Lactobacillus bambusae sp. nov., isolated from traditional fermented ma bamboo shoots in Taiwan.

A Gram-stain-positive strain, BS-W1T, was isolated from a traditional fermented ma bamboo shoots (Dendrocalamus latiflorus Munro) product of Taiwan. It was rod-shaped, non-motile, non-haemolytic, asporogenous, facultatively anaerobic, heterofermentative and did not exhibit catalase or oxidase activities. Comparative analysis of 16S rRNA, pheS, rpoA and gyrB gene sequences demonstrated that the novel strain BS-W1T was a member of the genus Lactobacillus. On the basis of 16S RNA gene sequence similarity, the type strains of Lactobacillus oryzae (94.4 % similarity), Lactobacillus acidifarinae (93.8 %), Lactobacillus namurensis (93.7 %) and Lactobacillus zymae (93.7 %) were the closest neighbours to strain BS-W1T. The pheS, rpoA and gyrB gene sequence similarities of strain BS-W1T to closely related these species were less than 80.2 %. DNA-DNA reassociation values with these type strains were 21.0-33.8 %. The DNA G+C content was 46.6 mol%. The average nucleotide identity values between BS-W1T and the closest relatives were lower than 70 %. Phenotypic and genotypic features demonstrated that the strain represents a novel species of the genus Lactobacillus, for which the name Lactobacillus bambusae sp. nov. is proposed. The type strain is BS-W1T (=BCRC 80970T=NBRC 112377T).

Single-pulsed electromagnetic field therapy increases osteogenic differentiation through Wnt signaling pathway and sclerostin downregulation.

Pulsed electromagnetic field (PEMF) therapy has been used for more than three decades to treat bone diseases. The main complaint about using PEMF is that it is time-consuming. Previously, we showed single-pulsed electromagnetic field (SPEMF) applied for 3 min daily increased osteogenic differentiation of mesenchymal stem cells and accelerated bone growth in a long bone defect model. In the current study, we investigated the mechanism of SPEMF to increase osteogenic differentiation in osteoblastic cells. We found that both short-term (SS) and long-term (SL) SPEMF treatment increased mineralization, while alkaline phosphatase (ALP) activity increased during the first 5 days of SPEMF treatment. SS treatment increased gene expression of Wnt1, Wnt3a, Wnt10b, Fzd9, ALP, and Bmp2. Also, SPEMF inhibited sclerostin after 5 days of treatment, and that inhibition was more significant with SL treatment. SL SPEMF increased expression of parathyroid hormone-related protein (PTHrP) but decreased expression of Sost gene, which encodes sclerostin. Together, the early osteogenic effect of SPEMF utilizes the canonical Wnt signaling pathway while the inhibitory effect of long-term SPEMF on sclerostin may be attributable to PTHrP upregulation. This study enhances our understanding of cellular mechanisms to support the previous finding and may provide new insight for clinical applications.

Comparative Study of Heavy Metal Blood Serum Level Between Organic and Conventional Farmers in Eastern Taiwan.

Numerous studies have indicated that organic fertilizers (OFer) might contain heavy metals (HMs) that present health risks to organic farmers (OFar). This study compared the concentrations of six HMs (Zn, Ni, Cd, Cu, Pb, Cr) in the blood of two distinct groups of farmers: 30 OFar from a designated organic area in eastern Taiwan, and 74 conventional farmers (CFar) from neighboring non-organic designated regions. The findings revealed that the OFar exhibited higher levels of Zn (1202.70 ± 188.74 µg/L), Cr (0.20 ± 0.09 µg/L), and Ni (2.14 ± 1.48 µg/L) in their blood compared to the CFar (988.40 ± 163.16 µg/L, 0.18 ± 0.15 µg/L, and 0.77 ± 1.23 µg/L), respectively. The disparities in Zn, Cr, and Ni levels were measured at 214.3 µg/L, 0.02 µg/L, and 1.37 µg/L, respectively. Furthermore, among the OFar, those who utilized green manures (GM) displayed significantly elevated blood levels of Zn (1279.93 ± 156.30 µg/L), Cr (0.24 ± 0.11 µg/L), and Ni (1.94 ± 1.38 µg/L) compared to individuals who exclusively employed chemical fertilizers (CFer) (975.42 ± 165.35 µg/L, 0.19 ± 0.16 µg/L, and 0.74 ± 1.20 µg/L), respectively. The differences in Zn, Cr, and Ni levels were measured at 304.51 µg/L, 0.05 µg/L, and 1.20 µg/L, respectively. As a result, OFar should be careful in choosing OFer and avoid those that may have heavy metal contamination.

