SIRT1, a class III histone deacetylase, regulates TNF-α-induced inflammation in human chondrocytes.

OBJECTIVE: The present study was performed to elucidate the possible role of SIRT1 signaling in joint inflammation in human articular chondrocytes. DESIGN: Real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR) and Western blotting were performed to detect gene products and proteins involved in tumor necrosis factor α (TNF-α)-induced inflammation and cartilage degradation in human primary chondrocytes. Matrix metalloproteinase (MMP)-2 and MMP-9 activity was evaluated by gelatin zymography. Overexpression and knockdown of SIRT1 were also performed to investigate whether SIRT1 is associated with the anti-inflammatory activity of resveratrol in chondrocytes. RESULTS: Resveratrol dose-dependently inhibited TNF-α-induced cyclooxygenase-2 (COX-2), MMP-1, MMP-3, MMP-13 and PGE(2) production in human chondrocytes. Moreover, MMP-2 and MMP-9 activity was increased by treatment with TNF-α; however, SIRT1 activation decreased the proinflammatory effects induced by TNF-α. In addition, treatment of SIRT1 activator and overexpression of SIRT1 inhibited the expression and activation of the main proinflammatory regulator NF-κB, which was increased by TNF-α. When SIRT1 was overexpressed in chondrocytes, the anti-inflammatory action of SIRT1 was similar to that exerted by resveratrol. CONCLUSIONS: SIRT1 activation deacetylates and inactivates NF-κB, and thereby, exerts an anti-inflammatory effect on chondrocytes, suggesting that SIRT1 activators could be explored as potential treatments for arthritis.

Comparison of polyvinyl alcohol coated nasal packing with non-absorbable nasal packing.

BACKGROUND: A number of different nasal packing materials are available for prevention of nasal bleeding after endoscopic sinus surgery. Polyvinyl alcohol (PVA) coated nasal packing is an expandable packing for use in rhinological surgery. This innovative surf- ace treatment helps to reduce the possibility of adherence to tissue and of blood clotting within the sponge. The present study investigated the effects of PVA coated packing and non-absorbable packing with respect to pain, healing site and postoperative bleeding following endoscopic sinus surgery. METHODOLOGY: Patients between 18-80 years of age undergoing sinus surgery were enrolled. Each patient`s ethmoid cavities were randomised to receive PVA coated packing material or the standard non-absorbable sinus packs. The remaining nasal packing material was removed on the 2nd day in the clinic. We determined visual analog scale score, bleeding time and wound healing status. A single rhinologist graded postoperative endoscopic appearance. Length of follow-up was 3 months. RESULTS: Thirty three patients were recruited. There was a significant difference in the bleeding time between the two groups, but pain and wound healing were not significantly different between the two groups. CONCLUSION: PVA-coated nasal packing presents comparable characteristics with traditional nasal packing.

Effects of electron-cyclotron-resonance-heating-induced internal kink mode on the toroidal rotation in the KSTAR Tokamak.

It is observed that the magnitude of the toroidal rotation speed is reduced by the central electron cyclotron resonance heating (ECRH) regardless of the direction of the toroidal rotation. The magnetohydrodynamics activities generally appear with the rotation change due to ECRH. It is shown that the internal kink mode is induced by the central ECRH and breaks the toroidal symmetry. When the magnetohydrodynamics activities are present, the toroidal plasma viscosity is not negligible. The observed effects of ECRH on the toroidal plasma rotation are explained by the neoclassical toroidal viscosity in this Letter. It is found that the neoclassical toroidal viscosity torque caused by the internal kink mode damps the toroidal rotation.

Skp1 forms multiple protein complexes, including RAVE, a regulator of V-ATPase assembly.

