A secreted form of chorismate mutase (Rv1885c) in Mycobacterium bovis BCG contributes to pathogenesis by inhibiting mitochondria-mediated apoptotic cell death of macrophages.

BACKGROUND: Mycobacterium tuberculosis is the causative agent of tuberculosis (TB), and its pathogenicity is associated with its ability to evade the host defense system. The secretory form of the chorismate mutase of M. tuberculosis (TBCM, encoded by Rv1885c) is assumed to play a key role in the pathogenesis of TB; however, the mechanism remains unknown. METHODS: A tbcm deletion mutant (B∆tbcm) was generated by targeted gene knockout in BCG to investigate the pathogenic role of TBCM in mice or macrophages. We compared the pathogenesis of B∆tbcm and wild-type BCG in vivo by measuring the bacterial clearance rate and the degree of apoptosis. Promotion of the intrinsic apoptotic pathway was evaluated in infected bone marrow-derived macrophages (BMDMs) by measuring apoptotic cell death, loss of mitochondrial membrane potential and translocation of pore-forming proteins. Immunocytochemistry, western blotting and real-time PCR were also performed to assess the related protein expression levels after infection. Furthermore, these findings were validated by complementation of tbcm in BCG. RESULTS: Deletion of the tbcm gene in BCG leads to reduced pathogenesis in a mouse model, compared to wild type BCG, by promoting apoptotic cell death and bacterial clearance. Based on these findings, we found that intrinsic apoptosis and mitochondrial impairment were promoted in B∆tbcm-infected BMDMs. B∆tbcm down-regulates the expression of Bcl-2, which leads to mitochondrial outer membrane permeabilization (MOMP), culminating in cytochrome c release from mitochondria. Consistent with this, transcriptome profiling also indicated that B∆tbcm infection is more closely related to altered mitochondrial-related gene expression than wild-type BCG infection, suggesting an inhibitory role of TBCM in mitochondrial dysfunction. Moreover, genetic complementation of B∆tbcm (C∆tbcm) restored its capacity to inhibit mitochondria-mediated apoptotic cell death. CONCLUSIONS: Our findings demonstrate the contribution of TBCM to bacterial survival, inhibiting intrinsic apoptotic cell death of macrophages as a virulence factor of M. tuberculosis complex (MTBC) strains, which could be a potential target for the development of TB therapy.

Dormancy-release and germination improvement of Korean bellflower (Campanula takesimana Nakai), a rare and endemic plant native to the Korean peninsula.

Korean bellflower (Campanula takesimana Nakai) is a rare and perennial herb with medicinal and ornamental values, is endemic to the Ulleung Island of Korea. In this study, we investigated the dormancy-release and germination characteristics of C. takesimana (Campanulaceae) seeds by subjecting them to varying temperatures (5, 10, 15, 20, and 25°C and diurnal/nocturnal temperatures of 15/6, 20/10, and 25/15°C), cold stratification periods (0, 4, 8, or 12 weeks at 5°C), and gibberellic acid (GA3) concentrations (0, 10, 100, or 1,000 mg·L-1 at 15/6°C and 25/15°C) to identify the ideal seed propagation conditions. The seeds were stimulated to germinate (at 25°C, 12-h photoperiod with fluorescent lamps at 40 ± 10 µmol∙m-2∙s-1) after cold stratification. To examine the germination characteristics, the seeds were tested for water imbibition and found to readily absorb water. The seeds exhibited underdeveloped embryos during dispersal, showed final germination of 37.00% ± 4.43 at 25°C and were not influenced by temperature. The seeds subjected to 0, 4, 8, or 12 weeks of cold stratification germinated at a success rate of 22.00% ± 4.76, 87.00% ± 6.80, 79.00% ± 2.52, and 77.00% ± 1.91, respectively. Additionally, the germination characteristics, which were based on final germination, mean germination time, and germination velocity (Timson index), were significantly greater in the seeds pretreated with 1,000 mg·L-1 GA3 at 25/15°C than in seeds pretreated with 0 mg·L-1 GA3. Overall, the seeds broke dormancy with GA3 and short-term cold stratification. Therefore, we concluded that C. takesimana seeds have non-deep, simple, morphophysiological dormancy, and pretreatment with cold stratification and GA3 is required for effective seed propagation.

