Hearing vulnerability after noise exposure in a mouse model of reactive oxygen species overproduction

Previous studies have convincingly argued that reactive oxygen species (ROS ) contribute to the development of several major types of sensorineural hearing loss, such as noise‐induced hearing loss (NIHL ), drug‐induced hearing loss, and age‐related hearing loss. However, the underlying molecular mechanisms induced by ROS in these pathologies remain unclear. To resolve this issue, we established an in vivo model of ROS overproduction by generating a transgenic (TG ) mouse line expressing the human NADPH oxidase 4 (NOX 4, NOX 4‐ TG mice), which is a constitutively active ROS ‐producing enzyme that does not require stimulation or an activator. Overproduction of ROS was detected at the cochlea of the inner ear in NOX 4 ‐TG mice, but they showed normal hearing function under baseline conditions. However, they demonstrated hearing function vulnerability, especially at high‐frequency sounds, upon exposure to intense noise, which was accompanied by loss of cochlear outer hair cells (OHC s). The vulnerability to loss of hearing function and OHC s was rescued by treatment with the antioxidant Tempol. Additionally, we found increased protein levels of the heat‐shock protein 47 (HSP 47) in models using HEK 293 cells, including H₂O₂ treatment and cells with stable and transient expression of NOX 4. Furthermore, the up‐regulated levels of Hsp47 were observed in both the cochlea and heart of NOX 4 ‐TG mice. Thus, antioxidant therapy is a promising approach for the treatment of NIHL . Hsp47 may be an endogenous antioxidant factor, compensating for the chronic ROS overexposure in vivo , and counteracting ROS ‐related hearing loss.

     

Heparin/heparan sulfate/CD44-v3 enhances cell migration in term placenta-derived immortalized human trophoblast cells

The function of CD44-v3 and heparin/heparan sulfate (HS) signaling was investigated during trophoblast cell migration to identify their role in the renewal of syncytial layer damage caused by increased hemodynamic turbulence in the intervillous space and maintenance of syncytial integrity in pre-eclampsia. We evaluated the effect of heparin/HS/CD44-v3-mediated processes during scratch wound closure in monolayer immortalized human trophoblast cells derived from term placenta (TCL-1 cells). Western blot analysis showed that these cultured human trophoblast cells express the epidermal growth factor receptor and CD44-v3 but do not express syndecan 4. An in vitro scratch wound healing assay showed enhanced migration of trophoblast cells in a dose-dependent manner in the presence of heparin compared with controls when cultured under serum-free conditions. Conversely, an anti-CD44 function-blocking antibody and CD44 siRNA suppressed the migration of trophoblast cells in the presence of heparin in a similar scratch assay. Furthermore, both heparin treatment and in vitro scratch wounding induced the phosphorylation of p21-activated kinase 1 (PAK1), whereas the anti-CD44-v3 antibody suppressed the heparin-induced phosphorylation of PAK1 in trophoblast cells. These results indicate that heparin/HS/CD44-v3-mediated signaling, in the absence of growth factor networks, enhances the direct repair of the damaged trophoblast layer through the migration of trophoblast cells. This renewed cell coverage may lead to the maintenance of syncytiotrophoblast cell function and an associated reduction in pathogenic soluble factors derived from the damaged trophoblast cells.     

Chitinase III in pomegranate seeds (Punica granatum Linn.): a high-capacity calcium-binding protein in amyloplasts