Sexuality and Quality of Life in Eastern Taiwan People With Schizophrenia.

OBJECTIVE: Patients with schizophrenia are living at the border of society and their sexuality is often neglected. The aim of the study is to explore the association among The Brief Psychiatric Rating Scale (BPRS), quality of life (QoL), Taiwanese Depression Questionnaire, and Sexual Desire Inventory in people with schizophrenia (PwS). METHODS: This study used a cross-sectional design with 277 psychiatric inpatients. A descriptive analysis, difference analysis, and logistic regression model were presented to identify relevant variables that may affect the probability of good QoL. RESULTS: The study showed that male PwS had higher scores of standard deviation (SD) than females in PwS. The study also showed that smoking, early illness onset age, and shorter illness duration demonstrated a significantly higher SD. The logistic regression analysis showed that BPRS, depression, and SD significantly affected the probability of QoL. By structural equation model, SD would be positively correlated with mental status and SD would indirectly influence QoL. CONCLUSION: Our results showed psychological and sociological factors interactions may contribute to the QoL and SD for PwS. This study also demonstrated a close relationship between SD, depression, and BPRS. These factors may predict the probability of good life quality for the PwS.

Severe cellular stress activates apoptosis independently of p53 in osteosarcoma.

Apoptosis induced by doxorubicin, bortezomib, or paclitaxel, targeting DNA, 26S proteasome, and microtubules respectively, was assessed in two osteosarcoma cells, p53 wild-type U2OS and p53-null MG63 cells. Doxorubicin-induced apoptosis only occurred in U2OS, not in MG63. In contrast, bortezomib and paclitaxel could drive U2OS or MG63 toward apoptosis effectively, suggesting that apoptosis induced by bortezomib or paclitaxel is p53-independent. The expressions of Bcl2 family members such as Bcl2, Bcl-xl, and Puma could be seen in U2OS and MG63 cells with or without doxorubicin, bortezomib, or paclitaxel treatment. In contrast, another member, Bim, only could be observed in U2OS, not in MG63, under the same conditions. Bim knockdown did not affect the doxorubicin-induced apoptosis in U2OS, suggested that a BH3-only protein other than Bim might participate in apoptosis induced by doxorubicin. Using a BH3-mimetic, ABT-263, to inhibit Bcl2 or Bcl-xl produced a limited apoptotic response in U2OS and MG63 cells, suggesting that this BH3-mimetic cannot activate the Bax/Bak pathway efficiently. Significantly, ABT-263 enhanced doxorubicin- and bortezomib-induced apoptosis synergistically in U2OS and MG63 cells. These results implied that the severe cellular stress caused by doxorubicin or bortezomib might be mediated through a dual process to control apoptosis. Respectively, doxorubicin or bortezomib activates a BH3-only protein in one way and corresponding unknown factors in another way to affect mitochondrial outer membrane permeability, resulting in apoptosis. The combination of doxorubicin with ABT-263 could produce synergistic apoptosis in MG63 cells, which lack p53, suggesting that p53 has no role in doxorubicin-induced apoptosis in osteosarcoma. In addition, ABT-263 enhanced paclitaxel to induce moderate levels of apoptosis.