Synthesis of α-Hydroxy Acids via Dehydrogenative Cross-Coupling of a Sustainable C2 Chemical (Ethylene Glycol) with Alcohols.

Ir(NHC) (NHC, N-heterocyclic carbene)-catalyzed dehydrogenative coupling of sustainable ethylene glycol and various bioalcohols can produce industrially valuable α-hydroxy acids (AHAs). This study is the first to report the sustainable synthesis of higher Cn AHAs, in addition to glycolic acid (C2 AHA) and lactic acid (C3 AHA). This catalytic system can be recycled to the seventh cycle while maintaining good yields. A reaction mechanism, including facile dehydrogenation of each alcohol and fast cross-coupling of dehydrogenated aldehydes forming products, was proposed based on 18O- and 2H-labeling experiments and electron spray ionization-mass spectrometry (ESI-MS) and NMR spectral analyses.

A Hepatitis B Virus-Derived Peptide Exerts an Anticancer Effect via TNF/iNOS-producing Dendritic Cells in Tumor-Bearing Mouse Model.

Recently, we reported a 6-mer hepatitis B virus (HBV)-derived peptide, Poly6, that exerts antiviral effects against human immunodeficiency virus type 1 (HIV-1). Here, we explored the immunotherapeutic potential of Poly6 via its administration into dendritic cells (DCs) in a mouse model. Our data revealed that Poly6 treatment led to enhanced production of tumor necrosis factor alpha (TNF-α) and inducible nitric oxide synthase (iNOS)-producing DCs (Tip-DCs) in a type 1 interferon (IFN-I)-dependent manner via the induction of mitochondrial stress. Poly6 treatment in mice implanted with MC38 cells, a murine colon adenocarcinoma line, led to attenuated tumor formation, primarily due to direct cell death induced by Tip-DC mediated nitric oxide (NO) production and indirect killing by Tip-DC mediated cluster of differentiation 8 (CD8) cytotoxic T lymphocyte (CTL) activation via CD40 activation. Moreover, Poly6 treatment demonstrated an enhanced anticancer effect with one of the checkpoint inhibitors, the anti PD-L1 antibody. In conclusion, our data reveal that Poly6 treatment elicits an antitumor immune response in mice, possibly through NO-mediated oncolytic activity via Tip-DC activation and Tip-DC mediated CTL activation. This suggests that Poly6 represents a potential adjuvant for cancer immunotherapy by enhancing the anticancer effects of immune checkpoint inhibitors.

First Report of Fire Blight of Apricot (Prunus armeniaca) Caused by Erwinia amylovora in Korea.

