Effects of maternal hyperhomocysteinemia induced by methionine intake on oxidative stress and apoptosis in pup rat brain

Maternal hyperhomocysteinemia is associated with a number of complications such as preeclampsia syndrome, thromboembolic events, repeated miscarriages, abruptio placentae, in utero fetal death, intrauterine fetal growth restriction and fetal neural tube defects. However, little is known about the mechanism of homocysteine on the degeneration of fetal brain. Thus, our study is aimed to investigate the effects of maternal hyperhomocysteinemia on oxidative stress and apoptosis in pup brain. Hyperhomocysteinemia was induced in female rats by way of administrating methionine dissolved in water at a dose of 1 g/kg body weight throughout the pregnancy. After delivery, level of lipid peroxidation (LPO; as malondialdehyde + 4-hydroxyalkenals) was determined in various fractions of pub brains. Furthermore, DNA fragmentation, levels of Bcl-2 protein and p53 mRNA expression were determined to evaluate apoptosis. Significant elevation was found in the levels of LPO in subcellular fractions of pup brains delivered from hyperhomocysteinemic mothers. DNA fragmentation, a hallmark of apoptosis was observed in the brain of pups of homocysteine group while significant reduction was seen in the levels of anti-apoptotic Bcl-2 levels. In addition, maternal hyperhomocysteinemia increased cerebral p53 mRNA expression above the control value. As a conclusion, we demonstrate and suggest that the pups of hyperhomocysteinemic mothers have an increased oxidative stress in brain tissues. The increased oxidative stress appears to cause apoptosis and cell death. These results may be significant to understand chronic pathology of the complications of hyperhomocysteinemia and congenital malformations of fetuses.     

14-3-3zeta interacts with human thromboxane receptors and is involved in the agonist-induced activation of the extracellular-signal-regulated kinase

Thromboxane receptor (TP) signaling results in a broad range of cellular responses including kinase activation and subsequent nuclear signaling events involved in cell transformation, proliferation, and cell survival. Proteins that may participate in the early signaling following receptor activation remain to be identified. We found that 14-3-3zeta is a novel protein interacting with TP intracellular loop 3 (i3) by yeast two-hybrid system. This interaction was further confirmed by GST pull-down and co-immunoprecipitation methods. Site-directed mutagenesis studies indicated that Pro-236 of the TP-i3 was involved in the binding to the 14-3-3zeta. Co-immunoprecipitation studies in the same cell lysate by TP antibody showed that TP binds not only with the 14-3-3zeta but also with the Raf-1. Our data also demonstrated that TP receptor activation induced by agonist rapidly recruited 14-3-3zeta and Raf-1 to form a complex with the TP on the plasma membrane. The significance of assembling this protein complex was examined by TP agonist-induced extracellular-signal-regulated kinase (ERK) phosphorylation in intact cells. TP agonist, I-BOP, induced ERK phosphorylation in HEK 293 cells expressing wild type TPalpha but significantly lower in those expressing TPalpha-P236V mutant. Attenuation of the expression of 14-3-3zeta by 14-3-3zeta siRNA decreased I-BOP-induced ERK phosphorylation indicating the involvement of the 14-3-3zeta in the signal transduction process. These results suggest that 14-3-3zeta may serve as a scaffold protein to form a protein complex consisting of TP, 14-3-3zeta, and Raf-1, and that this protein complex may be involved in the activation of ERK pathway following TP receptor activation.     

