The antioxidant xanthorrhizol prevents amyloid-β-induced oxidative modification and inactivation of neprilysin

Activity of neprilysin (NEP), the major protease which cleaves amyloid-β peptide (Aβ), is reportedly reduced in the brains of patients with Alzheimer’s disease (AD). Accumulation of Aβ generates reactive oxygen species (ROS) such as 4-hydroxynonenal (HNE), and then reduces activities of Aβ-degrading enzymes including NEP. Xanthorrhizol (Xan), a natural sesquiterpenoid, has been reported to possess antioxidant and anti-inflammatory proper- ties. The present study examined the effects of Xan on HNE- or oligomeric Aβ42-induced ox- idative modification of NEP protein. Xan was added to the HNE- or oligomeric Aβ42-treated SK-N-SH human neuroblastoma cells and then levels, oxidative modification and enzymatic activities of NEP protein were measured. Increased HNE levels on NEP proteins and re- duced enzymatic activities of NEP were observed in the HNE- or oligomeric Aβ42-treated cells. Xan reduced HNE levels on NEP proteins and preserved enzymatic activities of NEP in HNE- or oligomeric Aβ42-treated cells. Xan reduced Aβ42 accumulation and protected neurones against oligomeric Aβ42-induced neurotoxicity through preservation of NEP ac- tivities. These findings indicate that Xan possesses therapeutic potential for the treatment of neurodegenerative diseases, including AD, and suggest a potential mechanism for the neuroprotective effects of antioxidants for the prevention of AD

Drug Resistance Rates of Mycobacterium tuberculosis at a Private Referral Center in Korea

The goals of this study were to identify first-line drug resistance in new and previously treated tuberculosis (TB) cases and to determine risk factors for multidrug-resistant TB (MDR-TB) at a private referral center in Korea. All patients with culture-confirmed pulmonary TB over a 2-yr period between July 2002 and June 2004 were prospectively included in this study. In total, 637 patients were included; 512 (80.4%) were new cases, and 125 (19.6%) were previously treated cases. Resistance to at least one first-line drug was identified in 11.7% of new cases and 41.6% of previously treated cases. MDR-TB was detected in 3.9% of new cases and 27.2% of previously treated cases. The proportion of extensively drug-resistant TB among MDR-TB patients was 16.7% (9/54). Factors associated with MDR-TB included age under 45 yr, previous TB treatment, and the presence of cavitation on chest radiography. Rates of first-line drug resistance are high, particularly in previously treated patients, in the private sector in Korea. This underscores the need for an improved control program, coupled with early diagnosis of MDR-TB, to reduce the spread and development of resistance

Activated PKCε stimulates NEP activity to protect SK-N-SH cells against Aβ neurotoxicity.

<p><b>A</b>, Cells were untreated or treated with bryostatin (Bryo, 1 nM), phosphoramidon (PA, 10 µM)+Bryo, Ro 32-0432 (Ro, 2 µM)+Bryo, or PA+Ro+Bryo for 1 hr and incubated with monomeric Aβ 1–42 peptide (Abeta, 2.5 µg) for additional 4 hr. Aβ 1–42 peptide was precipitated from the reactions by 20% TCA and immunoblotted with anti-Aβ peptide antibody 6E10 (Mean ± SEM for three independent experiments, **P<0.01, compared with untreated; ^#P<0.05, compared with Bryo). <b>B</b>, Aβ degradation assay in cells incubated with control siRNA (Con siRNA) or HuD siRNA without or with treatment of bryostatin (Bryo, 1 nM) or Ro 32-0432 (Ro, 2 µM)+Bryo for 1 hr (Mean ± SEM for three independent experiments, ***P<0.001, compared with Con siRNA; ^#P<0.001, compared with HuD siRNA; ^{P<0.001, compared with HuD siRNA+Bryo). C, Aβ degradation assay in cells incubated with Con siRNA or NEP siRNA without or with treatment of 1 nM Bryo or 2 µM Ro+Bryo for 1 hr (Mean ± SEM for three independent experiments, ***P<0.001, compared with Con siRNA; ^#P<0.05, compared with NEP siRNA;}P<0.05, compared with NEP siRNA+Bryo). <b>D</b>, Cells were incubated in absence (untreated) or presence of DMSO (vehicle) or oligomeric Aβ1–42 peptide (1 µM) for 24 hr. Cells were co-treated with Abeta +0.5 or 1 nM Bryo or pre-treated with 2 µM Ro for 1 hr or phosphoramidon (PA, 10 µM) for 10 min prior to co-treatment with Abeta+Bryo (1 nM). The viability of the cells after treatment was determined by cell viability assay and results are expressed as a percentage of viable cells compared with untreated control cells (Mean ± SEM of three independent experiments, **P<0.01, compared with untreated; ^#P<0.05, compared with Abeta; ^$P<0.01, compared with Abeta+Bryo).</p