During a survey in May 2020, symptoms of blight were observed on apricot (Prunus armeniaca cv. undetermined) in an orchard (37°06´01.5″N 127°57´44.9″E) in Chungju, South Korea, where fire blight of apple occurred. Three apricot trees in the apple orchard were heavily diseased and showed severe shoot blight and stem canker symptoms. Bacterial isolates were recovered on King's medium B from leaves and twigs that were surface-sterilized with 70% alcohol. Colonies with uniform mucoid, smooth surfaces were collected. DNA from nine isolates did not yield an amplicon in a PCR assay for detection of Erwinia pyrifoliae using primer set CPS1/CPS2c (Kim et al. 2001). Each isolate was positive in PCR assays for E. amylovora using primer sets A/B (Bereswill et al. 1992) and AJ75/76 (Llop et al. 2000) that target pEA29. Sequencing of the PCR products resulted in 99.9% (929 bp out of 930 bp) and 100% (747 bp out of 747 bp) identity with sequences of E. amylovora FB20 (GeneBank: CP050240), respectively. Amplifications of the partial 16S rRNA (GeneBank: LC557153) and hrpN (GeneBank: LC575997) genes were performed, and the products were sequenced. The primers used to amplify 16S rRNA were 518F: 5'-CCAGCAGCCGCGGTAATACG-3' and 800R: 5'-TACCAGGGTATCTAATCC-3', and those for the hrpN genes were HRPN1: 5'-ATGAGTCTGAATACAAG-3' and HRPN3c: 5'-GCTTGCCAAGTGCCATA-3'. BLAST analyses showed 99.8% (1439 bp out of 1442 bp) and 100% (1136 bp out of 1136 bp) identities, respectively, to the sequences of E. amylovora FB20. The ability of the isolates to induce a hypersensitive reaction on tobacco (Nicotiana tabacum cv. Xanthi) leaves was also evaluated. Bacterial suspensions (1.5 ⅹ 108 CFU) of 2 isolates were injected into tobacco leaves, and after 48 h, both isolates caused a hypersensitive response. To confirm pathogenicity of isolates, 3-mm-deep holes in five immature apricot (cv. Goldcot) and five immature apple (cv. Fuji) fruits were inoculated with 10 µl bacterial suspension (1.5 ⅹ 108 CFU/ml). The inoculated fruits were placed in a humid plastic box. After 7 days at 27℃, severe necrosis and bacterial ooze were present at the inoculated sites in three repeated tests. No symptoms were observed on fruits inoculated with sterile water. To complete Koch's postulates, bacteria were reisolated from the inoculated apricot and apple fruits. PCR using the specific primer sets stated above confirmed the identity as E. amylovora. Thus, based on disease symptoms, sequences, and pathogenicity, the bacterium causing blight of apricot was identified as E. amylovora. Natural infections of E. amylovora on apricot trees have been reported in the Czech Republic and Hungary (Korba and Sillerova 2011; Vegh and Palkovics 2013). Fire blight was observed in the Czech Republic on apricot trees near pear seedlings, which are highly susceptible to E. amylovora (Korba and Sillerova 2011). Natural infections of E. amylovora on Japanese plum planted adjacent to an apple orchard with severe fire blight has been reported in the United States (Mohan and Thomson 1996). Moreover, susceptibility to fire blight has been reported for apricot and Japanese plum cultivars (Mohan and Bijman 1999). To our knowledge, this the first report of fire blight of apricot caused by E. amylovora in Korea. This report is important because it provides evidence that apricot may be an overlooked reservoir for E. amylovora, in addition to apple, pear, and other rosaceous plants, in Korea. An intensive survey for additional host plants for the fire blight pathogen will be continued in Korea. This work was supported by a grant from the Agenda program (PJ01530202) of Rural Development Administration, Republic of Korea.

Survival rates following medical intensive care unit admission from 2003 to 2013: An observational study based on a representative population-based sample cohort of Korean patients.

The decision as to whether patients should be admitted to a medical intensive care unit (ICU), in the absence of information concerning survival rates or prognostic factors in survival, is often challenging. We analyzed survival trends in relation to hospital discharge and examined patient and hospital characteristics associated with survival following ICU care, using a sample of nationwide claims data in Korea from 2002 through 2013. The Korean government implements a compulsory social insurance program that covers the country's entire population, and the Korean National Health Insurance Service-National Sample Cohort (NHIS-NSC) data from 2002 based on this program were used for this study. The NHIS-NSC is a stratified random sample of 1,025,340 subjects selected from around 46 million Koreans. We evaluated annual survival trends using the Kaplan-Meier test. Analyses of the relationship between survival and patient and hospital characteristics were performed using Cox regression analyses. Employing a multivariate model, variables were selected using the forward selection method to consider the multicollinearity of variables. A total of 32,553 patients admitted to an ICU between 2002 and 2013 were identified among the eligible beneficiaries. The number of patients who had histories of ICU admission steadily increased throughout the study period, and patients older than 80 years constituted a progressively increasing proportion of ICU admissions, from 7.3% in 2002 to 16.9% in 2007 to 23.1% in 2013. The mean number of mechanical equipment items applied consistently increased, while no difference was observed in the trend for overall 1-year survival in patients following ICU treatment across the study period: the 1-year survival rate ranged from 66.7% (year 2003) to 64.2% (year 2010). Advanced age, cancer, renal failure, pneumonia, and influenza were all associated with heightened risk of mortality within 1 year. Our results should prove useful to older patients and their clinicians in their decisions regarding whether to seek ICU care, with the goals of improving the end-of life care and optimizing resource utilization.

NOD2 signaling pathway is involved in fibronectin fragment-induced pro-catabolic factor expressions in human articular chondrocytes.