O(6)-methylguanine DNA-methyltransferase (MGMT) overexpression in melanoma cells induces resistance to nitrosoureas and temozolomide but sensitizes to mitomycin C

Alkylating agents play an important role in the chemotherapy of malignant melanomas. The activity of alkylating agents depends on their capacity to form alkyl adducts with DNA, in some cases causing cross-linking of DNA strands. However, the use of these agents is limited by cellular resistance induced by the DNA repair enzyme O(6)-methylguanine DNA-methyltransferase (MGMT) which removes alkyl groups from alkylated DNA strands. To determine to what extent the expression of MGMT in melanoma cells induces resistance to alkylating agents, the human cell line CAL77 Mer- (i.e., MGMT deficient) were transfected with pcMGMT vector containing human MGMT cDNA. Several clones expressing MGMT at a high level were selected to determine their sensitivity to chemotherapeutic drugs. Melanoma-transfected cells were found to be significantly less sensitive to nitrosoureas (carmustine, fotemustine, streptozotocin) and temozolomide with an increase of IC(50) values between 3 and 14 when compared to parent cells. No difference in cell survival rates between MGMT-proficient and -deficient cells was observed for melphalan, chlorambucil, busulphan, thiotepa and cisplatin which preferentially induce N(7) guanine lesions. Surprisingly, MGMT overexpression increased the sensitivity of CAL77 cells to mitomycin C by approximately 10-fold. Treatment of clonal cell lines with buthionine-[S,R]-sulfoximine (BSO), an inhibitor of gamma-glutamylcysteine synthetase which depletes cellular glutathione, completely reversed this unexpected increase in sensitivity to mitomycin C. This observation suggests that glutathione is involved in the sensitivity of MGMT-transfected cells to mitomycin C and may act synergistically with MGMT via an unknown mechanism.     

Anti-proliferation effect of 3-amino-2-imino-3,4-dihydro-2H-1,3-benzothiazin-4-one (BJ-601) on human vascular endothelial cells: G0/G1 p21-associated cell cycle arrest

The aim of this study was to examine the anti-proliferation effect of 3-amino-2-imino-3,4-dihydro-2H-1,3-benzothiazin-4-one (BJ-601) on human vascular endothelial cells and its possible molecular mechanism underlying. Our data showed that BJ-601 at a range of concentrations (0-40 microM) dose- and time-dependently decreased cell number in cultured human dermal microvascular endothelial cells (HDMVECs), but not human fibroblasts. The BJ-601-induced growth inhibition in HDMVECs was reversible. [3H]thymidine incorporation demonstrated that BJ-601 arrested the HDMVECs at the G0/G1 phase of the cell cycle. Western blot analysis revealed that BJ-601 (0-40 microM) dose-dependently increased the levels of the protein p21, but not of p27, p53, cyclins A, D1, D3 and E, cyclin-dependent kinase 2 (CDK2), and CDK4 in HDMVECs. Immunoprecipitation showed that the formation of the CDK2-p21 complex, but not CDK2-p27, CDK4-p21 and CDK4-p27 complexes, was increased in the BJ-601-treated HDMVECs. Kinase assay further demonstrated that CDK2, but not CDK4, kinase activity was decreased in a dose-dependent manner in the BJ-601-treated HDMVECs. Pretreatment of HDMVECs with a p21 antisense oligonucleotide, which blocked the expression of p21 protein, reversed the BJ-601-induced inhibition of [3H]thymidine incorporation into HDMVECs. Moreover, cotreatment of the endothelial cells with protein kinase C (PKC) inhibitor, staurosporine, prevented the BJ-601-induced decrease of [3H]thymidine incorporation into HDMVECs. Administration of BJ-601 dose-dependently inhibited capillary-like tube formation of HDMVECs in Matrigel. In conclusion, these data suggest that BJ-601 inhibits HDMVECs proliferation by increasing the level of p21 protein, which in turn inhibits CDK2 kinase activity, and finally causes retardation of the cell cycle at the G0/G1 phase.     