SCF ubiquitin ligases are composed of Skp1, Cdc53, Hrt1 and one member of a large family of substrate receptors known as F-box proteins (FBPs). Here we report the identification, using sequential rounds of epitope tagging, affinity purification and mass spectrometry, of 16 Skp1 and Cdc53-associated proteins in budding yeast, including all components of SCF, 9 FBPs, Yjr033 (Rav1) and Ydr202 (Rav2). Rav1, Rav2 and Skp1 form a complex that we have named 'regulator of the (H+)-ATPase of the vacuolar and endosomal membranes' (RAVE), which associates with the V1 domain of the vacuolar membrane (H+)-ATPase (V-ATPase). V-ATPases are conserved throughout eukaryotes, and have been implicated in tumour metastasis and multidrug resistance, and here we show that RAVE promotes glucose-triggered assembly of the V-ATPase holoenzyme. Previous systematic genome-wide two-hybrid screens yielded 17 proteins that interact with Skp1 and Cdc53, only 3 of which overlap with those reported here. Thus, our results provide a distinct view of the interactions that link proteins into a comprehensive cellular network.

Dynamic Time-Locking Mechanism in the Cortical Representation of Spoken Words.

Human speech has a unique capacity to carry and communicate rich meanings. However, it is not known how the highly dynamic and variable perceptual signal is mapped to existing linguistic and semantic representations. In this novel approach, we used the natural acoustic variability of sounds and mapped them to magnetoencephalography (MEG) data using physiologically-inspired machine-learning models. We aimed at determining how well the models, differing in their representation of temporal information, serve to decode and reconstruct spoken words from MEG recordings in 16 healthy volunteers. We discovered that dynamic time-locking of the cortical activation to the unfolding speech input is crucial for the encoding of the acoustic-phonetic features of speech. In contrast, time-locking was not highlighted in cortical processing of non-speech environmental sounds that conveyed the same meanings as the spoken words, including human-made sounds with temporal modulation content similar to speech. The amplitude envelope of the spoken words was particularly well reconstructed based on cortical evoked responses. Our results indicate that speech is encoded cortically with especially high temporal fidelity. This speech tracking by evoked responses may partly reflect the same underlying neural mechanism as the frequently reported entrainment of the cortical oscillations to the amplitude envelope of speech. Furthermore, the phoneme content was reflected in cortical evoked responses simultaneously with the spectrotemporal features, pointing to an instantaneous transformation of the unfolding acoustic features into linguistic representations during speech processing.

Production of transgenic recloned piglets harboring the human granulocyte-macrophage colony stimulating factor (hGM-CSF) gene from porcine fetal fibroblasts by nuclear transfer.

We used nuclear transfer (NT) to develop transgenic female pigs harboring goat beta-casein promoter/human granulocyte-macrophage colony stimulating factor (hGM-CSF). The expression of hGM-CSF was specific to the mammary gland, and the glycosylation-derived size heterogeneity corresponded to that of the native human protein. Although various cell types have been used to generate cloned animals, little is currently known about the potential use of fibroblasts derived from a cloned fetus as donor cells for nuclear transfer. The developmental potential of porcine cloned fetal fibroblasts transfected with hGM-CSF was evaluated in the present study. Cloned fetal fibroblasts were isolated from a recipient following the transplantation of NT embryos. The cells were transfected with both hGM-CSF and the neomycin resistance gene in order to be used as donor cells for NT. Reconstructed embryos were implanted into six sows during estrus; two of the recipient sows delivered seven healthy female piglets with the hGM-CSF gene (confirmed with PCR and fluorescent in situ hybridization) and microsatellite analysis confirmed that the clones were genetically identical to the donor cells. The expression of hGM-CSF was strong in the mammary glands of a transgenic pig that died a few days prior to parturition (110 d after AI). These results demonstrated that somatic cells derived from a cloned fetus can be used to produce recloned and transgenic pigs.

Skp1p and the F-box protein Rcy1p form a non-SCF complex involved in recycling of the SNARE Snc1p in yeast.

Skp1p-cullin-F-box protein (SCF) complexes are ubiquitin-ligases composed of a core complex including Skp1p, Cdc53p, Hrt1p, the E2 enzyme Cdc34p, and one of multiple F-box proteins which are thought to provide substrate specificity to the complex. Here we show that the F-box protein Rcy1p is required for recycling of the v-SNARE Snc1p in Saccharomyces cerevisiae. Rcy1p localized to areas of polarized growth, and this polarized localization required its CAAX box and an intact actin cytoskeleton. Rcy1p interacted with Skp1p in vivo in an F-box-dependent manner, and both deletion of its F box and loss of Skp1p function impaired recycling. In contrast, cells deficient in Cdc53p, Hrt1p, or Cdc34p did not exhibit recycling defects. Unlike the case for F-box proteins that are known to participate in SCF complexes, degradation of Rcy1p required neither its F box nor functional 26S proteasomes or other SCF core subunits. Importantly, Skp1p was the only major partner that copurified with Rcy1p. Our results thus suggest that a complex composed of Rcy1p and Skp1p but not other SCF components may play a direct role in recycling of internalized proteins.

