PCR-RFLP-based typing for differentiation of Tomato yellow leaf curl virus (TYLCV) genotypes from infected host plants in Korea.

A polymerase chain reaction (PCR) using two sets of primers designed from published Tomato yellow leaf curl virus (TYLCV) genomes was developed to distinguish from the TYLCV-IL groups. The specificity of the two sets of primers was proven by testing against control TYLCV genomes and the symptomatic leaves of 34 different tomato cultivars naturally infected with TYLCV in greenhouses. One set for TYLCV-IL strain-specific primers (TYLCV-UNI-F and TYLCV-UNI-R) amplified full-length genome fragments from all the 34 tomato cultivars. Another set for TYLCV-IL group-II strain-specific primers (TYLCV-GPII-F and TYLCV-GPII-R) amplified target DNA fragments from only 9 tomato cultivars. Digestion by BglII and EcoRV of the PCR amplicons produced restriction fragment length polymorphism pattern that distinguished the TYLCV-IL group-I with two fragments from the TYLCV-IL group-II with no digested fragment. PCR coupled with BglII and EcoRV digestion confirmed that the 9 tomato cultivars were infected with the TYLCV-IL group-II and the remained 25 tomato cultivars were infected with the TYLCV-IL group-I.

Cryptotanshinone from Salviae miltiorrhizae radix inhibits sodium-nitroprusside-induced apoptosis in neuro-2a cells.

The root of Salvia miltiorrhiza (Salviae miltiorrhizae radix), a herbal medicine has widely been used for the treatment of pain, miscarriage and oedema. In this study, we evaluated the neuroprotective effect of cryptotanshinone (CRT) from Salviae miltiorrhizae radix on sodium-nitroprusside (SNP)-induced apoptosis in neuro-2a (N2a) cells, and further investigated its action mechanism in signalling pathways. The effects of CRT against SNP-induced toxicity, mitochondrial membrane potential (MMP) changes, and oxidants/antioxidant defences and apoptotic signalling pathways were investigated in N2a cells. Cryptotanshinone significantly inhibited SNP-induced cell toxicity and the generation of reactive oxygen and nitrogen species (RONS), and improved MMP in N2a cells. Cryptotanshinone significantly suppressed SNP-induced peroxidation of lipid and protein, and the expression of Gclc mRNA. In the signalling pathway, CRT effectively blocked SNP-induced activation of NF-κB and ERK1/2 and JNK MAPK pathways through the elevation of Akt and cyclic AMP response element binding protein. Furthermore, CRT remarkably reduced the increase of mitochondrial Bax/Bcl-2 ratio, the release of cytochrome c from mitochondria to cytosol, and the activations of cytosolic procaspase-3 and nuclear inactive poly ADP (adenosine diphosphate)-ribose polymerase by SNP-induced apoptosis. These results indicate that CRT has neuroprotective effects against SNP-induced apoptosis in neuronal cells via the regulation of mitochondrial apoptotic cascades and antiapoptotic cellular signalling pathways.

Seismic Performance Evaluation of Corrosion-Damaged Reinforced Concrete Columns Controlled by Shear Based on Experiment and FEA.

