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Studies have highlighted the association between the cellular damage caused by reactive oxygen species and aging. The reducing sugar d-galactose causes aging-related changes and oxidative stress. Lipids are the first target of free radicals, and lipid peroxidation is related to aging. Walnut (Juglans regia Chandler) kernel contains antioxidant phenolic compounds, and chokeberry (Aronia melanocarpa) is one of the richest sources of polyphenols, including anthocyanins, among other fruits. Polyphenols from chokeberry exhibit antioxidant and anti-inflammatory activities. In this study, the additive antioxidative effect of walnut and chokeberry mixture was evaluated by oxidative stress index in d-galactose-induced aging model. Thirty-five Balb/c mice (8 weeks old) were divided into following five groups (n = 7 in each group): normal control (C), d-galactose control (D), d-galactose with chokeberry diet (CH), d-galactose with walnut diet (W), and d-galactose with walnut and chokeberry mixture diet (WCH). In all treatment diets groups, the levels of serum, hepatic, and kidney malonaldehyde were significantly lower than D group and the levels were approaching to control level. Moreover, the kidney malondialdehyde levels were significantly lower in WCH group compared with the control group. This study also confirmed the activities of antioxidant enzymes in liver, as the levels of superoxide dismutase, and glutathione peroxidase were significantly increased in CH group compared to in W or CH groups. The results of this study supported the additive effect of walnut and chokeberry on increment of antioxidant enzyme gene expression in liver and consequently the attenuation of lipid peroxidation in serum, liver, and kidney in d-galactose-induced aging-mouse model. Further studies are needed to investigate the detailed mechanism underlying the additive antioxidative effects in various tissues. [ABSTRACT FROM AUTHOR]

A recombinant target-specific signal amplifier was constructed by genetically fusing the enhanced ascorbate peroxidase 2 (APEX2) and an antibody-binding domain (ABD). The fusion protein APEX2–ABD possessed the peroxidase activity and the antibody-binding capability simultaneously and replaced the conventional HRP-conjugated secondary antibodies in a TSA assay for amplifying fluorescence signals. [ABSTRACT FROM AUTHOR]

Cartilage is one of the major structural elements in the human body and can be exposed to various vulnerable conditions such as osteoarthritis, rheumatoid arthritis, and unexpected joint injuries. Unfortunately, cartilage tissues are not efficiently repaired by innate biological systems. Although medical treatments may ameliorate the symptoms of the patients, the regeneration of the damaged cartilage is yet among the most challenging assignments to the scientist in the field. Cartilage tissues are built by deposition of complex extracellular matrices such as collagen, fibronectin, and laminin. Most of the components in the cartilage tissues are secreted and organized by chondrocytes. Recent studies have revealed a substantial amount of biological mechanisms for cartilage development and differentiation. However, we are still very far from a complete understanding of the chondrogenic procedures and signaling. Among the most intriguing points in chondrogenic signaling is integrin signaling. An increasing amount of data indicates that integrin signaling is critical for chondrocyte differentiation. However, some conflicting, not mutually exclusive per se, data have frequently been reported. In this review, we summarize the functions of integrin signaling for chondrogenesis and suggest future directions to further elucidate this challenging issue. [ABSTRACT FROM AUTHOR]

CD38, an ADP ribosyl cyclase, is a 45 kDa type II transmembrane protein having a short N-terminal cytoplasmic domain and a long C-terminal extracellular domain, expressed on the surface of various cells including macrophages, lymphocytes, and pancreatic β cells. It is known to be involved in cell adhesion, signal transduction and calcium signaling. In addition to its transmembrane form, CD38 is detectable in biological fluids in soluble forms. The mechanism by which CD38 is solubilized from the plasma membrane is not yet clarified. In this study, we found that lipopolysaccharide (LPS) induced CD38 upregulation and its extracellular release in J774 macrophage cells. Furthermore, it also increased CD38 expression at the mRNA level by activating the Janus kinase-signal transducers and activators of transcription (JAK-STAT) pathway. However, LPS decreased the levels of CD38 in the plasma membrane by releasing CD38 into the culture supernatant. LPS-induced CD38 release was blocked by the metalloproteinase-9 inhibitor indicating that MMP-9 solubilizes CD38. In conclusion, the present findings demonstrate a potential mechanism by which C38 is solubilized from the plasma membrane. [ABSTRACT FROM AUTHOR]