Figure 2. Activated PKCε stabilizes NEP mRNA.

<p><b>A</b>, Cells were untreated or incubated with HuD siRNA or control siRNA (Con siRNA) for 4 days and then treated with actinomycin D (ActD, 10 µg/ml) for 2, 4, 6, 8, and 10 hrs. Total RNA was isolated and NEP mRNA was quantified by real time RT-qPCR. NEP mRNA at each time point was compared with the initial mRNA level (100%). A nonlinear regression analysis was conducted to calculate the first-order decay constant (<i>k</i>). Average mRNA half-life (t_1/2) was calculated as 0.693/<i>k</i> and reported in the table (Mean ± SEM of the three independent experiments, *P<0.05, HuD siRNA+ActD compared with untreated; #P<0.05, HuD siRNA+ActD compared with Con siRNA-treated). <b>B</b>, mRNA stability assay from cells treated with ActD or ActD+bryostatin (Bryo, 0.5 nM), or pre-treated with Ro 32-0432 (Ro, 2 µM) for 30 min and then treated with ActD+Bryo for 2, 4, 6, 8, and 10 hrs. Average mRNA half-life (t_1/2) was calculated as 0.693/<i>k</i> and reported in the table (Mean ± SEM of the three independent experiments, **P<0.01, ActD+Bryo compared with ActD to assess the bryostatin effect; ^#P<0.01, ActD+Bryo compared with ActD+Ro+Bryo to assess the Ro 32-0432 effect). <b>C</b>, Quantitative RT-qPCR analysis showing remaining NEP mRNA from untreated cells or cells incubated with HuD siRNA or Con siRNA for 4 days (Mean ± SEM for three independent experiments, **P<0.01, compared with untreated). <b>D</b>, RIP analysis to detect NEP mRNA associated with HuD protein in untreated cells or cells treated with 0.5 nM Bryo or pre-treated with 2 µM Ro for 30 min and then treated with Bryo for 1 hr. Relative amounts of NEP mRNA bound to HuD were analyzed by real-time RT-qPCR (Mean ± SEM for three independent experiments, ***P<0.001, compared with untreated; ^#P<0.001, compared with Bryo-treated). <b>E</b>, RIP analysis to detect NEP mRNA associated with HuD protein in untreated cells or cells incubated with PKCε siRNA or Con siRNA, and then treated without/with Bryo for 1 hr (Mean ± SEM for three independent experiments, **P<0.01, compared with untreated; n.s. = not specific).</p

HuD protein specifically binds to NEP mRNA in human SK-N-SH cells.