Single pulsed electromagnetic field restores bone mass and microarchitecture in denervation/disuse osteopenic mice.

Pulsed electromagnetic fields (PEMFs) have been proposed to treat bone loss. However, time-consuming is the main complaint. A time-saving and effective treatment is of expectation. Previously, we showed a 3 min daily of single pulsed electromagnetic field (SPEMF) accelerated bone formation of long bone defect in mice. Here we compared the effect of SPEMF with PEMF for treating denervation/disuse osteopenic mice. Healthy mice were divided into 3 groups: intact mice (INT), INT + PEMF, and INT + SPEMF. Induced osteopenic mice were divided to osteopenia (IOP), IOP + PEMF, and IOP + SPEMF groups. The PEMF treated groups were subjected to daily 8 h PEMF(15 Hz, 18 G) exposure, while SPEMF treated groups were daily 3 min SPEMF(0.2 Hz, 1 T) exposure. BMD was evaluated every two weeks during the 12 weeks of treatment. Microarchitecture was evaluated on week 12. SPEMF significantly reversed bone loss in IOP mice as early as 6 weeks post-treatment, while PEMF reversed bone loss after 8 weeks. Bone volume was significantly increased in the IOP + PEMF and IOP + SPEMF group. Besides, bone volume and trabecular number of IOP + SPEMF mice were restored to the levels of INT mice in 12 weeks. Our finding suggests SPEMF increased BMD and restored microarchitecture of disuse osteopenic mice to healthy level.

A single-pulsed electromagnetic field enhances collagen synthesis in tendon cells.

Tendinopathy is a progressive pathology of tendon that is characteristic of imbalance between matrix synthesis and degeneration and is often caused by failure to adapt to mechanical loading. Non-steroidal anti-inflammatory medications (NSAIDS) are used as a conventional treatment to alleviate pain and swelling in the short term, but the ideal treatment for tendinopathy remains unclear. Here, we show a single pulsed electromagnetic field (SPEMF, 0.2 Hz) that up-regulated tenogenic gene expression (Col1a1, Col3a1, Scx, Dcn) and down-regulated inflammatory gene expression (Mmp1) in vitro. After five days of SPEMF stimulation (3 min/day), the collagen type I and total collagen synthesis protein levels were significantly increased. Under pro-inflammatory cytokine (IL-1ß) irritation, the decreased expression of Col1a1/Col3a1 was up-regulated by SPEMF treatment, and the increased expression of Mmp1 was also reversed. From the above, it can be inferred that SPEMF that enhances matrix synthesis and reduces matrix degeneration may counteract the imbalance in tendinopathy. SPEMF application may be developed as a potential future strategy for therapeutic intervention in tendon disorders.

(-)-Epigallocatechin gallate inhibition of osteoclastic differentiation via NF-kappaB.

People who regularly drink tea have been found to have a higher bone mineral density (BMD) and to be at less risk of hip fractures than those who do not drink it. Green tea catechins such as (-)-epigallocatechin gallate (EGCG) have been reported to increase osteogenic functioning in mesenchymal stem cells. However, its effect on osteoclastogenesis remains unclear. In this study, we investigated the effect of EGCG on RANKL-activation osteoclastogenesis and NF-kappaB in RAW 264.7, a murine preosteoclast cell line. EGCG (10-100 microM) significantly suppressed the RANKL-induced differentiation of osteoclasts and the formation of pits in murine RAW 264.7 cells and bone marrow macrophages (BMMs). EGCG appeared to target osteoclastic differentiation at an early stage but had no cytotoxic effect on osteoclast precursors. In addition, it significantly inhibited RANKL-induced NF-kappaB transcriptional activity and nuclear translocation. We conclude that EGCG inhibits osteoclastogenesis through its activation of NF-kappaB.

Transcription-independent and -dependent p53-mediated apoptosis in response to genotoxic and non-genotoxic stress.

