T-5224, a selective inhibitor of c-Fos/activator protein-1, attenuates lipopolysaccharide-induced liver injury in mice

The effect of T-5224, a selective inhibitor of c-Fos/activator protein (AP)-1, on lipopolysaccharide (LPS) induced liver injury was examined in mice. Administration of LPS (10?mg?kg^?1, i.p.) markedly increased serum levels of tumor necrosis factor-alpha (TNFα), high mobility group box 1 (HMGB1), alanine aminotransferase/aspartate aminotransferase (ALT/AST), liver tissue levels of macrophage-inflammatory protein-1 alpha (MIP-1α) and monocyte chemoattractant protein-1 (MCP-1), as well as hepatic necrosis and inflammation, leading to 67?% lethality. Administration of T-5224 (300?mg?kg^?1, p.o.) after intraperitoneal injection of LPS imparted appreciable protection against acute elevations in serum levels of TNFα, HMGB1, ALT/AST as well as in liver tissue levels of MIP-1α and MCP-1, and reduced the lethality (27?%). These data indicate that T-5224 ameliorates liver injury and improves survival through decreasing production of proinflammatory cytokines and chemokines in endotoxemic mice.     

Functional and molecular characterization of naturally occurring mutations in the oocyte-secreted factors bone morphogenetic protein-15 and growth and differentiation factor-9

Bone morphogenetic protein-15 (BMP-15) and growth and differentiation factor-9 (GDF-9) are oocyte-secreted factors that are critical local regulators of ovarian physiology. Recent studies have identified a number of mutations in these genes that cause increased fertility and infertility in heterozygous or homozygous ewes carrying the mutations, respectively. Interestingly, heterozygous ewes with a mutation in both BMP-15 and GDF-9 exhibit higher fertility than those having mutation in only one of the genes. Here, we have produced recombinant human BMP-15 and GDF-9 that carry the mutations identified in those sheep, i.e. I31D and S99I in BMP-15 and S77F in GDF-9. We found that when individually expressed, both BMP-15 mutations had no effect on the processing, secretion, and dimerization of the mature proteins or on the biological activity of the molecules. However, when mutant BMP-15 was co-expressed with wild-type GDF-9, the secretion of BMP-15 and GDF-9 was significantly reduced, suggesting that the mechanisms by which the BMP-15 mutations affect sheep fertility occurs at the level of protein secretion rather than dimerization and biological activity. Moreover, when mutant GDF-9 was co-expressed with mutant BMP-15, the secretion levels of both proteins were significantly lower than those of cells co-expressing wildtype GDF-9 and mutant BMP-15, suggesting a possible mechanism for the extreme fertility observed in the compound heterozygous mutant sheep.     

Catalase reaction by myoglobin mutants and native catalase: mechanistic investigation by kinetic isotope effect

The catalase reaction has been studied in detail by using myoglobin (Mb) mutants. Compound I of Mb mutants (Mb-I), a ferryl species (Fe(IV)=O) paired with a porphyrin radical cation, is readily prepared by the reaction with a nearly stoichiometric amount of m-chloroperbenzoic acid. Upon the addition of H2O2 to an Mb-I solution, Mb-I is reduced back to the ferric state without forming any intermediates. This indicates that Mb-I is capable of performing two-electron oxidation of H2O2 (catalatic reaction). Gas chromatography-mass spectroscopy analysis of the evolved O2 from a 50:50 mixture of H2(18)O2/H2(16)O2 solution containing H64D or F43H/H64L Mb showed the formation of 18O2 (m/e = 36) and 16O2 (m/e = 32) but not 16O18O (m/e = 34). This implies that O2 is formed by two-electron oxidation of H2O2 without breaking the O-O bond. Deuterium isotope effects on the catalatic reactions of Mb mutants and catalase suggest that the catalatic reactions of Micrococcus lysodeikticus catalase and F43H/H64L Mb proceed via an ionic mechanism with a small isotope effect of less than 4.0, since the distal histidine residue is located at a proper position to act as a general acid-base catalyst for the ionic reaction. In contrast, other Mb mutants such as H64X (X is Ala, Ser, and Asp) and L29H/H64L Mb oxidize H2O2 via a radical mechanism in which a hydrogen atom is abstracted by Mb-I with a large isotope effect in a range of 10-29, due to a lack of the general acid-base catalyst.     

