Affinity Improvement of a Cancer-Targeted Antibody through Alanine-Induced Adjustment of Antigen-Antibody Interface

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Antifibrotic effect of methylated quercetin derivatives on TGFβ-induced hepatic stellate cells

Quercetin (QCT) and isorhamnetin (ISO), natural flavonoids, were both shown to possess antifibrotic activity in in vivo and in vitro models of hepatic fibrosis. Although ISO is a direct metabolite of QCT differing by a methyl group, it has been reported to be absorbed more adequately and eliminated slower than QCT after oral administration. Our aim of the study was to investigate biological effect of mono-methylated QCT derivatives against fibrosis using rat hepatic stellate cells (HSC-T6). All test derivatives were synthesized from QCT. HSC-T6 cells were induced by TGFβ and treated with derivatives followed by cell proliferation assay, immunofluorescence staining of αSMA, and gene expression analysis of fibrosis markers. All compounds showed a dose- and time-dependent antiproliferation effect. ISO, 3-O-methylquercetin (3MQ), and rhamnetin (RHA) reduced αSMA mRNA; 3MQ prevented the augmentation of collagen I mRNA; and compounds, except azaleatin and 3MQ, reduced Timp1 mRNA expression in TGFβ-induced HSCs. In conclusion, each compound had singular effect against different features of fibrosis depending on the position of methyl group although the further mechanism of action of compounds during fibrosis development remains to be investigated. These findings suggest that antifibrotic effect of quercetin can be enhanced by adding methyl group on functionally important position.     

Heart rate and electroencephalogram changes caused by finger acupressure on planta pedis

Preliminary experiments were carried out to investigate the feasibility of using an electroencephalogram and heart rates to evaluate the efficacy of finger acupressure on the key points of planta pedis (both soles). Continuous electroencephalograms were recorded from 19 electrodes based on the International 10-20 electrode placement system on 22 university students (21+/-2.3 years). Spectral power changes were obtained at each electrode site. The power of the alpha1 frequency range (8-10 Hz) increased slightly during acupressure although no statistical significance was observed, while heart rates decreased in all subjects (p<0.05). Cerebral cortex asymmetry in the spectral power changes was not clearly observed during the right and left sole acupressure. This preliminary study suggests that a classification of subjects is necessary in understanding brain wave data during acupressure on soles.     

Evidence of ROS generation by mitochondria in cells with impaired electron transport chain and mitochondrial DNA damage

Mitochondrial damage is a well known cause of mitochondria-related diseases. A major mechanism underlying the development of mitochondria-related diseases is thought to be an increase in intracellular oxidative stress produced by impairment of the mitochondrial electron transport chain (ETC). However, clear evidence of intracellular free radical generation has not been clearly provided for mitochondrial DNA (mtDNA)-damaged cells. In this study, using the novel fluorescence dye, 2-[6-(4'-hydroxy)phenoxy-3H-xanthen-3-on-9-yl]benzoic acid (HPF), which was designed to detect hydroxyl radicals (*OH), intracellular free radical formation was examined in 143B cells (parental cells), 143B-rho(0) cells (mtDNA-lacking cells), 87 wt (cybrid), and cybrids of 4977-bp mtDNA deletion (common deletion) cells containing the deletion with 0%, 5%, 50% and >99% frequency (HeLacot, BH5, BH50 and BH3.12, respectively), using a laser confocal microscope detection method. ETC inhibitors (rotenone, 3-nitropropionic acid, thenoyltrifluoroacetone, antimycin A and sodium cyanide) were also tested to determine whether inhibitor treatment increased intracellular reactive oxygen species (ROS) generation. A significant increase in ROS for 143B-rho(0) cells was observed compared with 143B cells. However, for the 87 wt cybrid, no increase was observed. An increase was also observed in the mtDNA-deleted cells BH50 and BH3.12. The ETC inhibitors increased intracellular ROS in both 143B and 143B-rho(0) cells. Furthermore, in every fluorescence image, the fluorescence dye appeared localized around the nuclei. To clarify the localization, we double-stained cells with the dye and MitoTracker Red. The resulting fluorescence was consistently located in mitochondria. Furthermore, manganese superoxide dismutase (MnSOD) cDNA-transfected cells had decreased ROS. These results suggest that more ROS are generated from mitochondria in ETC-inhibited and mtDNA-damaged cells, which have impaired ETC.     

Norlichexanthone produced by cultured endolichenic fungus induced from Pertusaria laeviganda and its antioxidant activity

Endolichenic fungi, nonobligate microfungi that live in lichen, are promising as new bioresources of pharmacological compounds. We found that norlichexanthone isolated from the endolichenic fungus in Pertusaria laeviganda exhibited high antioxidant activity. Norlichexanthone produced by endolichenic fungus had the antioxidant activity with same level of ascorbic acid. This is the first report of high antioxidant activity of norlichexanthone.