Chitinases are a class of ubiquitous proteins that are widely distributed in plants. Defense is the major natural role for chitinases, primarily against fungal pathogens. Little is known regarding their non-defensive roles in seeds. In this study, a new class III chitinase from pomegranate seeds (pomegranate seed chitinase, PSC) was isolated and purified to homogeneity. The native state of PSC is a monomer with a molecular weight of approximately 30 kDa. This chitinase naturally binds calcium ions with high capacity and low affinity, suggesting that PSC is a calcium storage protein. Consistent with this idea, its amino acid sequence (inferred from cDNA) is rich in acidic amino acid residues, especially Asp, similar to reported calcium storage proteins. The presence of calcium considerably improves the stability of the protein but has little effect on its enzymatic activity. Transmission electron microscopy analyses indicate that, similar to phytoferritin, this enzyme is widely distributed in the stroma of amyloplasts of the embryonic cells, suggesting that amyloplasts in seeds could serve as an alternative plastid for calcium storage. Indeed, the transmission electron microscopy results showed that, within the embryonic cells, calcium ions are mainly distributed in the stroma of the amyloplasts, consistent with a role for PSC in calcium storage. Thus, the plant appears to have evolved a new plastid for calcium storage in seeds. During seed germination, the content of this enzyme decreases with time, suggesting that it is involved in the germination process.     

Novel quinazoline-quinoline alkaloids with cytotoxic and DNA topoisomerase II inhibitory activities

Two new synthetic analogues of luotonins A and F, 7-acetylaminoluotonin A (6) and 3-[3H(quinazolino-4-one)]quinoline (7) were synthesized. The new analogues, along with four natural quinazoline-quinoline alkaloids, luotonins A (1), B (2), E (3), F (4) and a synthetic deoxoluotonin F (5), showed cytotoxic activity (IC(50) 1.8-40.0 microg/mL) and DNA topoisomerase II inhibition at a concentration of 25 microM.     

Synthesis of non-natural C2-homo-ceramide and its apoptotic activity against HL-60 cells

Non-natural ceramide analogues, C2-homo-ceramide and C2-homo-dihydroceramide, were prepared from L-aspartic acid via L-homo-serine. The apoptotic activities of the synthesized ceramide analogues were examined in HL-60 human leukemia cells. C2-homo- and C2-bishomo-ceramide indicate low but considerable apoptotic activities in comparison with C2-ceramide.     

Requirement of domain-domain interaction for conformational change and functional ATP hydrolysis in myosin

Coordination between the nucleotide-binding site and the converter domain of myosin is essential for its ATP-dependent motor activities. To unveil the communication pathway between these two sites, we investigated contact between side chains of Phe-482 in the relay helix and Gly-680 in the SH1-SH2 helix. F482A myosin, in which Phe-482 was changed to alanine with a smaller side chain, was not functional in vivo. In vitro, F482A myosin did not move actin filaments and the Mg2+-ATPase activity of F482A myosin was hardly activated by actin. Phosphate burst and tryptophan fluorescence analyses, as well as fluorescence resonance energy transfer measurements to estimate the movements of the lever arm domain, indicated that the transition from the open state to the closed state, which precedes ATP hydrolysis, is very slow. In contrast, F482A/G680F doubly mutated myosin was functional in vivo and in vitro. The fact that a larger side chain at the 680th position suppresses the defects of F482A myosin suggests that the defects are caused by insufficient contact between side chains of Ala-482 and Gly-680. Thus, the contact between these two side chains appears to play an important role in the coordinated conformational changes and subsequent ATP hydrolysis.     

Main polyol dehydrogenase of Gluconobacter suboxydans IFO 3255, membrane-bound D-sorbitol dehydrogenase,that needs product of upstream gene,sldB, for activity

The D-sorbitol dehydrogenase gene, sldA, and an upstream gene, sldB, encoding a hydrophobic polypeptide, SldB, of Gluconobacter suboxydans IFO 3255 were disrupted in a check of their biological functions. The bacterial cells with the sldA gene disrupted did not produce L-sorbose by oxidation of D-sorbitol in resting-cell reactions at pHs 4.5 and 7.0, indicating that the dehydrogenase was the main D-sorbitol-oxidizing enzyme in this bacterium. The cells did not produce D-fructose from D-mannitol or dihydroxyacetone from glycerol. The disruption of the sldB gene resulted in undetectable oxidation of D-sorbitol, D-mannitol, or glycerol, although the cells produced the dehydrogenase. The cells with the sldB gene disrupted produced more of what might be signal-unprocessed SldA than the wild-type cells did. SldB may be a chaperone-like component that assists signal processing and folding of the SldA polypeptide to form active D-sorbitol dehydrogenase.     