Molecular basis of bone morphogenetic protein-15 signaling in granulosa cells

Bone morphogenetic protein-15 (BMP-15), an oocyte growth factor belonging to the transforming growth factor-beta superfamily, has recently been shown to be necessary for normal female fertility in mammals. We have previously demonstrated that BMP-15 regulates granulosa cell (GC) proliferation and differentiation; namely, BMP-15 promotes GC mitosis, suppresses follicle-stimulating hormone (FSH) receptor expression, and stimulates kit ligand expression. Although the role of BMP-15 in female reproduction has progressively deserved much attention, there is nothing known to date about the signaling pathway and receptors for BMP-15. Using rat primary GCs and a human GC cell line, COV434, we have now found that administration of BMP-15 causes a rapid and transient phosphorylation, thus activation, of the Smad1/5/8 pathway. BMP-15 also stimulated promoter activity of a selective BMP-responsive reporter construct, further demonstrating the stimulation of Smad1/5/8 signaling by BMP-15. In contrast, BMP-15 stimulation of Smad2 phosphorylation was very weak. To identify the receptors for BMP-15, we utilized recombinant extracellular domains of individual transforming growth factor-beta superfamily receptors and found that activin receptor-like kinase-6 extracellular domain most effectively co-immunoprecipitates with BMP-15, whereas BMP receptor type II extracellular domain was most effective in inhibiting BMP-15 bioactivity on FSH-induced progesterone production and GC thymidine incorporation. We also investigated whether activation of the MAPK pathway is necessary for BMP-15 biological activity and found that the addition of U0126, an inhibitor of ERK1/2 phosphorylation, suppresses BMP-15 activity on GC mitotsis but not on FSH-induced progesterone production, suggesting a selective signaling cascade in GC proliferation and differentiation.     

Multiple roles of Rev3, the catalytic subunit of polzeta in maintaining genome stability in vertebrates

Translesion DNA synthesis (TLS) and homologous DNA recombination (HR) are two major postreplicational repair (PRR) pathways. The REV3 gene of Saccharomyces cerevisiae encodes the catalytic subunit of DNA polymerase zeta, which is involved in mutagenic TLS. To investigate the role of REV3 in vertebrates, we disruped the gene in chicken DT40 cells. REV3(-/-) cells are sensitive to various DNA-damaging agents, including UV, methyl methanesulphonate (MMS), cisplatin and ionizing radiation (IR), consistent with its role in TLS. Interestingly, REV3(-/-) cells showed reduced gene targeting efficiencies and significant increase in the level of chromosomal breaks in the subsequent M phase after IR in the G(2) phase, suggesting the involvement of Rev3 in HR-mediated double-strand break repair. REV3(-/-) cells showed significant increase in sister chromatid exchange events and chromosomal breaks even in the absence of exogenous genotoxic stress. Furthermore, double mutants of REV3 and RAD54, genes involved in HR, are synthetic lethal. In conclusion, Rev3 plays critical roles in PRR, which accounts for survival on naturally occurring endogenous as well as induced damages during replication.     

Synovial sarcoma of the thyroid. Report of a case with aspiration cytology findings and gene analysis

BACKGROUND: Synovial sarcoma, generally known as a soft tissue tumor, can also occur in the head and neck region, including the thyroid gland. Cytologic findings are important to differentiate the tumor from other types of neoplasms arising in the thyroid gland. CASE: A 60-year-old man complained of hoarseness. A palpable neck tumor was detected, and a computed tomography scan showed a thyroid tumor accompanied by destruction of the thyroid and cricoid cartilage. The results of a preoperative fine needle aspiration biopsy showed numerous spindle cells with pale cytoplasm and oval nuclei with fine, granular chromatin, all of which suggested a medullary carcinoma. The extirpated thyroid tissue weighed approximately 120 g, and a grayish white, elastic, solid tumor (6.8 x 6.5 cm) was present in the left lobe. Histologically, fasciculation of spindle cells that had proliferated solidly and densely was observed. Also, the expression of a chimera gene, SYT-SSX, was detected in the tumor tissue. CONCLUSION: Synovial sarcoma of the thyroid is extremely rare, and its diagnosis by fine needle aspiration biopsy is generally considered very difficult. The detailed cytologic findings observed here might be helpful with the differential diagnosis of thyroid neoplasms.     