Core-shell nanoparticle arrays double the strength of steel.

Manipulating structure, defects and composition of a material at the atomic scale for enhancing its physical or mechanical properties is referred to as nanostructuring. Here, by combining advanced microscopy techniques, we unveil how formation of highly regular nano-arrays of nanoparticles doubles the strength of an Fe-based alloy, doped with Ti, Mo, and V, from 500 MPa to 1 GPa, upon prolonged heat treatment. The nanoparticles form at moving heterophase interfaces during cooling from the high-temperature face-centered cubic austenite to the body-centered cubic ferrite phase. We observe MoC and TiC nanoparticles at early precipitation stages as well as core-shell nanoparticles with a Ti-C rich core and a Mo-V rich shell at later precipitation stages. The core-shell structure hampers particle coarsening, enhancing the material's strength. Designing such highly organized metallic core-shell nanoparticle arrays provides a new pathway for developing a wide range of stable nano-architectured engineering metallic alloys with drastically enhanced properties.

Effects of Aerobic and Resistance Training Combined with Fortified Milk on Muscle Mass, Muscle Strength, and Physical Performance in Older Adults: A Randomized Controlled Trial.

OBJECTIVES: Fortified milk and resistance training (RT) increase muscle mass, muscle strength, and physical performance in older adults, but it remains unclear whether RT combined with aerobic training (AT) would have stronger effects on these outcomes. The purpose of this study was to examine the effects of aerobic and resistance training (ART) combined with fortified milk consumption on muscle mass, muscle strength, and physical performance in older adults. DESIGN: Open-labeled randomized controlled trial. SETTING: University of Tsukuba. PARTICIPANTS: Fifty-six older adults aged 65-79. INTERVENTION: Participants were randomly allocated into resistance training (RT + fortified milk, n = 28) and aerobic and resistance training (ART + fortified milk, n = 28) groups. All participants attended supervised exercise programs twice a week at University of Tsukuba and ingested fortified milk every day for 12 weeks. Skeletal muscle index ([SMI]: appendicular lean mass/height2) was assessed using dual-energy X-ray absorptiometry as a muscle mass measure. One-repetition maximum strength was measured using four kinds of resistance training machines (chest press, leg extension, leg curl, and leg press) as muscle strength measures. Sit-to-stand and arm curl tests were also assessed as physical performance measures. MEASUREMENTS: The primary measurements were muscle mass and strength. The secondary outcomes were physical performance, blood samples, habitual diet, habitual physical activity, and medication use. RESULTS: Although the muscle strength and physical performance measures significantly improved in both groups, SMI significantly improved in only the RT group. There was no significant difference in the change in SMI and muscle strength measures between the two groups. However, the change in sit-to-stand and arm curl measures in the ART group were significantly higher than those in the RT group. CONCLUSIONS: These results suggest that AT before RT combined with fortified milk consumption has similar effects on skeletal muscle mass and strength compared with RT alone, but it may be a more useful strategy to improve physical performance in older adults. Although the mechanism of our intervention is uncertain, our program would be an effective prevention for sarcopenia in older adults.

Arsenic trioxide-mediated growth inhibition in MC/CAR myeloma cells via cell cycle arrest in association with induction of cyclin-dependent kinase inhibitor, p21, and apoptosis.