Abstract: NAD is available in the extracellular environment and elicits immune modulation such as T cell apoptosis by being used as the substrate of cell surface ADP-ribosyl transferase. However, it is unclear whether extracellular NAD affects function of macrophages expressing cell surface ADP-ribosyl transferase. Here we show that extracellular NAD enhances Fcγ receptor (FcγR)-mediated phagocytosis in J774A.1 macrophages via the conversion into cyclic ADP-ribose (cADPR), a potent calcium mobilizer, by CD38, an ADP-ribosyl cyclase. Extracellular NAD increased the phagocytosis of IgG-coated sheep red blood cells (IgG-SRBC) in J774A.1 macrophages, which was completely abolished by pretreatment of 8-bromo-cADPR, an antagonist of cADPR, or CD38 knockdown. Extracellular NAD increased basal intracellular Ca2+ concentration, which also was abolished by pretreatment of 8-bromo-cADPR or CD38 knockdown. Moreover, the chelation of intracellular calcium abolished NAD-induced enhancement of phagocytosis of IgG-SRBC. Our results suggest that extracellular NAD act as a regulator for FcγR-mediated phagocytosis in macrophages. [Copyright &y& Elsevier]

Abstract: The ADP-ribose (ADPR) pyrophosphatase (ADPRase) NUDT5, a member of a superfamily of Nudix hydrolases, hydrolyzes ADP-ribose (ADPR) to AMP and ribose 5′-phosphate. Nitric oxide (NO) enhances nonenzymatic ADP-ribosylation of proteins such as β-actin and glyceraldehydes 3-phosphate dehydrogenase in the presence of free ADPR, suggesting a possibility that NUDT5 could also be ADP-ribosylated by its substrate, ADPR. Here, we show that NO stimulates nonenzymatic ADP-ribosylation of NUDT5 using ADP-ribose and consequently activates its ADPRase activity. We found that ADPRase activity in J774 macrophage cells is increased by the treatment with SNP, an exogenous NO generator or TNF-α/IFN-γ, endogenous NO inducers. Anti-NUDT5 antibody pulled down most of the ADPRase activity increased by NO, indicating that the ADPRase regulated by NO is NUDT5. Using recombinant human NUDT5, we also demonstrated that the increase of ADPRase activity is mediated via ADP-ribosylation at cysteine residue(s) in the presence of reductant. This result suggests that NO activates NUDT5 through ADP-ribosylation at cysteine residues of the enzyme in macrophages. [Copyright &y& Elsevier]

Abstract: ADP-ribose pyrophosphatase (ADPRase) hydrolyzes ADP-ribose to ribose-5-phosphate and AMP. The ADPRase activity have been assessed by coupling the reaction to alkaline phosphatase and colorimetrically measuring the amount of inorganic phosphate released from AMP that is one of the products of ADPRase. Another but less sensitive colorimetric method has been employed: the reaction mixture was treated with charcoal to adsorb the adenine-containing compounds such as AMP and ADPR and subsequently remaining ribose-5-phosphate was measured colorimetrically. However, the measurement of inorganic phosphate cannot be feasible to assay ADPRase in phosphate-containing samples and the determination of ribose-5-phosphate also is less sensitive. Here we develop a fluorescent assay for ADPRase that utilizes 1, N6-etheno ADP-ribose, a fluorescent analogue of ADP-ribose. This method measures fluorescent 1, N6-etheno adenosine that is produced by coupling the hydrolysis of 1, N6-etheno ADP-ribose to dephosphorylation with alkaline phosphatase. The fluorometric assay is comparable in sensitivity and useful for ADPRase assay in phosphate-containing samples. [Copyright &y& Elsevier]

An extracellular levanbiohydrolase gene, levM, from Microbacterium laevaniformans ATCC 15953 was cloned and its nucleotide sequence was determined. Nucleotide sequence analysis of this gene revealed a 1863 bp open reading frame coding for a protein of 621 amino acids. The deduced amino acid sequence of the levM gene exhibited 28–47% sequence identities with levanases, levanfructotransferases, and inulinases. The LevM was overexpressed by using a T7 promoter in Escherichia coli BL21 (DE3) and purified 24-fold from culture supernatant. The molecular weight of this enzyme was 68,800 Da based on the sodium dodecyl sulfate–polyacrylamide gel electrophoresis. The optimum pH and temperature of this enzyme for levan degradation was pH 6.0 and 30 °C, respectively. Thin-layer and high-performance liquid chromatography analyses proved that the enzyme produced mostly levanbiose from levan in an exo-acting manner. The recombinant enzyme also hydrolyzed inulin, 1-kestose, and nystose, indicating that the enzyme cleaves not only β-2,6-linkage of levan but also β-2,1-linkage of fructooligosaccharides. This is the first report on a gene encoding a levanbiohydrolase that produces levanbiose as a major degradation product. [Copyright &y& Elsevier]

