Alpha-tocopherol content and alpha-tocopherol transfer protein expression in leukocytes of children with acute leukemia

-Tocopherol (a form of vitamin E) is a fat-soluble vitamin that can prevent lipid peroxidation of cell membranes. This antioxidant activity of -tocopherol can help to prevent cardiovascular disease, atherosclerosis and cancer. We investigated the -tocopherol level and the expression of -tocopherol transfer protein (-TTP) in the leukocytes of children with leukemia. The plasma and erythrocyte -tocopherol levels did not differ between children with leukemia and the control group. However, lymphocytes from children with leukemia had significantly lower -tocopherol levels than lymphocytes from the controls (58.4±39.0 ng/mg protein versus 188.9±133.6, respectively; p0.05), despite the higher plasma -tocopherol/cholesterol ratio in the leukemia group (5.83±1.64 μmol/mmol versus 4.34±0.96, respectively; p0.05). No significant differences in the plasma and leukocyte levels of isoprostanes (the oxidative metabolites of arachidonic acid) were seen between the leukemia patients and controls. The plasma level of acrolein, a marker of oxidative stress, was also similar in the two groups. Investigation of -TTP expression by leukocytes using real-time PCR showed no difference between the two groups. These findings suggest that there may be comparable levels of lipid peroxidation in children with untreated leukemia and controls, despite the reduced -tocopherol level in leukemic leukocytes.     

Characterization of complement C3 as a glycyrrhizin (GL)-binding protein and the phosphorylation of C3alpha by CK-2, which is potently inhibited by GL and glycyrrhetinic acid in vitro

The physiological interaction between glycyrrhizin (GL) and serum complement C3, and the inhibitory effects of GL, glycyrrhetinic acid (GA), and a GA derivative (oGA) on the phosphorylation of C3 by casein kinase 2 (CK-2), were investigated in vitro. C3 was found to be a GL-binding protein (gbP), because (i) of its high affinity for a GL-affinity HPLC column; and (ii) both GL and GA induce conformational changes in C3. At least four trypsin-resistant fragments (p30, p25, p18, and p15) were detected when the (32)P-labeled C3alpha was digested with trypsin in the presence of 100 micro M GA. Two of these (p25 and p15) were immuno-precipitated with anti-C3a serum. Furthermore, it was found that C3a contains GL-binding domains, because (i) C3a (anaphylatoxin) could be selectively purified from the synovial fluids of patients with rheumatoid arthritis by GL-affinity column chromatography (HPLC); and (ii) purified human C3a has a high affinity for a GL-affinity column. In addition, C3alpha (p115) of C3 was effectively phosphorylated by CK-2 in the presence of poly-Arg (a CK-2 activator) in vitro. This phosphorylation was completely inhibited by 10 micro M oGA, 30 micro M GA, or 100 micro M GL. Taken together, these results suggest that the GL-induced inhibition of the physiological activities of C3a and C3alpha may be involved in the anti-inflammatory effect of GL in vivo.     

Selective cell death by water-soluble Fe-porphyrins with superoxide dismutase (SOD) activity

We investigated the effect on cell death of reactive oxygen species induced by [[5,10 (or 5,15)-bis(N-methyl-4-pyridyl)-15,20 (or 10,20) diphenyl]porphinato]iron (cis-FeMPy(2)P(2)P or trans-FeMPy(2)P(2)P) with SOD activity. The SOD activities of the cis-FeMPy(2)P(2)P and trans-FeMPy(2)P(2)P were measured using stopped-flow kinetic analysis. The cell viability of four cell lines treated with cis-Fe-porphyrin, trans-Fe-porphyrin, mitomycin c (MMC), or cisplatin was estimated by the alamar blue exclusion assay of the modified MTT method. The amount of cis-FeMPy(2)P(2)P and trans-FeMPy(2)P(2)P in the Walker 256 cultured for 24 h was 4.0 and 2.6 fmolcell(-1), respectively, indicating that the plasma membrane permeability of the Fe-porphyrins depended on their structure. Cis-FeMPy(2)P(2)P selectively killed Walker 256 and H-4-II-E as cancer cells but not FR and BRL-3A as normal cells and showed a significant cytotoxicity for the cancer cells compared with trans-FeMPy(2)P(2)P, MMC and cisplatin. We believe that cis-FeMPy(2)P(2)P as an SOD mimic converts intracellular O(2)(*-) to H(2)O(2) and that H(2)O(2) or *OH causes DNA damage and induces cell death. This result suggests that for the SOD mimic, O(2)(*-) may be useful as a target molecule to induce selective cell death between cancer and normal cells and that a metalloporphyrin having SOD activity is a new class of anticancer agents.     