We investigated the in vitro effect of As2O3 on proliferation, cell cycle regulation, and apoptosis in human myeloma cell lines. As2O3 significantly inhibited the proliferation of all of eight myeloma cell lines examined in a dose-dependent manner with IC50 of approximately 1-2 microM. DNA flow cytometric analysis indicated that As2O3 (2 microM) induced a G1 and/or a G2-M phase arrest in these cell lines. To address the mechanism of the antiproliferative effect of As2O3, we examined the effect of As2O3 on cell cycle-related proteins in MC/CAR cells in which both G1 and G2-M phases were arrested. Western blot analysis demonstrated that treatment with As2O3 (2 microM) for 72 h did not change the steady-state levels of CDK2, CDK4, cyclin D1, cyclin E, and cyclin B1 but decreased the levels of CDK6, cdc2, and cyclin A. The mRNA and protein levels of CDKI, p21 were increased by treatment with As2O3, but those of p27 were not. In addition, As2O3 markedly enhanced the binding of p21 with CDK6, cdc2, cyclin E, and cyclin A compared with untreated control cells. Furthermore, the activity of CDK6-associated kinase was reduced in association with hypophosphorylation of Rb protein. The activity of cdc2-associated kinase was decreased, which was accompanied by the up-regulation of cdc2 phosphorylation (cdc2-Tyr15 phosphorylation) resulting from reduction of cdc25B and cdc25C phosphatases. As2O3 also induced apoptosis in MC/CAR cells as evidenced by flow cytometric detection of sub-G1 DNA content and annexin V binding assay. This apoptotic process was associated with down-regulation of Bcl-2, loss of mitochondrial transmembrane potential (delta psi(m)), and an increase of caspase-3 activity. These results suggest that As2O3 inhibits the proliferation of myeloma cells, especially MC/CAR cells, via cell cycle arrest in association with induction of p21 and apoptosis.

An adenovirus expressing mutant p27 showed more potent antitumor effects than adenovirus-p27 wild type.

The main inhibitory action of p27, a cyclin-dependent kinase inhibitor (CDKI), arises from its binding with the cyclin E/cyclin-dependent kinase 2 (Cdk2) complex that results in G(1)-S arrest. Degradation of p27 is mediated by phosphorylation of Thr-187 of p27, which follows ubiquitination. In this study, we generated two adenoviruses expressing wild-type p27 (ad-p27wt) and mutant p27 (ad-p27mt), with mutation of Thr-187/Pro-188 (ACGCCC) to Met-187/Ile-188 (ATGATC), which was produced with the belief that mutant p27 would bind cyclin E/CDK2 more stably and show more potent antitumor effects. Ad-p27wt and ad-p27mt expressed p27 proteins that were indistinguishable by anti-p27 antibody. A pulse chase experiment showed that p27mt was more resistant to degradation than p27wt. In human lung cancer cell lines, ad-p27mt showed stronger growth inhibition than ad-p27wt. Both types of ad-p27 induced G(1)-S arrest and apoptosis; however, ad-p27mt induced stronger G(1)-S arrest and apoptosis. Intratumoral injection of ad-p27mt induced partial regression of established tumors and inhibited the growth of human lung cancer xenografts more strongly than ad-p27wt. From these results, we conclude that ad-p27mt has the potential to become a novel and powerful gene therapy tool.

Effect of a vitamin D3 analog, EB1089, on hematopoietic stem cells from normal and myeloid leukemic blasts.

The seco-steroid 1,25 dihydroxyvitamin D3 (1,25(OH)2D3) induces differentiation and inhibits clonal proliferation of HL-60 cells. We analyzed the effect of a novel vitamin D3 analog, EB1089, on normal myeloid and leukemic cells as well as CD34+ cells. EB1089 showed an extraordinary inhibition of clonal growth of HL-60 cells (ED50 = 5 x 10(-11) M) and AML blast cells (ED50 = 9 x 10(-10) M) compared to 1,25(OH)2D3 without suppression of growth of normal human bone marrow CFU-GM. The CD34+ cells from acute myeloid leukemia (AML) blasts were inhibited in a dose-dependent fashion by 1,25(OH)2D3 with an ED50 of 1.2 x 10(-9) M; and even more strikingly, 10(-10) M of EB1089 inhibited all clonal growth of human CD34+ leukemic colony-forming cells. In contrast, both EB1089 and 1,25(OH)2D3 (10(-8) M) showed little or only mild inhibition of CD34+ clongenic hematopoietic cells from normal human peripheral blood (PB); and in liquid culture, EB1089 stimulated the proliferation of normal human CD34+ cells about 2.5 times as compared to control cultures. In order to evaluate the potential use of EB1089 for purging leukemic cells from normal CD34+ progenitor cells for PB stem cell transplantation (PBSCT), normal human PB mononuclear cells (PBMNC) were contaminated with HL-60 cells, and then CD34+ cells purified and treated with EB1089. We found that CD34+ purification and EB1089 purging was able to eliminate approximately 100% of HL-60 leukemic cells with no toxicity to normal CD34+ hematopoietic progenitor cells. These data suggested that purification of CD34+ cells and ex vivo treatment with EB1089 might provide an effective therapeutic approach for PBSCT.