Design and functional analysis of actomyosin motor domain chimera proteins

To gain more structural and functional information on the actomyosin complexes, we have engineered chimera proteins carrying the entire Dictyostelium actin in the loop 2 sequence of the motor domain of Dictyostelium myosin II. Although the chimera proteins were unable to polymerize by themselves, addition of skeletal actin promoted polymerization. Electron microscopic observation demonstrated that the chimera proteins were incorporated into actin filaments, when copolymerized with skeletal actin. Copolymerization with skeletal actin greatly enhanced the MgATPase, while the chimera proteins without added skeletal actin hydrolyzed ATP at a very low rate. These results indicate that the actin part and the motor domain part of the chimera proteins are correctly folded, but the chimera proteins are structurally stressed so that efficient polymerization is inhibited.     

Distinct expression patterns and roles of aldehyde dehydrogenases in normal oral mucosa keratinocytes: differential inhibitory effects of a pharmacological inhibitor and RNAi-mediated knockdown on cellular phenotype and epithelial morphology

Aldehyde dehydrogenases (ALDHs), enzymes responsible for detoxification and retinoic acid biosynthesis, are considered a potent functional stem cell marker of normal and malignant cells in many tissues. To date, however, there are no available data on ALDH distributions and functions in oral mucosa. This study aims to clarify the levels and types of ALDH expression using immunohistochemistry with accompanying mRNA expression as well as an ALDEFLUOR assay, and to assess phenotypic and histological changes after manipulation of the ALDH activity of oral keratinocytes to increase the potency of a tissue-engineered oral mucosa by a specific ALDH inhibitor, diethylaminobenzaldehyde (DEAB), together with small interfering RNA of ALDH1A3 and ALDH3A1. Results showed the mRNA and cytoplasmic protein expression of ALDH1A3 and ALDH3A1 to be mostly localized in the upper suprabasal layer although no ALDH1A1 immunoreaction was detected throughout the epithelium. Oral keratinocytes with high ALDH activity exhibited a profile of differentiating cells. By pharmacological inhibition, the phenotypic analysis revealed the proliferating cell-population shifting to a more quiescent state compared with untreated cells. Furthermore, a well-structured epithelial layer showing a normal differentiation pattern and a decrease in Ki-67 immunopositive basal cells was developed by DEAB incubation, suggesting a slower turnover rate efficient to maintain undifferentiated cells. Histological findings of a regenerated oral epithelium by ALDH1A3 siRNA were similar to those when treated with DEAB while ALDH3A1 siRNA eradicated the epithelial regenerative capacity. These observations suggest the effects of phenotypic and morphological alterations by DEAB on oral keratinocytes are mainly consequent to the inhibition of ALDH1A3 activity.     

“Nojirimycin” as a Potent Inhibitor of Glucosidase

Glucose-6-phosphate dehydrogenase in a yeast, Hansenula mrakii IFO 0895 is induced when the cells are cultured in a medium containing lipid hydroperoxide. The enzyme was purified from H. mrakii to the homogeneous state on polyacrylamide gel electrophoresis. The molecular weight of the purified enzyme was estimated to be approximately 52kDa by SDS-PAGE and 130 kDa by Sephadex G-150column chromatography, respectively. The enzyme was specific to glucose-6-phosphate and NADP⁺, and K_mvalues for glucose-6-phosphate and NADP⁺ were 293µM and 24.1 µM, respectively. The enzyme activity was inhibited by diethylpyrocarbonate and 2, 4, 6-trinitrobenzene sulfonate, and by metal ions such as Zn^{2 +}, Cd^{2 +}, Cu^{2 +}, and Al^{3 +} . tert-Butyl hydroperoxide, a kind of lipid hydroperoxide, slightly(approximately 20%) increased the enzyme activity.