Degradation of nociceptin (orphanin FQ) by mouse spinal cord synaptic membranes is triggered by endopeptidase-24.11: an in vitro and in vivo study

We analyzed spinal metabolic pathway of nociceptin/orphanin FQ related to pain-transmission or modulation in the both in vitro and in vivo experiments. Nociceptin was degraded by spinal synaptic membranes. Major metabolites of nociceptin were free phenylalanine, nociceptin (1-13) and nociceptin (14-17). Both the degradation of nociceptin and the accumulation of the major cleavage metabolites, nociceptin (1-13) and nociceptin (14-17), were strongly inhibited by a metal chelator and also by specific inhibitors of endopeptidase-24.11, thiorphan and phosphoramidon. Furthermore, purified endopeptidase-24.11 hydrolyzed nociceptin at the cleavage site (Lys(13)-Leu(14) bond) identical to that by spinal synaptic membranes. Recently, we have found that nociceptin, injected intrathecally at small doses (fmol order) elicits a behavioral response consisting of scratching, biting and licking in mice. In the present study, we have examined the effect of peptidase inhibitors on the behavioral response elicited by intrathecal injection of nociceptin in mice. Phosphoramidon simultaneously injected with nociceptin additively enhanced nociceptin-induced behavioral response, whereas the nociceptin-induced behavioral response was unaffected by either bestatin, an aminopeptidase inhibitor or captopril, an angiotensin-converting enzyme inhibitor. However, the nociceptin effect was potentiated by combined injection of phosphoramidon and bestatin, indicating that inhibition of aminopeptidase may also contribute to inducing the behavioral response to nociceptin. These data suggest that endopeptidase-24.11 plays a major role in initial stage of nociceptin metabolism at the spinal cord level in mice.     

Cell surface antigens of Trypanosoma cruzi: possible correlation with the interiorization process in mammalian cells

Differences were observed in the pattern of the cell surface proteins of evolutive stages of Trypanosoma cruzi after radioiodination of the cells and subsequent analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Affinity chromatography revealed that surface glycoproteins also vary in the epimastigote and trypomastigote forms of the parasite. The cell surface antigens of the two forms were identified after radioiodination or biosynthetic labeling with [35S]methionine and incubation with different antisera. Both epimastigotes and trypomastigotes share two main antigens, possibly glycoproteins, of apparent molecular weight 95 000 and 80 000, respectively, which are recognized by all antisera tested. On the other hand, the trypomastigote form possesses somewhat more cell surface antigens recognized both by rabbit anti-trypomastigote and by human Chagasic sera. Sequential immunoprecipitations with heterologous and homologous antisera established that an 85 000 and some higher molecular weight antigens are specific to the trypomastigote form. Preincubation of the trypomastigotes with sera from Chagasic patients elicits a 60% inhibition of the parasite interiorization into mammalian cells in culture when compared with normal human serum or anti-epimastigote serum. This result suggests that the antigens specific to the trypomastigote form are involved in the interiorization process.     

A comparison of avian and murine retrovirus polyprotein cleavage activities

The murine leukemia virus Pr65^gag proteolytic activity (MuLV-PF) which processes Pr65^gag to murine gag-specific polypeptides and the avian tumor virus p15-associated protease (AMV-p15) which likewise processes the avian gag polyprotein, differ substantially in their detergent, pH, and salt requirements for optimal activity. These differences are consonant with reports that MuLV-PF is associated with a serine protease (Y. Yoshinaka and R. B. Luftig, 1977, Cell12, 709–719) while AMV-p15 has a thiol protease-like activity (K.J. Dittmar and K. Moelling, 1978, J. Virol.28, 106–118). In spite of these differences, in vitro cleavage of MuLP Pr65^gag (NH₂-p15-P12-p30-p10-COOH) by AMV-p15 can be achieved. The initial cleavage products observed are polypeptides of M_r 45,000 daltons (45K) and M_r 15K. The 15K polypeptide cross-reacts with MuLV p15 antisera while the 45K polypeptide possesses antigenic determinants of p30 and p12 but not p10. The 45K polypeptide thus differs from Pr40^gag, the intermediate cleavage product obtained after treatment of Pr65^gag with the murine leukemia virus proteolytic activity; Pr40^gag contains only p30 and p10 determinants (Y. Yoshinaka and R.B. Luftig, 1977, Biochem. Biophys. Res. Commun.79, 319–325). AMV-p15 further cleaves the 45K polypeptide to one with M_r 39K which has the group-specific antigenic determinant of p30 but not p12. At higher concentrations of AMV-p15, a more complete breakdown to polypeptides of M_r 12–15K, without any buildup of p30, is observed. These results suggest that there are at least four thiol protease-like sites on Pr65^gag: one very near the COOH terminus at p15 which makes it a possible in vivo cleavage site, and three other sites at the interior of polypeptides p10, p12, and p30.     