Differential effects of adenovirus-p16 on bladder cancer cell lines can be overcome by the addition of butyrate.

High frequency of p16 alteration and high local recurrence rate of bladder cancer make this cancer an ideal target for p16 gene therapy. However, a low transduction rate of p16 via adenoviral vector causes an inconsistent result. In this study, we have tested adenovirus-p16 in several bladder cancer cell lines and investigated a way of improving the low transduction rate. Adenovirus-p16 showed a strong antitumor effect on bladder cancer cell lines (253J and T24) with strong Coxackie-adenoviral receptor (CAR) expression but little antitumor effect on bladder cancer cell lines (J82 and HT1376) with little CAR expression. In this study, we suggest a simple way of overcoming the differential effects of the adenovirus. The addition of butyrate to media was found to increase the transduction rate of adenovirus remarkably and increase the antitumor effect of adenovirus-p16 in bladder cancer cell lines with little CAR expression. Butyrate effects were related with increased CAR expression on the cell surface as well as increased transgene expression from adenoviral vector. From these observations, application of adenovirus-p16 gene therapy with butyrate can overcome the obstacle of low gene transfer and enhance the antitumor effect of adenovirus-p16 in bladder cancer.

Locoregional response and increased natural killer activity after intratumoral injection of HLA-B7/beta2-microglobulin gene in patients with cancer.

The purpose of this study was to assess the therapeutic potential of injecting the gene for HLA-B7/beta2-microglobulin into the subcutaneous metastatic nodules of patients who are refractory to conventional treatments. The nine patients evaluated were divided into three groups and given escalating doses of DNA (20, 40, and 100 microg of the HLA-B7 plasmid DNA/lipid complex for each group) every 2 weeks. Biopsy specimens from the treated tumor nodules of all nine patients were positive for the presence of DNA and for HLA-B7 mRNA expression. Moreover, in six of the nine patients, immunohistology of tumor biopsy samples revealed the expression of recombinant HLA-B7 protein. Also, all nine patients showed an increase in NK activity in their circulating peripheral blood lymphocytes. In two lung cancer patients, one partial and one mixed response was observed after gene transfer. These responses were confined to the treated nodules and the untreated locoregional lymph nodes; the lung masses showed no regression. Remission durations were 14 and 6 weeks, respectively, and in a total of 35 cycles no significant toxicities were observed. Immunohistologic analysis revealed an increased infiltration of CD4+ T cells, macrophages, and NK cells after therapy. In two responding cases, direct intratumoral injection of an allogeneic class I gene could elicit an antitumor response in locoregional areas, possibly through the activation of NK cells.

Na+, K+-specific inhibition of protein and peptide hydrolyses by proteasomes from human hepatoma tissues.

Proteasomes were purified from human hepatoma tissues, and their sensitivities to Na+ and K+ were examined. At concentrations of 10 mM or more, these cations were found to inhibit completely polylysine-activated casein degradation by the purified proteasomes. They also strongly inhibited the hydrolyses of peptides, although to a lesser extent. On the other hand, they reversed the inhibitory and stimulatory effects of polylysine on the hydrolyses of Suc-Leu-Tyr-AMC and Cbz-Ala-Arg-Arg-MNA, respectively. These results suggest that Na+ and/or K+ may be involved in the regulation of intracellular protein breakdown by controlling the multicatalytic activity of proteasomes.

