Mutagenicity of TCDD in Big Blue transgenic rats

The compound 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) is a significant environmental contaminant resulting from such industrial processes as pulp and paper production. TCDD is a suspected human carcinogen and its ability to induce cancer in laboratory rodents is well documented. Its mechanism of tumor initiation, however, is not well understood and in vitro mutagenicity studies have yielded inconsistent results. In this study, Big Blue lacI transgenic rats were used to assess the mutagenicity of TCDD in both male and female animals. After 6 weeks of exposure to 2 microg/kg TCDD neither an increase in mutation frequency nor any change in mutation spectrum was observed in either male or female animals.     

Expression and differential intracellular localization of two major forms of human 8-oxoguanine DNA glycosylase encoded by alternatively spliced OGG1 mRNAs

We identified seven alternatively spliced forms of human 8-oxoguanine DNA glycosylase (OGG1) mRNAs, classified into two types based on their last exons (type 1 with exon 7: 1a and 1b; type 2 with exon 8: 2a to 2e). Types 1a and 2a mRNAs are major in human tissues. Seven mRNAs are expected to encode different polypeptides (OGG1-1a to 2e) that share their N terminus with the common mitochondrial targeting signal, and each possesses a unique C terminus. A 36-kDa polypeptide, corresponding to OGG1-1a recognized only by antibodies against the region containing helix-hairpin-helix-PVD motif, was copurified from the nuclear extract with an activity introducing a nick into DNA containing 8-oxoguanine. A 40-kDa polypeptide corresponding to a processed form of OGG1-2a was detected in their mitochondria using antibodies against its C terminus. Electron microscopic immunocytochemistry and subfractionation of the mitochondria revealed that OGG1-2a locates on the inner membrane of mitochondria. Deletion mutant analyses revealed that the unique C terminus of OGG1-2a and its mitochondrial targeting signal are essential for mitochondrial localization and that nuclear localization of OGG1-1a depends on the NLS at its C terminus.     

Involvement of N-acetylcysteine-sensitive pathways in ricin-induced apoptotic cell death in U937 cells

We have found that the antioxidant N-acetylcysteine (NAC) strongly inhibited ricin-induced apoptotic cell death in U937 cells (human myeloid leukemia), as judged by cytotoxicity, nuclear morphological change, and DNA fragmentation. Consistent with these observations, a significant depletion of cellular glutathione was observed in ricin-treated cells, and NAC prevented the decrease in cellular glutathione. On the other hand, among the caspase inhibitors tested, Z-Asp-CH2-DCB, which inhibited ricin cytotoxicity, also suppressed ricin-mediated glutathione depletion, while NAC did not affect the generation of caspase-3 like activity in ricin-treated cells. These results suggest that glutathione loss takes place downstream from caspase activation during the ricin-induced apoptotic process. Treatment with a specific inhibitor of glutathione biosynthesis, buthionine sulfoximine (BSO) failed to induce apoptosis, and had no effect on the overall extent of ricin-induced apoptosis, even though the glutathione level was decreased to less than 5% of the control level. However, NAC still protected against ricin-induced apoptosis in the BSO-treated cells. We conclude that glutathione loss is one of several apoptotic changes caused by ricin, but is not a sufficient factor for the progress of apoptosis. NAC may prevent ricin-induced apoptosis through maintaining an intracellular reducing condition by acting as a thiol supplier.     

Crystal structure of tobacco PR-5d protein at 1.8 A resolution reveals a conserved acidic cleft structure in antifungal thaumatin-like proteins

The crystal structure of tobacco PR-5d, an antifungal thaumatin-like protein isolated from cultured tobacco cells, was determined at the resolution of 1.8 A. The structure consists of 208 amino acid residues and 89 water molecules with a crystallographic R-factor of 0.169. The model has good stereochemistry, with respective root-mean-square deviations from the ideal values for bond and angle distances of 0.007 A and 1.542 degrees. Of the homologous PR-5 proteins, only those with antifungal activity had a common motif, a negatively charged surface cleft. This cleft is at the boundary between domains I and II, with a bottom part consisting of a three-stranded antiparallel beta-sheet in domain I. The acidic residues located in the hollow of the cleft form the beta-sheet region. Sequence and secondary structure analyses showed that the amino acid residues comprising the acidic cleft of PR-5d are conserved among other antifungal PR-5 proteins. This is the first report on the high-resolution crystal structure of an antifungal PR-5 protein. This structure provides insight into the function of pathogenesis-related proteins.     