Changes in the peptidergic innervation in the carotid body of rats chronically exposed to hypercapnic hypoxia: an effect of arterial CO2 tension

The abundance of neuropeptide Y (NPY)-, vasoactive intestinal polypeptide (VIP)-, substance P (SP)-, and calcitonin gene-related peptide (CGRP)-immunoreactive nerve fibers in the carotid body was examined in chronically hypercapnic hypoxic rats (10% O2 and 6-7% CO2 for 3 months), and the distribution and abundance of these four peptidergic fibers were compared with those of previously reported hypocapnic- and isocapnic hypoxic carotid bodies to evaluate the effect of arterial CO2 tension. The vasculature in the carotid body of chronically hypercapnic hypoxic rats was found to be enlarged in comparison with that of normoxic control rats, but the rate of vascular enlargement was smaller than that in the previously reported hypocapnic- and isocapnic hypoxic carotid bodies. In the chronically hypercapnic hypoxic carotid body, the density per unit area of parenchymal NPY fibers was significantly increased, and that of VIP fibers was unchanged, although the density of NPY and VIP fibers in the previously reportetd chronically hypocapnic and isocapnic hypoxic carotid bodies was opposite to that in hypercapnic hypoxia as observed in this study. The density of SP and CGRP fibers was decreased. These results along with previous reports suggest that different levels of arterial CO2 tension change the peptidergic innervation in the carotid body during chronically hypoxic exposure, and altered peptidergic innervation of the chronically hypercapnic hypoxic carotid body is one feature of hypoxic adaptation.     

Sequence-specific protection of plasmid DNA from restriction endonuclease hydrolysis by pyrrole-imidazole-cyclopropapyrroloindole conjugates

The pyrrole-imidazole (Py-Im) triamide-cyclopropa pyrroloindole (CPI) conjugates ImPyImLDu86 (7) and ImImPyLDu86 (14) were synthesized and their alkylating activities and inhibitory effects on DNA hydrolysis by restriction endonucleases were examined. Sequencing gel analysis demonstrated that conjugates 7 and 14 specifically alkylated DNA at 5'-CGCGCG-3' and 5'-PyGGCCPu-3', respectively. Agarose gel electrophoresis indicated that incubation of a supercoiled plasmid, pSPORT I (4109 bp), with conjugate 7 effectively inhibited its hydrolysis by BssHII (5'-G_CGCGC-3'), whereas conjugate 14 had no effect on this hydrolysis. These results suggest that conjugate 7 sequence-specifically inhibits the hydrolysis of DNA by BssHII. Sequence-specific alkylation by the Py-Im triamide-CPI conjugates was further confirmed by inhibition of the Eco52I (5'-C_GGCCG-3') hydrolysis of conjugate 14-treated pQBI PGK (5387 bp). In clear contrast, hydrolysis of pQB1 PGK by DraI (3'-TTT_AAA-3') was not inhibited by 5 micro M conjugate 14. That ImImPy did not inhibit the hydrolysis of pQB1 PGK indicates that covalent bond formation is necessary for inhibition. A similar experiment, using linear pQBI PGK, achieved the same extent of protection of the DNA with approximately half the concentration of conjugate 14 as was required to protect supercoiled DNA from hydrolysis.     

Enhanced induction of human WT1-specific cytotoxic T lymphocytes with a 9-mer WT1 peptide modified at HLA-A*2402-binding residues

The Wilms' tumor gene WT1 is overexpressed in most types of leukemias and various kinds of solid tumors, including lung and breast cancer, and participates in leukemogenesis and tumorigenesis. WT1 protein has been reported to be a promising tumor antigen in mouse and human. In the present study, a single amino-acid substitution, M-->Y, was introduced into the first anchor motif at position 2 of the natural immunogenic HLA-A*2402-restricted 9-mer WT1 peptide (CMTWNQMNL; a.a. 235-243). This substitution increased the binding affinity of the 9-mer WT1 peptide to HLA-A*2402 molecules from 1.82 x 10(-5) to 6.40 x 10(-7) M. As expected from the increased binding affinity, the modified 9-mer WT1 peptide (CYTWNQMNL) elicited WT1-specific cytotoxic T lymphocytes (CTL) more effectively than the natural 9-mer WT1 peptide from peripheral blood mononuclear cells (PBMC) of HLA-A*2402-positive healthy volunteers. CTL induced by the modified 9-mer WT1 peptide killed the natural 9-mer WT1 peptide-pulsed CIR-A*2402 cells, primary leukemia cells with endogenous WT1 expression and lung cancer cell lines in a WT1-specific HLA-A*2402-restricted manner. These results showed that this modified 9-mer WT1 peptide was more immunogenic for the induction of WT1-specific CTL than the natural 9-mer WT1 peptide, and that CTL induced by the modified 9-mer WT1 peptide could effectively recognize and kill tumor cells with endogenous WT1 expression. Therefore, cancer immunotherapy using this modified 9-mer WT1 peptide should provide efficacious treatment for HLA-A*2402-positive patients with leukemias and solid tumors.     

Detection of a processed pseudogene of the human MBL-associated serine protease,MASP1

Southern hybridization analysis of the MASP1 gene using an intron-specific probe detected a single band. An exon-specific probe detected several bands. PCR of genomic DNA using several exon-specific primer sets of MASP1 produced short and long products. Sequence of the shorter products corresponded to the processed pseudogene of MASP1. By fluorescence in situ hybridization, this pseudogene (MASP1P1) was mapped to 1p34.