Simulation study on the disordered state of an Alzheimer's beta amyloid peptide Abeta(12 36) in water consisting of random-structural, beta-structural, and helical clusters

The monomeric Alzheimer's beta amyloid peptide, Abeta, is known to adopt a disordered state in water at room temperature, and a circular dichroism (CD) spectroscopy experiment has provided the secondary-structure contents for the disordered state: 70% random, 25% beta-structural, and 5% helical. We performed an enhanced conformational sampling (multicanonical molecular dynamics simulation) of a 25-residue segment (residues 12-36) of Abeta in explicit water and obtained the conformational ensemble over a wide temperature range. The secondary-structure contents calculated from the conformational ensemble at 300 degrees K reproduced the experimental secondary-structure contents. The constructed free-energy landscape at 300 degrees K was not plain but rugged with five clearly distinguishable clusters, and each cluster had its own characteristic tertiary structure: a helix-structural cluster, two beta-structural clusters, and two random-structural clusters. This indicates that the contribution from the five individual clusters determines the secondary-structure contents experimentally measured. The helical cluster had a similarity with a stable helical structure for monomeric Abeta in 2,2,2-trifluoroethanol (TFE)/water determined by an NMR experiment: The positions of helices in the helical cluster were the same as those in the NMR structure, and the residue-residue contact patterns were also similar with those of the NMR structure. The cluster-cluster separation in the conformational space indicates that free-energy barriers separate the clusters at 300 degrees K. The two beta-structural clusters were characterized by different strand-strand hydrogen-bond (H-bond) patterns, suggesting that the free-energy barrier between the two clusters is due to the H-bond rearrangements.     

Molecular analysis of RANKL-independent cell fusion of osteoclast-like cells induced by TNF-alpha, lipopolysaccharide, or peptidoglycan

Focusing on the final step of osteoclastogenesis, we studied cell fusion from tartrate-resistant acid phosphatase (TRAP)-positive mononuclear cells into multinuclear cells. TRAP-positive mononuclear cells before generation of multinuclear cells by cell fusion were differentiated from RAW264.7 cells by treatment with receptor activator of nuclear factor kappa B ligand (RANKL), and then the cells were treated with lipopolysaccharide (LPS), followed by culturing for further 12 h. LPS-induced cell fusion even in the absence of RANKL. Similarly, tumor necrosis factor (TNF)-alpha and peptidoglycan (PGN) induced cell fusion, but M-CSF did not. The cell fusion induced by RANKL, TNF-alpha, and LPS was specifically blocked by osteoprotegerin (OPG), anti-TNF-alpha antibody, and polymyxin B, respectively. LPS- and PGN-induced cell fusion was partly inhibited by anti-TNF-alpha antibody but not by OPG. When TRAP-positive mononuclear cells fused to yield multinuclear cells, phosphorylation of Akt, Src, extracellular signal-regulated kinase (ERK), p38MAPK (p38), and c-Jun NH2-terminal kinase (JNK) was observed. The specific chemical inhibitors LY294002 (PI3K), PP2 (Src), U0126 (MAPK-ERK kinase (MEK)/ERK), and SP600125 (JNK) effectively suppressed cell fusion, although SB203580 (p38) did not. mRNA of nuclear factor of activated T-cells c1 (NFATc1) and dendritic cell-specific transmembrane protein (DC-STAMP) during the cell fusion was quantified, however, there was no obvious difference among the TRAP-positive mononuclear cells treated with or without M-CSF, RANKL, TNF-alpha, LPS, or PGN. Collectively, RANKL, TNF-alpha, LPS, and PGN induced cell fusion of osteoclasts through their own receptors. Subsequent activation of signaling pathways involving PI3K, Src, ERK, and JNK molecules was required for the cell fusion. Although DC-STAMP is considered to be a requisite for cell fusion of osteoclasts, cell fusion-inducing factors other than DC-STAMP might be necessary for the cell fusion.     