Sulfated sialic acid-polymers inhibit the cytotoxic action of bee and snake venom

Colominic acid is an 2,8-linked sialic acid polymer produced by Escherichia coli. We found that synthetic sulfated-colominic acids (SC) remarkably inhibited the cytotoxicity of bee and snake venom toward mouse fibroblast cells, but colominic acids showed no inhibition themselves, indicating the important role of sulfate groups in the inhibitory activity of SC. Other sulfated carbohydrates such as chondroitin sulfates, heparin and heparan sulfate showed no inhibition. SC also exhibited potent inhibition of melittin, a highly basic peptide, which is a major cytotoxic component of bee venom. SC did not inhibit phospholipase A₂ activity in bee venom. This suggests that the inhibition of bee and snake venom by SC is due to inhibition of melittin and cardiotoxin, which is a cytolytic peptide in snake venom, respectively. SC with a higher sulfur content and a larger molecular mass showed more potent activity. The interaction between SC and melittin basically seems an ionic one, however, the conformation of SC is also likely important. For the binding of SC to melittin leading loss of its cytotoxic activity, the sulfate groups of SC must be properly arranged to interact with lysine and arginine residues of melittin molecules, which play an important role in the cytolytic activity. A higher molecular mass of SC substituted with more sulfate groups is required for more obvious inhibition of the cytotoxic activity.     

Attachment of oligonucleotide probes to poly carbodiimide-coated glass for microarray applications

Oligonucleotide-based DNA microarrays are becoming increasingly useful tools for the analysis of gene expression and single nucleotide polymorphisms (SNPs). Here, we present a method that permits the manufacture of microarrays from non-modified oligonucleotides on a poly carbodiimide-coated glass surface by UV-irradiation. The use of UV-irradiation facilitates an increase in the level of signal intensity, but it does not affect signal discrimination by the oligonucleotides immobilized on the surface. The signal intensity obtained for an array fabricated using non-modified oligonucleotides with UV-irradiation is ~7-fold greater than that without UV-irradiation. The detection of SNPs was tested to ascertain whether this technique could discriminate specific hybridization signals without causing significant UV-irradiation-induced damage to the immobilized oligonucleotides. We found that this immobilization method provides greater hybridization signals and a better match/mismatch ratio of SNPs than do the established aminosilane techniques. Application of this technology to manufacturing DNA microarrays for sequence analysis is discussed.     

The Induction of Intercellular Adhesion Molecule-1 on Human Umbilical Vein Endothelial Cells by a Heat-Stable Component of <Emphasis Type="Italic">Porphyromonas gingivalis</Emphasis>

Live Porphyromonas gingivalis enhanced the expression of intercellular adhesion molecule-1 (ICAM-1) on the surface of human umbilical vein endothelial cells (HUVECs) in a bacterial dose-dependent manner. Inactivation of P. gingivalis by ultraviolet (UV), heat (56°C, 30 min), or sonication did not alter its stimulatory activity. ICAM-1 expression began to increase at 4 h after stimulation, reached a maximum at 12 h, and remained at the maximum for at least the next 8 h. This time course was similar to that of expression by Escherichia coli LPS. Furthermore, the effect of UV-inactivated P. gingivalis was not inhibited by boiling or polymyxin B treatment. In addition, the effect of P. gingivalis strain W83 on ICAM-1 expression was stronger than that of strain ATCC 33277. Our results suggested that some unidentified, heat-stable proteins, polysaccharides, or lipids may be the stimulatory factor(s), although the participation of LPS could not be completely ruled out. The ability of P. gingivalis to stimulate ICAM-1 expression on endothelial cells may play an important role in the pathogenesis of periodontal disease.     

The role of the calcium-sensing receptor in epidermal differentiation

Calcium regulates the proliferation and differentiation of keratinocytes both in vivo and in vitro. Elevated extracellular Ca2+ concentration ([Ca2+]o) raises the intracellular free calcium ([Ca2+]i) and activates differentiation-related genes. Cells lacking the calcium-sensing receptor (CaR) fail to respond to [Ca2+]o and to differentiate, indicating a role for CaR in keratinocyte differentiation. These concepts derived from in vitro experiments have been tested and confirmed in two mouse models.     

Insect cytokine growth-blocking peptide triggers a termination system of cellular immunity by inducing its binding protein

Growth-blocking peptide (GBP) is a 25-amino acid cytokine found in lepidopteran insects that possesses diverse biological activities such as stimulation of immune cells (plasmatocytes), cell proliferation, and larval growth regulation. We found another novel function of GBP that induces a hemolysis of another class of blood cells (oenocytoids). In the lysate of oenocytoids we identified a GBP-binding protein that shows a specific affinity for GBP. The characterization of purified GBP-binding protein and its cDNA demonstrated it as a 49.5-kDa novel protein with a C-terminal region displaying limited homology to several insect lipoproteins. Results of Northern and Western blotting indicated that the GBP-binding protein should be synthesized only in blood cells. Immunoelectron microscopic analyses confirmed that indirect immunoreactive signals were mostly localized in oenocytoids. Kinetic and biological analyses of interaction between GBP and the binding protein showed their strong binding was followed by clearance of GBP from hemolymph, thus indicating that this protein might function as an inhibitory factor against GBP. Based on these results, we propose that insect cytokine GBP shows multifunctions even in cellular immunity: it serves to stimulate immune cells and afterward silences its own action by inducing the binding protein through specific hemolysis.