We previously reported that p53-mediated apoptosis is determined by severity of DNA damage, not by the level of p53, in doxorubicin-treated prostate cancer cells. In addition to doxorubicin, our results here indicated that camptothecin and bortezomib, which are a topoisomerase 1 poison and a 26 S proteasome inhibitor, respectively, could also induce apoptosis in a p53-dependent manner in prostate cancer. Then, we examined whether p53-mediated apoptosis induced by genotoxic and non-genotoxic stress occur in the same or a different way. By using dominant negative p53 to compete with wild-type p53 in transcription activity, we demonstrated that p53-mediated apoptosis in response to doxorubicin- or camptothecin-induced genotoxic stress is transcription-independent. In contrast, p53-mediated apoptosis from bortezomib-induced stress is transcription-dependent. Interestingly, we also found that doxorubicin-induced p21 expression is activated by p53 in transcription-dependent manner, while camptothecin-induced p21 expression is p53-independent. We then investigated the p53 ratio of nucleus to cytosol corresponding to low and high dose doxorubicin, camptothecin, or bortezomib treatment. The results suggested that p53 translocation from cytoplasm to nucleus actively drives cells toward apoptosis in either transcription-dependent or -independent manner for responding to non-genotoxic or genotoxic stress, respectively.

P53 enhances apoptosis induced by doxorubicin only under conditions of severe DNA damage.

We previously demonstrated that Bim is the main BH3-only protein replacing Bak/Bax from Bcl-xl to activate apoptosis in a p53-independent manner in response to doxorubicin in prostate cancer. However, the comparison of doxorubicin treatment between LNCaP cells carrying p53-wild type and PC3 cells carrying p53-null suggested that p53 might be essential for maximizing apoptosis. Inhibition of ATM did not affect doxorubicin-induced apoptosis. Overexpression of p53 did not affect ABT-263-induced apoptosis and nevertheless, the combination of doxorubicin with ABT-263 induced higher apoptotic responses than did doxorubicin or ABT-263 alone. These results advocated that doxorubicin-induced DNA damage controls p53 function for intensifying apoptosis. Indeed, overexpression of p53 only enhanced apoptosis under conditions of severe DNA damage induced by high concentrations of doxorubicin in LNCaP cells. Immunofluorescence staining showed vague γH2AX foci and enlarged nuclei in LNCaP cells in response to high concentrations of doxorubicin, en route to apoptosis. In addition, our results revealed that the apoptosis in response to DNA replication stress induced by CFS-1686, a catalytic inhibitor of topoisomerase, is p53-independent. Interestingly, the combination of doxorubicin with CFS-1686 generated DNA damage and replication stress simultaneously, resulting in a synergistic apoptotic effect in prostate cancer cells. Thus, we concluded that p53 is a sensor for enhanced apoptosis in response to DNA damage stress, not DNA replication stress, at least in prostate cancer.

Bim directly antagonizes Bcl-xl in doxorubicin-induced prostate cancer cell apoptosis independently of p53.

Doxorubicin and other anthracycline compounds exert their anti-cancer effects by causing DNA damage and initiating cell cycle arrest in cancer cells, followed by apoptosis. DNA damage generally activates a p53-mediated pathway to initiate apoptosis by increasing the level of the BH3-only protein, Puma. However, p53-mediated apoptosis in response to DNA damage has not yet been validated in prostate cancers. In the current study, we used LNCaP and PC3 prostate cancer cells, representing wild type p53 and a p53-null model, to determine if DNA damage activates p53-mediated apoptosis in prostate cancers. Our results revealed that PC3 cells were 4 to 8-fold less sensitive than LNCaP cells to doxorubicin-inuced apoptosis. We proved that the differential response of LNCaP and PC3 to doxorubicin was p53-independent by introducing wild-type or dominant negative p53 into PC3 or LNCaP cells, respectively. By comparing several apoptosis-related proteins in both cell lines, we found that Bcl-xl proteins were much more abundant in PC3 cells than in LNCaP cells. We further demonstrated that Bcl-xl protects LNCaP and PC3 cells from doxorubicin-induced apoptosis by using ABT-263, an inhibitor of Bcl-xl, as a single agent or in combination with doxorubicin to treat LNCaP or PC3 cells. Bcl-xl rather than p53, likely contributes to the differential response of LNCaP and PC3 to doxorubicin in apoptosis. Finally, co-immunoprecipitation and siRNA analysis revealed that a BH3-only protein, Bim, is involved in doxorubicin-induced apoptosis by directly counteracting Bcl-xl.