Enzymatic activities of uridine and thymidine phosphorylase in normal and cancerous uterine cervical tissues

In this study, the preliminary analyses were conducted of enzymatic activities of uridine phosphorylase (UP) and thymidine phosphorylase (TP) in normal tissues and cancer tissues of the uterine cervix. The study was performed on 27 patients of cervical cancer, treated first in our hospital. Normal cervical tissues obtained from 15 patients undergoing hysterectomy for benign diseases were used as controls. The supernatant of the homogenated cervical tissues and the stroma (5-FU and ribose-1-P or deoxyribose-1-P) were analyzed by high performance liquid chromatography, and then the UP and TP activities calculated. TP activity was significantly greater than UP activity (P < 0.0001). Both UP and TP showed significantly greater activity in cancer tissues than in normal tissues (P < 0.0001). In the TP activity of the cancer tissues, there was no significant difference among the histological types, while the TP activity tended to be significantly higher in the cases with lymph node metastasis. These results showed that the TP-mediated route seemed important as the 5FU metabolic pathway in the uterine cervical tissues, and TP enzymatic activity might be associated with lymph node metastasis.     

Vasculitis induced by immunization with Bacillus Calmette-Guérin followed by atypical mycobacterium antigen: a new mouse model for Kawasaki disease

Kawasaki disease causes systemic vasculitis. The development of skin lesions at the vaccination site with Bacillus Calmette-Guérin (BCG) is an important diagnostic symptom. We hypothesized that infection with ubiquitous microorganisms immunogenically related to BCG might induce an immunopathologic reaction leading to the development of Kawasaki disease. Mice were first inoculated with BCG, and then secondarily inoculated 4 weeks later with crude extract from Mycobacterium intracellulare (cMI), an abundant atypical mycobacterium. Animals inoculated with BCG followed by cMI developed coronary arteritis with infiltration of inflammatory cells, whereas control animals inoculated with only cMI or BCG did not, suggesting that the immune response to the mycobacteria induced autoimmunity to the vascular wall. Intravenous injection with antibodies to peroxiredoxin II, a modulator of vascular remodeling and a suggested target for autoimmune vasculitis, also resulted in coronary arteritis, but only after prior inoculation with BCG. Tumor necrosis factor-alpha, MCP1 and interferon-gamma production were significantly higher in the animals inoculated with BCG than in the control groups (P<0.05). BCG immunization was required for the development of coronary arteritis, suggesting that these cytokines might play important roles. The results indicate that BCG induces primary autoimmunity and stimulates cytokine induction, and that atypical mycobacterial infection boosts the autoimmunity resulting in coronary arteritis.     

Improved immunocytochemical detection of daunomycin

Improved immunocytochemical (ICC) detection of the anthracycline anticancer antibiotic daunomycin (DM) has been achieved by use of hydrogen peroxide oxidation prior to ICC staining for DM. The new method greatly enhanced the localization of DM accumulation in cardiac, smooth and skeletal muscle of rats after a single i.v. dose of the drug. DM accumulated in the nuclei as well as in the sarcoplasm, where it occurred in the form of small granules, which were particularly evident in cardiac muscle cells. The distribution of the granules coincided with that of mitochondria. Uptake of DM in nuclei and mitochondria of heart muscle cells may help to improve our understanding of the cardiac toxicity of DM and related anthracyclin antibiotics. A number of ELISA tests were carried out in order to elucidate the mechanims of H₂O₂−assisted antigen retrieval. A possible mechanism is that DM is reduced and converted to its semiquinone and/or hydroquinone derivative in vivo. Oxidation by hydrogen peroxide acts to convert these derivatives back to the native antigen. The improved ICC methodology using oxidation to recreate native antigens from reduced metabolites may be helpful also with respect to the localization of other drugs.     

Association of cathepsin E with tumor growth arrest through angiogenesis inhibition and enhanced immune responses