The nucleotide-binding and oligomerization domain (NOD) is an innate pattern recognition receptor that recognizes pathogen- and damage-associated molecular patterns. The 29-kDa amino-terminal fibronectin fragment (29-kDa FN-f) is a matrix degradation product found in the synovial fluids of patients with osteoarthritis (OA). We investigated whether NOD2 was involved in 29-kDa FN-f-induced pro-catabolic gene expression in human chondrocytes. The expression of mRNA and protein was measured using quantitative real-time polymerase chain reaction (qrt-PCR) and Western blot analysis. Small interfering RNAs were used for knockdown of NOD2 and toll-like receptor 2 (TLR-2). An immunoprecipitation assay was performed to examine protein interactions. The NOD2 levels in human OA cartilage were much higher than in normal cartilage. NOD1 and NOD2 expression, as well as pro-inflammatory cytokines, including interleukin-1beta (IL-1ß) and tumor necrosis factor-alpha (TNF-α), were upregulated by 29-kDa FN-f in human chondrocytes. NOD2 silencing showed that NOD2 was involved in the 29-kDa FN-f-induced expression of TLR-2. Expressions of IL-6, IL-8, matrix metalloproteinase (MMP)-1, -3, and -13 were also suppressed by TLR-2 knockdown. Furthermore, NOD2 and TLR-2 knockdown data demonstrated that both NOD2 and TLR-2 modulated the expressions of their adaptors, receptorinteracting protein 2 (RIP2) and myeloid differentiation 88, in 29-kDa FN-f-treated chondrocytes. 29-kDa FN-f enhanced the interaction of NOD2, RIP2 and transforming growth factor beta-activated kinase 1 (TAK1), an indispensable signaling intermediate in the TLR-2 signaling pathway, and activated nuclear factor-κB (NF-κB), subsequently leading to increased expressions of pro-inflammatory cytokines and cartilagedegrading enzymes. These results demonstrate that 29-kDa FN-f modulated pro-catabolic responses via cross-regulation of NOD2 and TLR-2 signaling pathways. [BMB Reports 2019; 52(6): 373-378].

Fibronectin fragment-induced expression of matrix metalloproteinases is mediated by MyD88-dependent TLR-2 signaling pathway in human chondrocytes.

INTRODUCTION: Fibronectin fragments (FN-fs) are increased in the cartilage of patients with osteoarthritis (OA) and have a potent chondrolytic effect. However, little is known about the cellular receptors and signaling mechanisms that are mediated by FN-fs. We investigated whether the 29-kDa amino-terminal fibronectin fragment (29-kDa FN-f) regulates cartilage catabolism via the Toll-like receptor (TLR)-2 signaling pathway in human chondrocytes. METHODS: Small interfering RNA was used to knock down TLR-2 and myeloid differentiation factor 88 (MyD88). TLR-2 was overexpressed in chondrocytes transfected with a TLR-2 expression plasmid. The expression levels of matrix metalloproteinase (MMP)-1, MMP-3, and MMP-13 were analyzed using quantitative real-time reverse transcription polymerase chain reactions, immunoblotting, or enzyme-linked immunosorbent assay. The effect of TLR-2 on 29-kDa FN-f-mediated signaling pathways was investigated by immunoblotting. RESULTS: TLR-2, TLR-3, TLR-4, and TLR-5 mRNA were significantly overexpressed in OA cartilage compared with normal cartilage, whereas no significant difference of TLR-1 mRNA expression was found. 29-kDa FN-f significantly increased TLR-2 expression in human chondrocytes in a dose- and time-dependent manner. Knockdown of TLR-2 or MyD88, the latter a downstream adaptor of TLR-2, significantly inhibited 29-kDa FN-f-induced MMP production at the mRNA and protein levels. Conversely, TLR-2 overexpression led to enhanced MMP production by 29-kDa FN-f. In addition, TLR-2 knockdown apparently inhibited 29-kDa FN-f-mediated activation of phosphorylated nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor, alpha, and p38, but not of c-Jun N-terminal kinase or extracellular signal-regulated kinase. Exposure to synovial fluid (SF) from affected joints of patients with OA elevated MMP-1, MMP-3, and MMP-13 expression markedly in primary chondrocytes without reducing cell viability. However, TLR-2 knockdown in chondrocytes significantly suppressed SF-induced MMP induction. CONCLUSIONS: Our data demonstrate that the MyD88-dependent TLR-2 signaling pathway may be responsible for 29-kDa FN-f-mediated cartilage catabolic responses. Our results will enhance understanding of cartilage catabolic mechanisms driven by cartilage degradation products, including FN-f. The modulation of TLR-2 signaling activated by damage-associated molecular patterns, including 29-kDa FN-f, is a potential therapeutic strategy for the prevention of cartilage degradation in OA.