CFS-1686 causes cell cycle arrest at intra-S phase by interference of interaction of topoisomerase 1 with DNA.

CFS-1686 (chemical name (E)-N-(2-(diethylamino)ethyl)-4-(2-(2-(5-nitrofuran-2-yl)vinyl)quinolin-4-ylamino)benzamide) inhibits cell proliferation and triggers late apoptosis in prostate cancer cell lines. Comparing the effect of CFS-1686 on cell cycle progression with the topoisomerase 1 inhibitor camptothecin revealed that CFS-1686 and camptothecin reduced DNA synthesis in S-phase, resulting in cell cycle arrest at the intra-S phase and G1-S boundary, respectively. The DNA damage in CFS-1686 and camptothecin treated cells was evaluated by the level of ATM phosphorylation, γH2AX, and γH2AX foci, showing that camptothecin was more effective than CFS-1686. However, despite its lower DNA damage capacity, CFS-1686 demonstrated 4-fold higher inhibition of topoisomerase 1 than camptothecin in a DNA relaxation assay. Unlike camptothecin, CFS-1686 demonstrated no activity on topoisomerase 1 in a DNA cleavage assay, but nevertheless it reduced the camptothecin-induced DNA cleavage of topoisomerase 1 in a dose-dependent manner. Our results indicate that CFS-1686 might bind to topoisomerase 1 to inhibit this enzyme from interacting with DNA relaxation activity, unlike campothecin's induction of a topoisomerase 1-DNA cleavage complex. Finally, we used a computer docking strategy to localize the potential binding site of CFS-1686 to topoisomerase 1, further indicating that CFS-1686 might inhibit the binding of Top1 to DNA.

Synthesis of aminoalkoxy substituted 4,5-diphenylisoxazole derivatives as potential anti-osteoporotic agents.

Certain 4,5-diarylisoxazole derivatives have been found to possess broad biological effects, including antiinflammatory and anticancer activities. Recently, we have reported preparation of certain isoflavone derivatives and investigated for their anti-osteoporotic and antiproliferative activities in a detailed SAR study. The present report describes the conversion of isoflavones into novel 4,5-diphenylisoxazole derivatives by the treatment with NH2OH. Alkylation followed by amination of these 4,5-diphenylisoxazoles gave the desired aminoalkoxy substituted 4,5-diphenylisoxazole derivatives. These compounds were evaluated in vitro for the osteogenic differentiation and quantification of mineralization. Although 5-isopropoxy-2-[4-(4-methoxyphenyl)isoxazol-5-yl]phenol (3) exhibited approximately 2.8-fold more activity than the positive Ipriflavone in the promotion of osteoblast activity (277% mineralization), the low cell viability (6%) and high cytotoxicity (68%) prompted us to further pursue more suitable candidates. A series of aminoalkyl side chains were introduced with aims to decrease cytotoxicity. Among them, 5-{4-isopropoxy-2-[4-(pyrrolidin-1-yl)butoxy]phenyl}-4-(4- methoxyphenyl)isoxazole (7a) exhibited approximately 2-fold more activity than the positive Ipriflavone in the promotion of osteoblast activity (194% mineralization) with comparable cell viability (71% v.s. 77%). Compound 7a was non cytotoxic against hADSCs and therefore, was selected as a lead for further structural optimization.