Cathepsin E (CE) is an intracellular aspartic proteinase implicated in various physiological and pathological processes, yet its actual roles in vivo remain elusive. To assess the physiological significance of CE expression in tumor cells, human CE was stably expressed in human prostate carcinoma ALVA101 cells expressing very little CE activity. Tumor growth in nude mice with xenografted ALVA101/hCE cells was slower than with control ALVA101/mock cells. Angiogenesis antibody array and ELISA assay showed that this was partly due to the increased expression of some antiangiogenic molecules including interleukin 12 and endostatin in tumors induced by CE expression. In vitro studies also demonstrated that, among the cathepsins tested, CE most efficiently generated endostatin from the non-collagenous fragment of human collagen XVIII at mild acidic pH. Histological examination revealed that tumors formed by ALVA101/hCE cells were partitioned by well-developed membranous structures and covered with thickened, well-stratified hypodermal tissues. In addition, both the number and extent of activation of tumor-infiltrating macrophages were more profound in ALVA101/hCE compared to ALVA101/mock tumors. The chemotactic response of macrophages to ALVA101/hCE cells was also higher than that to ALVA/mock cells. These results thus indicate that CE expression in tumor cells induces tumor growth arrest via inhibition of angiogenesis and enhanced immune responses.     

Impaired hippocampal long-term potentiation and failure of learning in β1,4-N-acetylgalactosaminyltransferase gene transgenic mice

Gangliosides (sialic acid-containing glycosphingolipids) play important roles in many physiological functions, including synaptic plasticity in the hippocampus, which is considered as a cellular mechanism of learning and memory. In the present study, three types of synaptic plasticity, long-term potentiation (LTP), long-term depression (LTD) and reversal of LTP (depotentiation, DP), in the field excitatory post-synaptic potential in CA1 hippocampal neurons and learning behavior were examined in β1,4-N-acetylgalactosaminyltransferase (β1,4 GalNAc-T; GM2/GD2 synthase) gene transgenic (TG) mice, which showed a marked decrease in b-pathway gangliosides (GQ1b, GT1b and GD1b) in the brain and isolated hippocampus compared with wild-type (WT) mice. The magnitude of the LTP induced by tetanus (100 pulses at 100?Hz) in TG mice was significantly smaller than that in control WT mice, whereas there was no difference in the magnitude of the LTD induced by three short trains of low-frequency stimulation (LFS) (200 pulses at 1?Hz) at 20?min intervals between the two groups of mice. The reduction in the LTP produced by delivering three trains of LFS (200 pulses at 1?Hz, 20?min intervals) was significantly greater in the TG mice than in the WT mice. Learning was impaired in the four-pellet taking test (4PTT) in TG mice, with no significant difference in daily activity or activity during the 4PTT between TG and WT mice. These results suggest that the overexpression of β1,4 GalNAc-T resulted in altered synaptic plasticity of LTP and DP in hippocampal CA1 neurons and learning in the 4PTT, and this is attributable to the shift from b-pathway gangliosides to a-pathway gangliosides.     

Synthesis and evaluation of novel pyrimidine-based dual EGFR/Her-2 inhibitors

A structure-activity relationship study of 4-anilinopyrimidines for dual EGFR/Her-2 inhibitor has resulted in the identification of 4-anilino-5-alkenyl or 5-alkynyl-6-methylpyrimidine derivatives that have exhibited effective inhibitory activity against both enzymes. The presence of 5-alkenyl or 5-alkynyl moiety bearing terminal hydrophilic group played important role for inhibition of these enzymes. Selected compounds in the series demonstrated some activity against Her-2 dependent cell line (BT474).     

The COP1 ortholog PPS regulates the juvenile-adult and vegetative-reproductive phase changes in rice

Because plant reproductive development occurs only in adult plants, the juvenile-to-adult phase change is an indispensable part of the plant life cycle. We identified two allelic mutants, peter pan syndrome-1 (pps-1) and pps-2, that prolong the juvenile phase in rice (Oryza sativa) and showed that rice PPS is an ortholog of Arabidopsis thaliana CONSTITUTIVE PHOTOMORPHOGENIC1. The pps-1 mutant exhibits delayed expression of miR156 and miR172 and the suppression of GA biosynthetic genes, reducing the GA(3) content in this mutant. In spite of its prolonged juvenile phase, the pps-1 mutant flowers early, and this is associated with derepression of RAP1B expression in pps-1 plants independently of the Hd1-Hd3a/RFT1 photoperiodic pathway. PPS is strongly expressed in the fourth and fifth leaves, suggesting that it regulates the onset of the adult phase downstream of MORI1 and upstream of miR156 and miR172. Its ability to regulate the vegetative phase change and the time of flowering suggests that rice PPS acquired novel functions during the evolution of rice/monocots.