Characteristic location and growth patterns of functioning pituitary adenomas: correlation with histological distribution of hormone-secreting cells in the pituitary gland.

OBJECTIVE: To evaluate the correlation between the magnetic resonance imaging findings of functional pituitary adenomas and histological distribution of hormone-secreting cells in pituitary gland. METHODS: Forty-nine patients with pathologically confirmed functional micro and macro pituitary adenomas were retrospectively reviewed for its location and growth direction. RESULTS: Micro-prolactin, micro-adrenocorticotropic hormone (ACTH), and micro-growth hormone (GH) producing adenomas showed specific location (P-value <.01). Macro-GH and macro-thyroid-stimulating hormone producing adenomas showed specific growth direction (P-value <.05), whereas macro-prolactin and macro-ACTH producing adenomas did not. CONCLUSION: The functional pituitary microadenomas' location and macroadenomas' growth pattern correlate well with histological distribution of hormone-secreting cells in pituitary gland.

TRPM2 mediates the lysophosphatidic acid-induced neurite retraction in the developing brain.

Intracellular Ca(2+) signal is a key regulator of axonal growth during brain development. As transient receptor potential (TRP) channels are permeable to Ca(2+) and mediate numerous brain functions, it is conceivable that many TRP channels would regulate neuronal differentiation. We therefore screened TRP channels that are involved in the regulation of neurite growth. Among the TRP channels, the Trpm2 level was inversely associated with neurite growth. TRPM2 was highly expressed in embryonic brain. Pharmacological perturbation or knockdown of TRPM2 markedly increased the axonal growth, whereas its overexpression inhibited the axonal growth. Addition of ADP ribose, an endogenous activator of TRPM2, to PC12 cells significantly repressed the axonal growth. TRPM2 was actively involved in the neuronal retraction induced by cerebrospinal fluid-rich lysophosphatidic acid (LPA). More importantly, neurons isolated from the brain of Trpm2-deficient mice have significantly longer neurites with a greater number of spines than those obtained from the brain of wild-type mice. Therefore, we conclude that TRPM2 mediates the LPA-induced suppression of axonal growth, which provides a long-sought mechanism underlying the effect of LPA on neuronal development.

MicroRNA-127-5p regulates matrix metalloproteinase 13 expression and interleukin-1ß-induced catabolic effects in human chondrocytes.

OBJECTIVE: MicroRNAs (miRNAs), small noncoding RNA molecules, are involved in the pathogenesis of various diseases such as cancer and arthritis. The aim of this study was to determine whether miR-127-5p regulates interleukin-1ß (IL-1ß)-induced expression of matrix metalloproteinase 13 (MMP-13) and other catabolic factors in human chondrocytes. METHODS: Expression of miR-127-5p and MMP-13 by normal and osteoarthritic (OA) human cartilage was determined using real-time polymerase chain reaction. The effect of miR-127-5p on MMP-13 expression was evaluated using transient transfection of human chondrocytes or chondrogenic SW-1353 cells with miR-127-5p or its antisense inhibitor (anti-miR-127-5p). MMP-13 protein production was quantified by enzyme-linked immunosorbent assay, and the involvement of miR-127-5p in IL-1ß-mediated catabolic effects was examined by immunoblotting. MicroRNA-127-5p binding with the putative site in the 3'-untranslated region (3'-UTR) of MMP-13 messenger RNA (mRNA) was validated by luciferase reporter assay. RESULTS: There was a significant reduction in miR-127-5p expression in OA cartilage compared with normal cartilage. Up-regulation of MMP-13 expression by IL-1ß was correlated with down-regulation of miR-127-5p expression in human chondrocytes. MicroRNA-127-5p suppressed IL-1ß-induced MMP-13 production as well as the activity of a reporter construct containing the 3'-UTR of human MMP-13 mRNA. In addition, mutation of the miR-127-5p binding site in the 3'-UTR of MMP-13 mRNA abolished miR-127-5p-mediated repression of reporter activity. Conversely, treatment with anti-miR-127-5p remarkably increased reporter activity and MMP-13 production. Interestingly, the IL-1ß-induced activation of JNK, p38, and NF-κB and expression of MMP-1 and cyclooxygenase 2 were significantly inhibited by miR-127-5p. CONCLUSION: MicroRNA-127-5p is an important regulator of MMP-13 in human chondrocytes and may contribute to the development of OA.

