FasL)–Fas interaction. Moreover, the rare B lymphomas that had escaped rejection in TCR-transgenic mice had down-regulated their sensitivity to Fas-mediated apoptosis. Although these results suggest that FasL-Fas interaction is important, Id-specific CD4⁺ T cells could eliminate Id⁺ B-lymphoma cells in vivo by other mechanisms, since three independent ways of blocking FasL-Fas–mediated killing failed to abrogate tumor protection in TCR-transgenic mice. These results suggest that there are several redundant pathways by which Id-specific CD4⁺ T cells eliminate Id⁺ B-lymphoma cells in vivo, of which FasL-Fas interaction is only one.Supported by grants from the Norwegian Cancer Society, the Research Council of Norway, and the Multiple Myeloma Research Foundation.     

Oleuropein prevents oxidative myocardial injury induced by ischemia and reperfusion

The potential protective effects of oleuropein, a dietary antioxidant of olive oil, has been investigated in the isolated rat heart. The organs were subjected to 30 minutes of no-flow global ischemia and then reperfused. At different time intervals, the coronary effluent was collected and assayed for creatine kinase activity as well as for reduced and oxidized glutathione. In addition, the extent of lipid peroxidation was evaluated by measuring thiobarbituric acid reactive substance concentration in cardiac muscle. Pretreatment with 20 microg/g oleuropein before ischemia resulted in a significant decrease in creatine kinase and reduced glutathione release in the perfusate. The protective effect of oleuropein against the post-ischemic oxidative burst was investigated by measuring the release, in the coronary effluent, of oxidized glutathione, a sensitive marker of heart's exposure to oxidative stress. Reflow in ischemic hearts was accompanied by a prompt release of oxidized glutathione; in ischemic hearts pretreated with oleuropein, this release was significantly reduced. Membrane lipid peroxidation was also prevented by oleuropein. The reported data provide the first experimental evidence of a direct cardioprotective effect of oleuropein in the acute events that follow coronary occlusion, likely because of its antioxidant properties. This finding strengthens the hypothesis that the nutritional benefit of olive oil in the prevention of coronary heart disease can be also related to the high content of oleuropein and its derivatives. Moreover, our data, together with the well documented antithrombotic and antiatherogenic activity of olive oil polyphenols, indicate these antioxidants as possible therapeutic tools for the pharmacological treatment of coronary heart disease as well as in the case of cardiac surgery, including transplantation.     

Structure and function of vertebrate CMP-sialic acid synthetases

Activation of sugars into nucleotide sugars is critical for their entry into biosynthetic pathways. In eukaryotic cells, the activation of the acidic nine-carbon sugar sialic acid to CMP-sialic acid takes place in the cell nucleus, whereas all other nucleotide sugars are made in the cytoplasm. Molecular cloning of vertebrate CMP-sialic acid synthetases confirmed the nuclear localization and introduced new molecular tools for directly exploring the functional mechanisms of the enzymes, as well as the physiological relevance of their nuclear transport. Although major advances have been made in understanding structure-function relationships and defining elements involved in the nuclear transport, the riddle surrounding the physiological relevance of nuclear localization awaits resolution.     

Determination of tetracationic zinc(II) phthalocyanine derivative RLP068 in rabbit serum by liquid chromatography-tandem mass spectrometry

The development and validation of a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the determination of the tetracationic zinc(II) phthalocyanine derivative RLP068 in rabbit serum is described. The dodecadeuterated product (RLP068-D12) was used as co-eluting internal standard. RLP068 was isolated from serum samples by solid-phase extraction using weak cationic exchange cartridges (WCX). An oxidative derivatisation was used in order to simplify the peculiar HPLC and MS behaviour of the analyte and thus increasing sensitivity. Liquid Chromatography was carried out on a Polaris C18 Ether column (50 mm x 2.0 mm) with an isocratic run of 0.5% aqueous TFA/methanol. Detection was achieved by means of a Bruker Esquire 3000+ Ion Trap Mass Spectrometer equipped with an ESI source working in positive mode. A Multiple Reaction Monitoring method following the transitions 297.1 --> 282.1 for the analyte and 300.1 --> 282.1 + 285.1 for the internal standard was used. The analytical method was validated over the concentration range 2-65 ng/mL. lower limits of detection (LLOD) and quantification (LLOQ) were respectively 1 and 2 ng/mL. The method is innovative and applicable to pharmacokinetic studies.     