The 65-kDa protein derived from the internal translational start site of the clpA gene blocks autodegradation of ClpA by the ATP-dependent protease Ti in Escherichia coli.

The ATP-dependent protease Ti consists of two different components: ClpA containing ATP-cleaving sites and ClpP having serine active sites for proteolysis. The clpA gene has dual translational start sites and therefore encodes two polypeptides with sizes of 84 and 65 kDa (referred to as ClpA84 and ClpA65, respectively). Here we show that ClpA84, but not ClpA65, is degraded in vitro by ClpP in the presence of ATP. The ClpP-mediated hydrolysis of ClpA84 could be prevented by casein, which is an excellent substrate of protease Ti (i.e. ClpA84/ClpP complex). Thus, it appears that free form of ClpA84 competes with casein for the degradation by ClpA/ClpP complex. Furthermore, ClpA65 inhibited the auto-degradation of ClpA84 by the complex. These results suggest that ClpA65 may play an important role in the control of the ClpA84 level and in turn in the regulation of ATP-dependent protein breakdown in E. coli.

ATP-dependent degradation of SulA, a cell division inhibitor, by the HslVU protease in Escherichia coli.

HslVU is an ATP-dependent protease consisting of two multimeric components, the HslU ATPase and the HslV peptidase. To gain an insight into the role of HslVU in regulation of cell division, the reconstituted enzyme was incubated with SulA, an inhibitor of cell division in Escherichia coli, or its fusion protein with maltose binding protein (MBP). HslVU degraded both proteins upon incubation with ATP but not with its nonhydrolyzable analog, ATPgammaS, indicating that the degradation of SulA requires ATP hydrolysis. The pulse-chase experiment using an antibody raised against MBP-SulA revealed that the stability of SulA increased in hsl mutants and further increased in lon/hsl double mutants, indicating that SulA is an in vivo substrate of HslVU as well as of protease La (Lon). These results suggest that HslVU in addition to Lon plays an important role in regulation of cell division through degradation of SulA.

Mutational analysis of the ATP-binding site in HslU, the ATPase component of HslVU protease in Escherichia coli.

HslU is the ATPase component of the ATP-dependent HslVU protease in Escherichia coli. To gain an insight into the structure and function of HslU, site-directed mutagenesis was performed to generate a mutation in the ATP-binding site of the ATPase (i.e., to replace the Lys63 with Thr). Unlike the wild-type HslU, the mutant form (referred to as HslU/K63T) could not hydrolyze ATP or support the ATP-dependent hydrolysis of N-carbobenzoxy-Gly-Gly-Leu-7-amido-4-methyl coumarin by HslV. The wild-type HslU (a mixture of monomer and dimer) formed a multimer containing 6-8 subunits in the presence of either ATP or ADP, indicating that ATP-binding, but not its hydrolysis, is required for oligomerization of HslU. However, HslU/K63T remained as a monomer whether or not the adenine nucleotides were present. Furthermore, ATP or ADP could protect HslU, but not HslU/K63T, from degradation by trypsin. These results suggest that the mutation in the ATP-binding site results in prevention of the binding of the adenine nucleotides to HslU and hence in impairment of both oligomerization and ATPase function of HslU.

Mutagenesis of two N-terminal Thr and five Ser residues in HslV, the proteolytic component of the ATP-dependent HslVU protease.

HslVU in E. coli is a new type of ATP-dependent protease consisting of two heat shock proteins: the HslU ATPase and the HslV peptidase that has two repeated Thr residues at its N terminus, like certain beta-type subunit of the 20S proteasomes. To gain an insight into the catalytic mechanism of HslV, site-directed mutagenesis was performed to replace each of the Thr residues with Ser or Val and to delete the first or both Thr. Also each of the five internal Ser residues in HslV were replaced with Ala. The results obtained by the mutational analysis revealed that the N-terminal Thr acts as the active site nucleophile and that certain Ser residues, particularly Ser124 and Ser172, also contribute to the peptide hydrolysis by the HslVU protease. The mutational studies also revealed that both Thr, Ser103, and Ser172, but not Ser124, are involved in the interaction of HslV with HslU and hence in the activation of HslU ATPase as well as in the HslVU complex formation.