Carbon monoxide protects against ovariectomy-induced bone loss by inhibiting osteoclastogenesis.

Carbon monoxide (CO) has been shown to have remarkable therapeutic value at low dosage by suppressing inflammation via inhibitory effects on macrophages, which are also precursors of osteoclasts (OC). The objective of the present study was to determine whether CO limits bone loss through its effects on osteoclastogenesis. Intraperitoneal injection of CO-releasing molecule 2 (CORM2) into mice with reduced bone mass due to ovariectomy (OVX) resulted in significantly elevated bone mass. Increased serum levels of collagen-type I fragments, tartrate-resistant acid phosphatase 5b, and reactive oxygen species (ROS) due to OVX were also decreased when treated with CORM2. In vitro, CORM2 inhibited receptor activator of nuclear factor-κB ligand (RANKL)-induced OC formation without affecting bone resorption. CORM2 reduced long-lasting ROS levels and nuclear factor-κB (NF-κB) activation in response to RANKL. Inhibition of NADPH oxidase partially reduced the inhibitory effect of CO. CO induced increase of peroxiredoxin 1 (PRX1) in BMM. Down-regulation of PRX1 reduced the inhibitory effect of CO on OC formation and sustained the ROS levels induced by RANKL, suggesting that CO reduces generation of ROS and scavenges ROS to inhibit osteoclastogenesis. These data suggest that the inhibitory effect of CO on osteoclastogenesis is caused by impaired RANKL signaling through defective NF-κB activation and reduced levels of long-lasting ROS. These changes result in decreased bone loss. Our data highlight the potential utility of CO for ameliorating bone loss induced by loss of ovarian function.

Elevation of fibrinogen due to loss of ovarian function enhances actin ring formation and leads to increased bone resorption.

The aim of the present study was to evaluate the effect of fibrinogen on number and function of osteoclasts (OC) consequently resulting in bone loss. It was hypothesized that the enhanced level of released fibrinogen due to loss of ovarian function caused bone loss by acting on OCs. Bone loss was induced by ovariectomy (OVX) in mice and analyzed by micro-CT. The effect of fibrinogen on OCs was evaluated by tartrate-resistant acid phosphatase, annexin V, actin staining, pit formation observed on dentine slices, and Western blotting. Exogenous fibrinogen increased OC survival, actin ring formation, and bone resorption in vitro. The effect of fibrinogen was dependent on ß(3)-integrin, which is a marker for mature OCs. Fibrinogen induced the activation of transforming oncogene from Ak strain (Akt), Ras-related C3 botulinum toxin substrate 1 (Rac1), and Rho family of GTPase (Rho) and the degradation of the Bcl-2 interacting mediator of cell death (Bim) in a manner similar to macrophage colony-stimulating factor (M-CSF). OVX increased plasma fibrinogen and serum M-CSF together with elevated actin ring formation and bone loss. The increased fibrinogen level due to loss of ovarian function may contribute, at least partly, to bone loss through the enhanced number and activity of OCs.

Absence of herpes virus entry mediator (HVEM) increases bone mass by attenuating receptor activator of nuclear factor-κB ligand (RANKL)-induced osteoclastogenesis.