<p><b>A</b>, RIP assay showing HuD-bound NEP mRNA in cultured SK-N-SH cells. Cells were lysed and immunoprecipitated with normal rabbit immunoglobulin G (NRIgG), HuD antibody, or antibody solution with HuD peptide and then total RNA was isolated from immunoprecipitates and used for RT-qPCR with specific human NEP primers. As a control, human GAPDH mRNA was amplified from RT-qPCR of total RNA (Mean ± SEM of three independent experiments, ***P<0.001, compared with NRIgG). <b>B</b>, RNA-EMSA assay using recombinant HuD protein and a biotin-labeled oligoriboprobe corresponding to the candidate ARE sequence in 3′-UTR region of NEP mRNA and the 3′-UTR of GAPDH mRNA as a negative control, respectively. Unlabeled oligoriboprobe was used as a competitor for the inhibition reaction.</p

Figure 3. Activated PKCε enhances HuD binding to NEP mRNA and increases NEP protein expression.

<p><b>A</b>, Immunoblot analyses to detect HuD and NEP protein levels in untreated cells or cells incubated with control siRNA (Con siRNA) or HuD siRNA (Mean ± SEM, **P<0.01, compared with untreated). <b>B</b>, Relative expression of NEP protein was measured by immunoblot analysis after treatment with bryostatin (Bryo, 0.5 nM) or actinomycin D (ActD, 10 µg/ml), or pre-treatment with ActD for 1 hr prior to bryostatin (ActD+Bryo) for 24 hr (Mean ± SEM of three independent experiments, **P<0.01, ***P<0.001, compared with untreated control). <b>C</b>, Immunoblot analyses to detect PKCε and NEP protein levels in untreated cells or cells incubated with Con siRNA or PKCε siRNA (Mean ± SEM, *P<0.05, **P<0.01, compared with untreated).</p

PKCε activation recovers NEP membrane localization inhibited by oligomeric Aβ peptides.

<p><b>A</b>, Cells were untreated or treated with oligomeric Aβ (Abeta; 1 µM) or Abeta+bryostatin (Bryo, 1 nM), or pre-incubated with Ro 32-0432 (Ro, 2 µM) for 30 min before Abeta+Bryo treatment for 1 hr, and then used for biotin-labeling. Phosphorylated and biotinylated NEP proteins were detected by immunoblot from immunoprecipitated NEP protein and were compared (Mean ± SEM of the three independent experiments, *P<0.05, **P<0.01, compared with untreated; ^#P<0.05, compared with Abeta; ^{P<0.05, compared with Abeta+Bryo). B, NEP activity assay from untreated cells or cells treated with 0.5 or 1 µM Abeta, or Abeta +1 nM Bryo, or pre-incubated with 2 µM Ro for 30 min before Abeta+Bryo treatment for 1 hr (Mean ± SEM for three independent experiments, *P<0.05, **P<0.01, compared with untreated; ^#P<0.01, compared with Abeta;}P<0.01, compared with Abeta+Bryo).</p

PKCε activation rescues oligomeric Aβ-mediated inhibition of HuD-NEP mRNA interaction and NEP protein expression.

<p><b>A</b>, RIP analysis to detect NEP mRNA associated with HuD protein in untreated cells or cells treated with Aβ (Abeta, 1 µM), Aβ+bryostatin (Bryo, 0.5 nM) or pre-treated with Ro 32-0432 (Ro, 2 µM) for 30 min and then treated with Abeta+Bryo for 6 hr. Relative amounts of NEP mRNA bound to HuD were analyzed by real-time RT-qPCR (Mean ± SEM for three independent experiments, **P<0.01, compared with untreated; ^#P<0.01, compared with Abeta; ^{P<0.01, compared with Abeta+Bryo). B, Relative change in NEP protein expression was determined based on immunoblot analyses of untreated cells or cells treated with 1 µM Abeta or Abeta +0.5 nM Bryo, or pre-treated with 2 µM Ro for 1 hr prior to Abeta+Bryo for 24 hr (Mean ± SEM of the three independent experiments, *P<0.05, compared with untreated; ^#P<0.05, compared with Abeta;}P<0.05, compared with Abeta+Bryo).</p