The sh3bgr ( SH3 domain b inding g lutamate- r ich) gene encodes a small protein containing a thioredoxin-like fold, SH3 binding domain, and glutamate-rich domain. Originally, it was suggested that increased expression of Sh3bgr may cause the cardiac phenotypes in Down's syndrome. However, it was recently reported that the overexpression of Sh3bgr did not cause any disease phenotypes in mice. In this study, we have discovered that Sh3bgr is critical for sarcomere formation in striated muscle tissues and also for heart development. Sh3bgr is strongly expressed in the developing somites and heart in Xenopus . Morpholino mediated-knockdown of sh3bgr caused severe malformation of heart tissue and disrupted segmentation of the somites. Further analysis revealed that Sh3bgr specifically localized to the Z-line in mature sarcomeres and that knockdown of Sh3bgr completely disrupted sarcomere formation in the somites. Moreover, overexpression of Sh3bgr resulted in abnormally discontinues thick firmaments in the somitic sarcomeres. We suggest that Sh3bgr does its function at least partly by regulating localization of Enah for the sarcomere formation. In addition, we provide the data supporting Sh3bgr is also necessary for proper heart development in part by affecting the Enah protein level. [ABSTRACT FROM AUTHOR]

Objective: SIRT1, a class III histone/protein deacetylase, is known to interfere with the nuclear factor-kappaB (NF-kappaB) signaling pathway and thereby has an anti-inflammatory function. Because of the central role of NF-kappaB in cytokine-mediated pancreatic beta-cell damage, we postulated that SIRT1 might work in pancreatic beta-cell damage models.Research Design and Methods: RINm5F (RIN) cells or isolated rat islets were treated with interleukin-1beta and interferon-gamma. SIRT1 was activated by resveratrol, a pharmacological activator, or ectopic overexpression. The underlying mechanisms of SIRT1 against cytokine toxicity were further explored.Results: Treatment of RIN cells with cytokines induced cell damage, and this damage was well correlated with the expression of the inducible form of nitric oxide (NO) synthase (iNOS) and NO production. However, SIRT1 overexpression completely prevented cytokine-mediated cytotoxicity, NO production, and iNOS expression. The molecular mechanism by which SIRT1 inhibits iNOS expression appeared to involve the inhibition of the NF-kappaB signaling pathway through deacetylation of p65. In addition, SIRT1 activation by either resveratrol or adenoviral-directed overexpression of SIRT1 could prevent cytokine toxicity and maintain normal insulin-secreting responses to glucose in isolated rat islets.Conclusions: This study will provide valuable information not only into the mechanisms underlying beta-cell destruction but also into the regulation of SIRT1 as a possible target to attenuate cytokine-induced beta-cell damage. [ABSTRACT FROM AUTHOR]

Abstract: Peroxisome proliferator-activated receptor γ (PPARγ) ligands have been identified as a potential source of therapy for human cancers. However, PPARγ ligands have a limitation for breast cancer therapy, since estrogen receptor α (ERα) negatively interferes with PPARγ signaling in breast cancer cells. Here we show that ERα inhihits PPARγ transactivity and ERα-mediated inhibition of PPARγ transactivity is blocked by tamoxifen, an estrogen receptor blocker. The activation of ERα with 17-β-estradiol blocked PPRE transactivity induced by troglitazone, a PPARγ ligand, indicating the resistance of ERα-positive breast cancer cells to troglitazone. Indeed, troglitazone inhibited the growth of ERα-negative MDA-MB-231 cells more than that of ERα-positive MCF-7 cells. Combination of troglitazone with tamoxifen led to a marked increase in growth inhibition of ERα-positive MCF-7 cells compared to either agent alone. Our data indicates that troglitazone enhances the growth inhibitory activity of tamoxifen in ERα-positive MCF-7 cells. [Copyright &y& Elsevier]

Abstract: Peroxisome proliferator-activated receptor γ (PPARγ) ligands inhibit cell proliferation and induce apoptosis in cancer cells. Here we wished to determine whether the PPARγ ligand induces apoptosis and cell cycle arrest of the MDA-MB-231 cell, an estrogen receptor α negative breast cancer cell line. The treatment of MDA-MB-231 cell with PPARγ ligands was shown to induce inhibition of cell growth in a dose-dependent manner as determined by MTT assay. Cell cycle analysis showed a G1 arrest in MDA-MB-231 cells exposed to troglitazone. An apoptotic effect by troglitazone demonstrated that apoptotic cells elevated by 2.5-fold from the control level at 10μM, to 3.1-fold at 50μM and to 3.5-fold at 75μM. Moreover, troglitazone treatment, applied in a dose-dependent manner, caused a marked decrease in pRb, cyclin D1, cyclin D2, cyclin D3, Cdk2, Cdk4 and Cdk6 expression as well as a significant increase in p21 and p27 expression. These results indicate that troglitazone causes growth inhibition, G1 arrest and apoptotic death of MDA-MB-231 cells. [Copyright &y& Elsevier]