Herpes virus entry mediator (HVEM), which is constitutively expressed at a high level on myeloid lineage cells, is also expressed on bone marrow-derived macrophages, suggesting that it may play a role in bone metabolism by affecting osteoclasts (OC) derived from bone marrow-derived macrophages. To address this question, we evaluated bone mass by micro-computed tomography and the number and activity of OC by tartrate-resistant acid phosphatase (TRAP) and pit formation on dentine slices, comparing HVEM-knockout mice with wild-type mice. The absence of HVEM led to a higher bone mass and to decreased levels of serum collagen type I fragments and serum TRACP5b in vivo. In vitro HVEM deficiency resulted in a reduced number and activity of OC and an impaired receptor activator of nuclear factor-κB ligand signaling through reduced activation of nuclear factor-κB and of nuclear factor of activated T-cells cytoplasmic 1. Exogenous soluble HVEM decreased expression of TRAP, whereas soluble LIGHT (a ligand of HVEM) increased it, indicating the occurrence of a positive signaling through HVEM during osteoclastogenesis. Our findings indicate that HVEM regulates bone remodeling via action on OC. The higher bone mass in the femurs of HVEM-knockout mice could be, at least in part, due to attenuated osteoclastogenesis and bone resorption resulting from decreased receptor activator of nuclear factor-κB ligand signaling in the OC.

Platinum nanoparticles reduce ovariectomy-induced bone loss by decreasing osteoclastogenesis.

Platinum nanoparticles (PtNP) exhibit remarkable antioxidant activity. There is growing evidence concerning a positive relationship between oxidative stress and bone loss, suggesting that PtNP could protect against bone loss by modulating oxidative stress. Intragastric administration of PtNP reduced ovariectomy (OVX)- induced bone loss with a decreased level of activity and number of osteoclast (OC) in vivo. PtNP inhibited OC formation by impairing the receptor activator of nuclear factor-κB ligand (RANKL) signaling. This impairment was due to a decreased activation of nuclear factor-κB and a reduced level of nuclear factor in activated T-cells, cytoplasmic 1 (NFAT2). PtNP lowered RANKL-induced long lasting reactive oxygen species as well as intracellular concentrations of Ca(2+) oscillation. Our data clearly highlight the potential of PtNP for the amelioration of bone loss after estrogen deficiency by attenuated OC formation.

Dammarenediol-II production confers TMV tolerance in transgenic tobacco expressing Panax ginseng dammarenediol-II synthase.

Panax ginseng is one of the famous medicinal plants. Ginsenosides, a class of tetracyclic triterpene saponins, are mainly responsible for its pharmacological activity. Most ginsenosides are composed of dammarenediol-II aglycone with various sugar moieties. Dammarenediol-II synthase is the first enzyme in the biosynthesis of ginsenosides. Here, we report that transgenic tobacco expressing the P. ginseng dammarenediol-II synthase gene (PgDDS) produced dammarenediol-II, and conferred resistance to Tobacco mosaic virus (TMV). Upon infection with TMV, lesions developed more rapidly in transgenic tobacco plants, and their size was smaller than those of wild-type plants. Transgenic tobacco plants showed a low level of both the viral titer and mRNA accumulation of TMV coat protein (CP) compared with the wild type. The production of dammarenediol-II in transgenic tobacco stimulated the expression of tobacco pathogenesis-related genes (PR1 and PR2) under both virus-untreated and -treated conditions. When the leaves of wild-type plants were inoculated with a mixture of TMV and dammarenediol-II, the leaves exhibited a reduced viral concentration and TMV-CP expression than those receiving TMV treatment alone. When the leaves of P. ginseng were infected with TMV, transcription of PgDDS was significantly increased. Transgenic P. ginseng plants harboring a ß-glucuronidase (GUS) gene driven by the PgDDS promoter were constructed. The GUS expression was activated when the transgenic ginseng plants were treated with TMV. These results indicate that the medicinally important dammarenediol-II can be ectopically produced in tobacco, and the production of dammarenediol-II in tobacco plants allows them to adopt a viral defense system.

Tobacco NtLTP1, a glandular-specific lipid transfer protein, is required for lipid secretion from glandular trichomes.