It is extremely important to investigate the effect of the seismic performance of corrosion-damaged reinforced concrete (RC) members, in terms of strength and deformability, on the seismic performance of the entire building. This will allow a more accurate assessment of the seismic performance of RC structures with corroded members, including beams and columns. However, current methods of evaluating the seismic performance of RC structures fail to fully consider the influence of reinforcement corrosion and other performance deterioration of RC members. The main objective of this study is to propose a practical method of evaluating the seismic performance of RC structures with corrosion-damaged members, identifying factors contributing to structural performance deterioration based on strength and deformability for direct, quantitative evaluation of seismic performance. To achieve the aforementioned objective, the authors examined the effects of reinforcement corrosion on the structural behavior of RC beams and factors contributing to structural performance deterioration. Past experiments verified the strong correlation between the half-cell potential (HCP) before and after reinforcement corrosion and the reduction factor based on energy absorption capacity. However, current research evaluates the correlation between the extent of corrosion and structural performance deterioration of RC beam members, which are not members that resist lateral force. As such, the results cannot be directly applied to the evaluation of the seismic performance of RC structures containing corrosion-damaged members. To achieve this study's main purpose of proposing a practical method of evaluating the seismic performance of RC structures comprised of corrosion-damaged members, analytical methods including structural experiments should be applied to corrosion-damaged lateral resisting members, namely, column members of the shear failure type with non-seismic details. This study performed cyclic loading tests on columns of the shear failure type having reinforcement corrosion to examine the correlation between HCP before and after corrosion and seismic performance deterioration. At the same time, finite element analysis (FEA) was carried out in consideration of the weakened bonding between steel and concrete, so as to analyze the correlation between structural performance deterioration before and after corrosion of shear columns. Through a comparison of the experimental findings and FEA results, this study proposed a seismic performance reduction factor in relation to the extent of corrosion of shear columns.

Genetic diversity of tomato-infecting Tomato yellow leaf curl virus (TYLCV) isolates in Korea.

Epidemic outbreaks of Tomato yellow leaf curl virus (TYLCV) diseases occurred in greenhouse grown tomato (Solanum lycopersicum) plants of Busan (TYLCV-Bus), Boseong (TYLCV-Bos), Hwaseong (TYLCV-Hwas), Jeju Island (TYLCV-Jeju), and Nonsan (TYLCV-Nons) in Korea during 2008-2009. Tomato disease by TYLCV has never occurred in Korea before. We synthesized the full-length genomes of each TYLCV isolate from the tomato plants collected at each area and determined their nucleotides (nt) sequences and deduced the amino acids of six open reading frames in the genomes. TYLCV-Bus and -Bos genomes shared higher nt identities with four Japanese isolates -Ng, -Omu, -Mis, and -Miy. On the other hand, TYLCV-Hwas, -Jeju, and -Nons genomes shared higher nt identities with five Chinese isolates TYLCV-AH1, -ZJ3, -ZJHZ12, -SH2, -Sh10, and two Japanese isolates -Han and -Tosa. On the basis of a neighbor-joining tree, five Korean TYLCV isolates were separated into three clades. TYLCV-Bus and -Bos formed the first clade, clustering with four Japanese isolates TYLCV-Mis, -Omu, -Ng, and -Miy. TYLCV-Jeju and -Nons formed the second clade, clustering with two Chinese isolates -ZJHZ212 and -Sh10. TYLCV-Hwas was clustered with two Japanese isolates -Han and -Tosa and three Chinese isolates -AH1, -ZJ3, and -SH2. Two fragments that had a potentially recombinant origin were identified using the RDP, GENECONV, BootScan, MaxChi, Chimaera, SiScan, and 3Seq methods implemented in RDP3.41. On the basis of RDP analysis, all TYLCV isolates could originated from the interspecies recombination between TYLCV-Mld[PT] isolated from Portugal as a major parent and TYLCTHV-MM isolated from Myanmar as a minor parent.

Antithrombotic effects by oral administration of novel proteinase fraction from earthworm Eisenia andrei on venous thrombosis model in rats.

A novel proteinase fraction, SPP-501, was purified from the earthworm, Eisenia andrei, and its antithrombotic effects compared with those of urokinase and t-PA (tissue type-plasminogen activator) in a thrombosis model, induced by the insertion of a stainless wire coil into the inferior vena cava. SPP-501, urokinase and t-PA were administrated once a day for 14 days. On the oral administration of SPP-501, as well as urokinase and t-PA, the thrombus weight was dramatically decreased. The euglobulin lysis time (ELT) was also shortened by SPP-501, but urokinase and t-PA failed to dissolve the euglobulin clot. Conversely, urokinase and t-PA produced detectable fibrinogen/fibrin degradation products (FDP), but SPP-501 did not. Thrombin induced platelet aggregation was desensitized in the SPP-501 treatment groups. With a high dose of SPP-501 (45 mg/kg), the APTT (activated partial thromboplastin time) was prolonged. These results suggest that SPP-501 shows both antithrombotic and fibrinolytic activities when orally administered.