XRCC3 and Rad51 modulate replication fork progression on damaged vertebrate chromosomes

The mechanisms by which the progression of eukaryotic replication forks is controlled after DNA damage are unclear. We have found that fork progression is slowed by cisplatin or UV treatment in intact vertebrate cells and in replication assays in vitro. Fork slowing is reduced or absent in irs1SF CHO cells and XRCC3(-/-) chicken DT40 cells, indicating that fork slowing is an active process that requires the homologous recombination protein XRCC3. The addition of purified human Rad51C-XRCC3 complex restores fork slowing in permeabilized XRCC3(-/-) cells. Moreover, the requirement for XRCC3 for fork slowing can be circumvented by addition of human Rad51. These data demonstrate that the recombination proteins XRCC3 and Rad51 cooperatively modulate the progression of replication forks on damaged vertebrate chromosomes.     

Killing activity of human umbilical cord blood-derived TCRValpha24<Superscript>+</Superscript> NKT cells against normal and malignant hematological cells in vitro: a comparative study with NK cells or OKT3 activated T lymphocytes or with adult peripheral blood NKT cells

PURPOSE: We aimed to determine the effects of human umbilical cord blood (UCB)-derived natural killer T (NKT) cells as immunological effectors against hematological malignancies, as well as auto- or allo-dendritic cells (DCs) or EB transformed cell lines (EBCLs). MATERIALS: TCRValpha24(+) Vbeta11(+) UCB- or PB-NKT cells were isolated by sorting and activated by alpha-galactosylceramide-pulsed autologous DCs. UCB-NK cells were induced from CD34(+) cells by stem cell factor plus IL-15. UCB-T cells were primarily activated by anti-CD3 monoclonal antibody. All those effectors were cultured with IL-2 (100 U/ml), and their cytotoxic activities were evaluated by (51)Cr-release assay. UCB-NKT cells were cultured with IL-12, IL-18 or higher dose of IL-2 (1000 U/ml), and again tested for the cytotoxicity against selected targets. RESULTS: UCB-NKT cells exhibited a pattern of killing activity against various hematological malignancies similar to that of UCB-NK cells, but could not kill K562, which was a vulnerable target for NK cells. The level of activity was quite similar to that of PB-NKT cells. In contrast, OKT-3-activated UCB-T lymphocytes showed a stronger and wider spectrum of killing compared with UCB-NK or NKT cells. IL-12, IL-18 or a higher dose of IL-2 upregulated the activity; however several targets, including fresh leukemic cells, still remained resistant. NKT cells killed auto- or allo-DCs at a level similar to that of T cells, but could not kill allo-EBCLs, which were efficiently killed by T cells. While NK cells showed only marginal or no killing against DC or EBCLs. DISCUSSION: The anti-cancer activity of human NKT cells depends on the concentrations or the combination of Th1-cytokines. Basically, those cells might not be contributing to the immune surveillance of hematological malignancies, as shown by a relatively low cytotoxicity against malignant cells, together with the quite strong killing against auto-DCs.     

Distribution of the Parathyroid Hormone-Related Peptide and Its Receptor in the Saccus Vasculosus and Choroid Plexus in the Red Stingray (Dasyatis akajei: Elasmobranch)

1. The exact role of the parathyroid hormone-related peptide (PTHrP) is not fully understood. We used immunohistochemistry to localize the PTHrP and its receptor in the brain of the red stingray, particularly in the saccus vasculosus (SV) and choroid plexus.2. Immunoreactive PTHrP and its receptor were detected in the epithelial cells of the SV and the choroid plexus. In addition, the neuronal perikarya in the nucleus of the SV located in the hypothalamus is positive for the PTHrP.3. No PTHrP-containing neurons were detected in the choroid plexus. Extracts of SV and choroid plexus showed positive reactions against the PTHrP and its receptor antibody in Western blot analysis.4. High levels of immunoreactive PTHrP were detected in the plasma equivalent to those present in human humoral malignant hypercalcemia. In contrast, the immunoreactive PTHrP concentration in the cerebrospinal fluid was below detectable levels.5. Our results suggest that the regulation of the PTHrP in the SV differs from that in the choroid plexus in the red stingray.