Abstract: Diabetes mellitus is characterized by cytokine-induced insulitis and a deficit in β-cell mass. Ligands for peroxisome proliferator-activated receptor-γ (PPAR-γ) have been shown to have anti-inflammatory effects in various experimental models. We questioned whether activation of endogenous PPAR-γ by either PPAR-γ ligands or adenoviral-directed overexpression of PPAR-γ (Ad-PPAR-γ) could inhibit cytokine-induced β-cell death in RINm5F (RIN) cells, a rat insulinoma cell line. Treatment of RIN cells with interleukin-1β (IL-1β) and interferon-γ (IFN-γ) induced β-cell damage through NFκB-dependent signaling pathways. Activation of PPAR-γ by PPAR-γ ligands or Ad-PPAR-γ inhibited IL-1β and IFN-γ-stimulated nuclear translocation of the p65 subunit and DNA binding activity. NFκB target gene expression and their product formation, namely inducible nitric oxide synthase and cyclooxygenase-2 were decreased by PPAR-γ activation, as established by real-time PCR, Western blots and measurements of NO and PGE2. The mechanism by which PPAR-γ activation inhibited NFκB-dependent cell death signals appeared to involve the inhibition of IκBα degradation, evidenced by inhibition of cytokine-induced NFκB-dependent signaling events by Ad-IκBα (S32A, S36A), non-degradable IκBα mutant. IκBβ mutant, Ad-IκBβ (S19A, S23A) was not effective in preventing cytokine toxicity. Furthermore, a protective effect of PPAR-γ ligands was proved by assaying for normal insulin secreting capacity in response to glucose in isolated rat pancreatic islets. The β-cell protective function of PPAR-γ ligands might serve to counteract cytokine-induced β-cell destruction. [Copyright &y& Elsevier]

Abstract: Background: Adenovirus type 7 (Ad7) is frequently responsible for severe respiratory infections, especially in young infants. Since the Ad7 epidemics have been associated with severe childhood pneumonia and significant mortality in Korea, 1995–1999, continuous surveillance was necessary for Ad7 related diseases. Objectives: To characterize epidemiologic features of Ad7 in 1995–2004, genetic diversity of Ad7 were studied by determining genome types (GTs) and the fiber diversity. Study design: A total of 139 Ad7 strains were obtained from Korean children with pneumonia. Serotype specificity was confirmed by microneutralization assay. GTs were determined by restriction analysis with 12 enzymes. The variable region of the fiber was sequenced. Results: Two GTs, Ad7d (N =98, 71%) and Ad7l (N =41, 29%) have been identified. In 1995–1996 and 2001–2002, Ad7d was dominant accounting for 98–100% of all Ad7; in 1999–2000, Ad7l was the prevalent GT accounting for 100% of all Ad7; in 1997–1998 and 2003–2004, both GTs circulated concurrently. The change in the relative predominance of GT occurred in 2 or 3 years. The Lys substitution for Arg at codon 280 of the fiber was identified in 31 Ad7d strains (32%) while no variations were observed among Ad7l. It was noteworthy that two fiber variants of Ad7d were not concurrently prevalent on any time after 1996. The shift in predominant fiber variants of Ad7d was also observed in 2–3 years. Conclusions: Our data demonstrated that the two GTs, Ad7d and Ad7l circulated in an alternating manner between outbreaks of Ad7 associated childhood pneumonia over 10 consecutive years in Korea. Fiber diversity at position 280 within Ad7d appeared to contribute to the annual distribution of Ad7d. This observation necessitates further studies to demonstrate an association between fiber variation and host cell specificity or neutralization antibody recognition. [Copyright &y& Elsevier]

Abstract: Dimethylsulfoxide (DMSO) has been known to differentiate HL60 cells into neutrophil like cells. Here, we provide an evidence for the involvement of tumor suppressor PTEN, an antagonist of phosphatidylinositol 3-kinase (PI3K) in the DMSO-induced differentiation of HL60 cells. DMSO upregulated PTEN with unaffecting the expression of PI3K. The upregulation of PTEN by DMSO lead to the decrease of Akt phosphorylation, a downstream of PI3K. The DMSO-induced upregulation of PTEN might be mediated by NF-κB activation, which was evidenced by the blockage of DMSO-induced PTEN upregulation with an NF-κB inhibitor, pyrrolidine dithiocarbamate (PDTC). [Copyright &y& Elsevier]