Hydrogen Peroxide- and Nitric Oxide-mediated Disease Control of Bacterial Wilt in Tomato Plants.

Reactive oxygen species (ROS) generation in tomato plants by Ralstonia solanacearum infection and the role of hydrogen peroxide (H2O2) and nitric oxide in tomato bacterial wilt control were demonstrated. During disease development of tomato bacterial wilt, accumulation of superoxide anion (O2 (-)) and H2O2 was observed and lipid peroxidation also occurred in the tomato leaf tissues. High doses of H2O2and sodium nitroprusside (SNP) nitric oxide donor showed phytotoxicity to detached tomato leaves 1 day after petiole feeding showing reduced fresh weight. Both H2O2and SNP have in vitro antibacterial activities against R. solanacearum in a dose-dependent manner, as well as plant protection in detached tomato leaves against bacterial wilt by 10(6) and 10(7) cfu/ml of R. solanacearum. H2O2- and SNP-mediated protection was also evaluated in pots using soil-drench treatment with the bacterial inoculation, and relative 'area under the disease progressive curve (AUDPC)' was calculated to compare disease protection by H2O2 and/or SNP with untreated control. Neither H2O2 nor SNP protect the tomato seedlings from the bacterial wilt, but H2O2+ SNP mixture significantly decreased disease severity with reduced relative AUDPC. These results suggest that H2O2 and SNP could be used together to control bacterial wilt in tomato plants as bactericidal agents.

Inhibitory effects of the root extract of Dipsacus asperoides C.Y. Cheng et al T.M.Ai on collagen-induced arthritis in mice.

ETHNOPHARMACOLOGICAL RELEVANCE: Dipsaci radix, the dried root of Dipsacus asperoides C.Y. Cheng et al T.M.Ai is used as a medicinal plant in oriental clinics for the treatment of bone diseases and functions by strengthening bone and healing bone fractures. AIM OF THE STUDY: This study investigated the therapeutic efficacy of Dipsaci radix in treating rheumatoid arthritis using a type II collagen (CII)-induced arthritis (CIA) mouse model. MATERIALS AND METHODS: Arthritis was induced in male DBA/1 mice by immunization with CII. Dipsaci radix water (DR-W) extract at 50mg/kg and 100mg/kg was orally administered from days to after the induction of arthritis. Arthritic score, serum levels of anti-CII IgG2a, the inflammatory mediator prostaglandin E(2) (PGE(2)), and inflammatory cytokines (TNF-α, IL-1ß and IL-6), and histological changes in the ankle joint were analyzed in CIA mice. RESULTS: Arthritic induction increased the arthritic score, as well as serum levels of anti-CII IgG2a antibody, PGE(2), TNF-α, IL-1ß and IL-6 in mice. However, administration of DR-W extract in CIA mice significantly reduced arthritic scores and serum levels of anti-CII IgG2a antibody, PGE(2), TNF-α, IL-1ß and IL-6 compared with those in vehicle-treated CIA mice. Furthermore, histopathological improvement in joint architecture was also observed in DR-W extract-treated CIA mice. CONCLUSIONS: DR-W extract has anti-inflammatory and anti-arthritic effects in arthritic mice. This suggests that Dipsaci radix might be used as a therapeutic agent for the treatment of human arthritis.

25-Methoxyhispidol A, a novel triterpenoid of Poncirus trifoliata, inhibits cell growth via the modulation of EGFR/c-Src signaling pathway in MDA-MB-231 human breast cancer cells.