Radioiodination of new protein antigens on the surface of Plasmodium knowlesi schizont-infected erythrocytes

Schizont-infected red blood cells (SI-RBCs) from Plasmodium knowlesi-infected rhesus monkeys (Macaca mulatta) have been radioiodinated and the ¹²⁵I-proteins and ¹²⁵I-antigens compared with those of uninfected RBCs. New ¹²⁵I-proteins of M_r 230 000, 180 000, 165 000, 155 000, 135 000, 107 000, 72 000 and 65 000 were shown to be parasite-dependent components of SI-RBCs, the M_r 230 000 and 72 000 bands being quantitatively the major new components. New ¹²⁵I-antigens of SI-RBCs that were absent from uninfected cells and were only immunoprecipitated by sera from infected monkeys had M_r 230 000, 200 000, 180 000, 165 000, 155 000, 135 000, 130 000, 107 000, 72 000, 65 000 and 47 000. The following approaches were used to test which new radioiodinated components are on the SI-RBC outer membrane: analysis of radioactivity in haemoglobin; electron microscopic analysis of the integrity and purity of the SI-RBCs; treatment of intact ¹²⁵I-SI-RBCs with trypsin to ascertain the sensitivity of new proteins to exogenous protease; immunoprecipitation of antigen-antibody complexes after addition of antibody to intact ¹²⁵I-SI-RBCs. Although each test has inherent disadvantages, the accumulated evidence suggests that many, if not all of the new antigens identified for the first time in this report are on the SI-RBC outer membrane. The SICA variant antigen on P. knowlesi SI-RBCs was not identified by this approach.     

Isolation of a Plasmodium falciparum rhoptry protein

A monoclonal antibody raised against the malaria parasite Plasmodium falciparum recognised a protein of 140000 molecular weight which was synthesized during schizogony. The protein has been purified by monoclonal antibody affinity chromatography from extracts of parasitized red cells. Antibodies against the protein have been used to determine its subcellular location. The protein is not expressed on the merozoite surface and has been located in the rhoptries, the apical organelles of the merozoite.     

Disulfide structure of murine Ia alloantigens

The tryptic (T) and T insoluble chymotryptic (TIC) peptide maps from 35S-cysteine (Cys) labeled, disulfide-linked I-Ak dimer were compared to those from 35S-Cys labeled I-Ak alpha and beta chains which were not covalently linked. These comparisons indicated that the alpha and beta chains found in the covalent I-Ak dimer were not a specialized subset of I-A alpha and beta chains. Furthermore, these data, along with the knowledge that alkylation of spleen cells prior to and during detergent solubilization prevents the formation of disulfide-linked I-Ak dimer, indicate that covalent dimer formation is an inefficient and artifactual process. Comparison of the T and TIC peptide maps of reduced and nonreduced 35S-Cys labeled I-Ak alpha and beta chains suggests that the I-Ak alpha chain contains one intrachain disulfide bond, whereas the I-Ak beta chain contains two intrachain disulfide bonds. Examination of the T and TIC peptide maps of the reduced and nonreduced 35S-Cys labeled I-Ak dimer identifies the Cys-containing peptides which are involved in the formation of the artifactual I-Ak dimer interchain (alpha-beta) disulfide bond. Comparison of 35S-Cys labeled I-Ak and I-Ek alpha and beta chains by T and TIC peptide mapping reveals considerably more homology between the two alpha-chains and between the two beta-chains than is observed using other 3H-amino acid precursors, thus indicating that the I-Ak and I-Ek alloantigens are homologous in their amino acid sequences adjacent to the Cys resides. The reasons for the inability to induce formation of interchain (alpha-beta) disulfide bonds in I-Ek molecules are discussed.