Glandular trichomes are the phytochemical factories of plants, and they secrete a wide range of commercially important natural products such as lipids, terpenes and flavonoids. Herein, we report that the Nicotiana tabacum LTP1 (NtLTP1) gene, which is specifically expressed in long glandular trichomes, plays a role in lipid secretion from trichome heads. NtLTP1 mRNA is abundantly transcribed in trichomes, but NtLTP3, NtLTP4 and NtLTP5 are not. In situ hybridization revealed that NtLTP1 mRNAs accumulate specifically in long trichomes and not in short trichomes or epidermal cells. X-gluc staining of leaves from a transgenic plant expressing the NtLTP1 promoter fused to a GUS gene revealed that NtLTP1 protein accumulated preferentially on the tops of long glandular trichomes. GFP fluorescence from transgenic tobacco plants expressing an NtLTP1-GFP fusion protein was localized at the periphery of cells and in the excreted liquid droplets from the glandular trichome heads. In vitro assays using a fluorescent 2-p-toluidinonaphthalene-6-sulfonate probe indicated that recombinant NtLTP1 had lipid-binding activity. The overexpression of NtLTP1 in transgenic tobacco plants resulted in the increased secretion of trichome exudates, including epicuticular wax. In transgenic NtLTP1-RNAi lines, liquid secretion from trichomes was strongly reduced, but epicuticular wax secretion was not altered. Moreover, transgenic tobacco plants overexpressing NtLTP1 showed increased protection against aphids. Taken together, these data suggest that NtLTP1 is abundantly expressed in long glandular trichomes, and may play a role in lipid secretion from long glandular trichomes.

Combined treatment of photodynamic therapy and bevacizumab for choroidal neovascularization secondary to age-related macular degeneration.

PURPOSE: To evaluate the outcome of a combined photodynamic therapy and intravitreal injection of bevacizumab in choroidal neovascularization secondary to age-related macular degeneration. METHODS: Photodynamic therapy (PDT) was administered to 28 eyes followed by 3 consecutive bevacizumab injections. Patients were followed-up for more than 12 months. At baseline, 1, 3, 6, and 12 months post PDT, visual acuity (VA) and central macular thickness were measured using optical coherence tomography. RESULTS: The mean VA was significantly improved from logarithm of the minimal angle of resolution 0.86 at baseline to 0.69 at 1 month (p = 0.011), 0.63 at 3 months (p = 0.003), 0.64 at 6 months (p = 0.004) and 0.60 at 12 months (p < 0.001). Central macular thickness decreased significantly from 328.3 µm at baseline to 230.0 µm at 6 months and 229.9 µm at 1 year (p < 0.001). Reinjection mean number was 0.4 for 6 months and 0.8 for 12 months. By 1 year, retreatment was performed in 10 eyes (36%). CONCLUSIONS: PDT combined with three consecutive intraviteal bevacizumab injections was effective in improving VA and reducing central macular thickness.

Attenuation of the hypersensitive response by an ATPase associated with various cellular activities (AAA) protein through suppression of a small GTPase, ADP ribosylation factor, in tobacco plants.

ATPase associated with various cellular activities (AAA) proteins are commonly distributed among eukaryotes, and are involved in a multitude of cellular functions. NtAAA1 is one such example, being involved in pathogen response in tobacco plants. When its activity was suppressed in RNAi transgenic tobacco plants, an elevated resistance to the pathogenic bacterium Pseudomonas syringae was observed in comparison with the wild type. As AAA proteins function through interaction with specific partners, NtAAA1-interacting proteins were screened by the yeast two-hybrid assay, and one particular gene encoding a small GTPase, an ADP ribosylation factor, was identified and designated as NtARF. Its specific binding to NtAAA1 was confirmed by in vitro pull-down assay, and their interaction was predominant between active forms of NtARF and NtAAA1, each bound to GTP and ATP, respectively. Their physical interaction in vivo around the plasma membrane was shown by fluorescence resonance energy transfer assays, suggesting their role in membrane trafficking. Transgenic tobacco plants constitutively expressing NtARF under the control of a cauliflower mosaic virus 35S promoter exhibited spontaneous and wound-induced lesion formation, and enhanced resistance to pathogen attack. Expression of NtAAA1 in leaves of NtARF transgenic plants attenuated lesion and suppressed pathogen resistance. In wild-type tobacco plants, transcripts of NtAAA1 and NtARF could be induced by ethylene and salicylic acid, respectively. These results suggest that NtAAA1 balances plant resistance through suppression of NtARF, and that the molecular basis for the known antagonistic actions of ethylene and salicylic acid in defense response could be partly attributable to these two proteins.