The fruit of Poncirus trifoliata (Rutaceae) has been used a medicinal food and traditional medicine. Recently we reported the isolation of 25-methoxyhispidol A (25-MHA) as a novel triterpenoid from the immature fruit of P. trifoliata with the potential growth inhibition of cancer cells. However, the molecular mechanisms on the anti-proliferative activity in cancer cells remain to be elucidated. In the present study, we investigated the anti-proliferative activity and mechanisms of actions mediated by 25-MHA in estrogen receptor (ER)-negative MDA-MB-231 human breast cancer cells. 25-MHA exhibited the growth inhibitory activity against MDA-MB-231 cells with the cell cycle arrest in the G0/G1 phase. The cell cycle arrest in the G0/G1 by 25-MHA was well correlated with the downregulation of cyclin D1, cyclin dependent kinase (CDK4), CDK2, cyclin A, phosphorylated retinoblastoma protein (pRb), and induction of cdk inhibitor p21(WAF1/Cip1) protein. 25-MHA also suppressed the activation of c-Src/epidermal growth factor receptor (EGFR)/Akt signaling, and consequently led to the inactivation of mTOR and its downstream signal molecules including 4E-binding protein (4E-BP) and p70 S6 kinase. These findings suggest that 25-MHA-mediated inhibitory activity of human breast cancer cell growth might be related with the cell cycle arrest and modulation of signal transduction pathways.

Pinoresinol from the fruits of Forsythia koreana inhibits inflammatory responses in LPS-activated microglia.

The activation of microglia plays an important role in a variety of brain disorders by the excessive production of inflammatory mediators such as nitric oxide (NO), prostaglandin E(2) (PGE(2)) and proinflammatory cytokines. We investigated here whether pinoresinol isolated from the fruits of Forsythia koreana Nakai inhibits the inflammatory responses in LPS-activated microglia. Pinoresinol inhibited the production of NO, PGE(2), TNF-alpha, IL-1beta and IL-6 in LPS-activated primary microglia. Also, pinoresinol attenuated mRNA and protein levels of inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2) and proinflammatory cytokines in LPS-activation. However, most of these inhibitory effects of pinoresinol have been mediated by extracellular-signal-regulated kinase (ERK) 1/2 mitogen-activated protein kinase (MAPK) phosphorylation and the NF-kappaB dependent. The results suggest that pinoresinol attenuates inflammatory responses of microglia and could be potentially useful in modulation of inflammatory status in brain disorders.

Growth inhibition and G1 cell cycle arrest mediated by 25-methoxyhispidol A, a novel triterpenoid, isolated from the fruit of Poncirus trifoliata in human hepatocellular carcinoma cells.

Poncirus trifoliata (Rutaceae) extracts have been known to possess anti-allergic, anti-inflammatory and antiviral activities. However, other biological activities, especially, the anticancer potential of extracts of P. trifoliata or its constituents, have not been fully investigated yet. In this study, we have evaluated the antiproliferative effects of a novel triterpenoid, 25-methoxyhispidol A, isolated from the fruit of P. trifoliata against SK-HEP-1 human hepatocellular carcinoma cells. Flow cytometric analysis indicated that 25-methoxyhispidol A arrests the cell cycle in the G1 phase at the earlier time and subsequently induces apoptosis of the cancer cells. Further study revealed that the cell cycle arrest in the G1 phase by 25-methoxyhispidol A correlated well with the inhibition of phosphorylation of the retinoblastoma (Rb) protein, and with the down-regulation of cyclin D1 and cyclin-dependent kinase cdk4 and the induction of cdk inhibitor p21 (WAF1/Cip1) protein. These findings suggest the potential of 25-methoxyhispidol A isolated from the fructus of P. trifoliata as an antitumor agent against human hepatocarcinoma cells by arresting the cell cycle and inducing apoptosis.