Preparation of DNA chips using polyamine-oligonucleotide conjugates

A convenient and efficient method for three-dimensional immobilizing oligonucleotides on glass was developed using oligonucleotide derivatives bearing a polyamine linker (PA-oligo conjugates). Polyamine (polylysine, poly(lysine, phenylalanine), polyethyleneimine) residues stipulate durable fixation of such conjugates to the glass surface with a high yield (90-95%). A DNA fragment (414-mer) is hybridized specifically to an immobilized oligonucleotide.     

Fluorescent ligands for the histamine H2 receptor: synthesis and preliminary characterization

3-[3-(Piperidinomethyl)phenoxy]alkyl, N-cyano-N′-[ω-[3-(1-piperidinylmethyl)phenoxy]alkyl]guanidine and 2-(5-methyl-4-imidazolyl)methyl thioethyl derivatives containing fluorescent functionalities were synthesized and the histamine H₂ receptor affinity was evaluated using the H₂ antagonist [¹²⁵I]-aminopotentidine. The compounds exhibited weak to potent H₂ receptor affinity with pK_i values ranging from <4 to 8.85. The highest H₂ receptor affinity was observed for N-cyano-N′-[ω-[3-(1-piperidinylmethyl)phenoxy]alkyl]guanidines substituted with methylanthranilate (13), cyanoindolizine (6) and cyanoisoindole (11) moieties via an ethyl or propyl linker.     

P130Cas-associated protein (p140Cap) as a new tyrosine-phosphorylated protein involved in cell spreading

Integrin-mediated cell adhesion stimulates a cascade of signaling pathways that control cell proliferation, migration, and survival, mostly through tyrosine phosphorylation of signaling molecules. p130Cas, originally identified as a major substrate of v-Src, is a scaffold molecule that interacts with several proteins and mediates multiple cellular events after cell adhesion and mitogen treatment. Here, we describe a novel p130Cas-associated protein named p140Cap (Cas-associated protein) as a new tyrosine phosphorylated molecule involved in integrin- and epidermal growth factor (EGF)-dependent signaling. By affinity chromatography of human ECV304 cell extracts on a MBP-p130Cas column followed by mass spectrometry matrix-assisted laser desorption ionization/time of flight analysis, we identified p140Cap as a protein migrating at 140 kDa. We detected its expression in human, mouse, and rat cells and in different mouse tissues. Endogenous and transfected p140Cap proteins coimmunoprecipitate with p130Cas in ECV304 and in human embryonic kidney 293 cells and associate with p130Cas through their carboxy-terminal region. By immunofluorescence analysis, we demonstrated that in ECV304 cells plated on fibronectin, the endogenous p140Cap colocalizes with p130Cas in the perinuclear region as well as in lamellipodia. In addition p140Cap codistributes with cortical actin and actin stress fibers but not with focal adhesions. We also show that p140Cap is tyrosine phosphorylated within 15 min of cell adhesion to integrin ligands. p140Cap tyrosine phosphorylation is also induced in response to EGF through an EGF receptor dependent-mechanism. Interestingly expression of p140Cap in NIH3T3 and in ECV304 cells delays the onset of cell spreading in the early phases of cell adhesion to fibronectin. Therefore, p140Cap is a novel protein associated with p130Cas and actin cytoskeletal structures. Its tyrosine phosphorylation by integrin-mediated adhesion and EGF stimulation and its involvement in cell spreading on matrix proteins suggest that p140Cap plays a role in controlling actin cytoskeleton organization in response to adhesive and growth factor signaling.     

Apoptotic cascade initiated by angiotensin II in neonatal cardiomyocytes: role of DNA damage

Angiotensin II contributes to ventricular remodeling by promoting both cardiac hypertrophy and apoptosis; however, the mechanism underlying the latter phenomenon is poorly understood. One possibility that has been advanced is that angiotensin II activates NADPH oxidase, generating free radicals that trigger apoptosis. In apparent support of this notion, it was found that angiotensin II-mediated apoptosis in the cardiomyocyte is blocked by the NADPH oxidase inhibitor diphenylene iodonium. However, three lines of evidence suggest that peroxynitrite, rather than superoxide, is responsible for angiotensin II-mediated DNA damage and apoptosis. First, the inducible nitric oxide inhibitor aminoguanidine prevents angiotensin II-induced DNA damage and apoptosis. Second, based on ligation-mediated PCR, the pattern of angiotensin II-induced DNA damage resembles peroxynitritemediated damage rather than damage caused by either superoxide or nitric oxide. Third, angiotensin II activates p53 through the phosphorylation of Ser15 and Ser20, residues that are commonly phosphorylated in response to DNA damage. It is proposed that angiotensin II promotes the oxidation of DNA, which in turn activates p53 to mediate apoptosis.