Abbreviations: AAPH: 2,2?-azobis (2-methylpropionamidine) dihydrochloride; DPPH: 2,2-diphenyl-1-picrylhydrazyl; FL: fluorescein sodium salt; HPLC-PDA: high-performance liquid chromatography with photodiode array; LC-ESI-MS: liquid chromatography with electrospray ionization mass spectrometry; ORAC: oxygen radical absorbance capacity; PB: phosphate buffer; ROS: reactive oxygen species; TLC: thin-layer chromatography     

Hsp105α suppresses Adriamycin-induced cell death via nuclear localization signal-dependent nuclear accumulation

Heat shock protein 105 (Hsp105) is a molecular chaperone, and the isoforms Hsp105α and Hsp105β exhibit distinct functions with different subcellular localizations. Hsp105β localizes in the nucleus and induces the expression of the major heat shock protein Hsp70, whereas cytoplasmic Hsp105α is less effective in inducing Hsp70 expression. Hsp105 shuttles between the cytoplasm and the nucleus; the subcellular localization is governed by the relative activities of the nuclear localization signal (NLS) and nuclear export signal (NES). Here, we show that nuclear accumulation of Hsp105α but not Hsp105β is involved in Adriamycin (ADR) sensitivity. Knockdown of Hsp105α induces cell death at low ADR concentration, at which ADR is less effective in inducing cell death in the presence of Hsp105α. Of note, Hsp105 is localized in the nucleus under these conditions, even though Hsp105β is not expressed, indicating that Hsp105α accumulates in the nucleus in response to ADR treatment. The exogenously expressed Hsp105α but not its NLS mutant localizes in the nucleus of ADR-treated cells. In addition, the expression level of the nuclear export protein chromosomal maintenance 1 (CRM1) was decreased by ADR treatment of cells, and CRM1 knockdown caused nuclear accumulation of Hsp105α both in the presence and absence of ADR. These results indicating that Hsp105α accumulates in the nucleus in a manner dependent on the NLS activity via the suppression of nuclear export. Our findings suggest a role of nuclear Hsp105α in the sensitivity against DNA-damaging agents in tumor cells.     

Tau-tubulin kinase phosphorylates tau at Ser-208 and Ser-210, sites found in paired helical filament-tau

Hyperphosphorylated tau protein is known to be a major component of the paired helical filaments (PHFs) that accumulate in the brain of Alzheimer's patients. The kinase that phosphorylated Ser-208 and Ser-210 in PHF-tau had remained unknown. We used anti-pS208 and anti-pS210 antibodies and Western blots to confirm that the tau-tubulin kinase (TTK) phosphorylates tau at Ser-208 and at Ser-210. Using partial amino acid sequences of purified bovine brain TTK, a mouse cDNA of TTK was isolated and the sequence was determined. Its 963 bp coding region is composed of 320 amino acids and encodes a 36 kDa protein indistinguishable in size from authentic bovine brain TTK. Our immunoblot analysis demonstrated that TTK is ubiquitously distributed in the rat tissues, and that it is developmentally regulated in the rat brain.     

Solution structure of human acidic fibroblast growth factor and interaction with heparin-derived hexasaccharide

Fibroblast growth factors (FGFs) bind to extracellular matrices, especially heparin-like carbohydrates of heparansulfate proteoglycans which stabilize FGFs to protect against inactivation by heat, acid, proteolysis and oxidation. Moreover, binding of FGFs to cell surface proteoglycans promotes to form oligomers, which is essential for receptor oligomerization and activation. In the present study, we determined the solution structure of acidic FGF using a series of triple resonance multi-dimensional NMR experiments and simulated annealing calculations. Furthermore, we prepared the sample complexed with a heparin-derived hexasaccharide which is a minimum unit for aFGF binding. From the chemical shift differences between free aFGF and aFGF-heparin complex, we concluded that the major heparin binding site was located on the regions 110–131 and 17–21. The binding sites are quite similar to those observed for bFGF-heparin hexasaccharide complex, showing that both FGFs recognize heparin- oligosaccharides in a similar manner.     

MHC-restricted protection of cats against FIV infection by adoptive transfer of immune cells from FIV-vaccinated donors

The role of cellular immunity in vaccine protection against FIV infection was evaluated using adoptive cell transfer studies. Specific-pathogen-free cats received two adoptive transfers of washed blood cells from either vaccinated or unvaccinated donors with varying MHC compatibility at 1-week intervals, and a homologous FIV(Pet) challenge 1 day after the first adoptive transfer. FIV-specific CTL, IFN-gamma production, and proliferation responses were detected in the PBMC from the vaccinated donors. Seven of eleven (64%) recipients of cells from half-matched/vaccinated donors remained negative for FIV-antibodies after FIV challenge and four of those were completely protected. Two of two recipients of cells from MHC-identical/vaccinated donors were completely protected. All recipients of cells from unrelated/vaccinated, half-matched/unvaccinated, or unrelated/unvaccinated donors were unprotected. Thus, protection mediated by adoptive transfer of immunocytes from vaccinated cats was MHC-restricted, occurred in the absence of antiviral humoral immunity, and correlated with the transfer of cells with FIV-specific CTL and T-helper activities.