(-)-Epigallocatechin-3-gallate improves bone microarchitecture in ovariectomized rats.

OBJECTIVE: Previously, we reported that (-)-epigallocatechin-3-gallate (EGCG), a green tea polyphenol, increased the osteogenic differentiation of murine bone marrow mesenchymal stem cells by increasing the messenger RNA expression of osteogenesis-related genes, alkaline phosphatase activity, and, eventually, mineralization. The present study further investigated the effects of EGCG on bone microstructure change and possible mechanisms in ovariectomy (OVX)-induced osteopenic rats. METHODS: Rats subjected to OVX were administered EGCG systemically for 12 weeks. Proximal tibial bone mineral densities before and after treatment were compared between groups. Changes in the microarchitecture of both the proximal tibia and the third lumbar spine were compared between EGCG-treated and nontreated groups using micro-CT (µCT). Bone histology and immunohistochemistry in the proximal tibia were evaluated. RESULTS: Results showed that EGCG 3.4 mg/kg/day (estimated peak serum concentration, 10 µmol/L) hampered the decrease in bone mineral density (from 7.97% to 3.96%) and improved the parameters of µCT measurements, including bone volume (from 18% to 27%), trabecular thickness (from 0.17 to 0.22 mm), trabecular number (from 1.13 to 1.37 mm(-1)), and trabecular separation (from 0.91 to 0.69 mm), compared with nontreated ovariectomized rats. Similar improvements in bone volume (from 30% to 49%) and trabecular thickness (from 0.14 to 0.26 mm) were also found in the third lumbar spine. Bone volume in the tibial cortex also increased after EGCG treatment (from 9% to 28%). A higher trabecular number and greater trabecular volume were also seen in histology, further confirming the results of µCT. The immunolocalized bone morphogenetic protein 2 brown-stained area increased from 31% in the OVX group to 53% in the OVX + 10 EGCG group (P < 0.01). Serial biochemistry data revealed no significant systemic toxic effect of EGCG. CONCLUSIONS: Intraperitoneal treatment with EGCG 3.4 mg/kg/day for 3 months can mitigate bone loss and improve bone microarchitecture in ovariectomized rats, and increased expression of bone morphogenetic protein 2 may contribute to this effect.

Discovery of indeno[1,2-c]quinoline derivatives as inhibitors of osteoclastogenesis induced by receptor activator of NF-κB ligand (RANKL).

Certain indeno[1,2-c]quinolines were synthesized and evaluated for antiosteoclastogenic activities. Among them, 6,9-dimethoxy-11H-indeno[1,2-c]quinolin-11-one (8a) and 9-methoxy-6-(methylthio)-11H-indeno[1,2-c]quinolin-11-one (16a) inhibited RANKL-induced osteoclast formation in Raw 264.7 cells with an IC(50) of 2.00 and 2.58 µM, respectively. Compound 8a was only weakly active in the inhibition of the RANKL-induced NFAT activation, while 16a was inactive. These results indicated that the antiosteoclastogenic effect of 8a is only partly related while 16a is not related to the suppression of NFAT.

Synthesis and anti-osteoporotic evaluation of certain 3-amino-2-hydroxypropoxyisoflavone derivatives.

We report herein the synthesis and anti-osteoporotic evaluation of certain 3-amino-2-hydroxypropoxyisoflavone derivatives. The results indicated that 3-(3,4-dimethoxyphenyl)-7-(oxiran-2-ylmethoxy)-4H-chromen-4-one (4) and 3-{4-[3-(cyclohexylamino)-2-hydroxypropoxy]phenyl}-7-methoxy-4H-chromen-4-one (5a) exhibited significant inhibitory effects on osteoclast activity (TRAP activity in RAW 264.7 with an ED(50) of 0.56 and 2.28 microM respectively). Both compounds have also exhibited very strong osteogenic effects, approximately a 10-fold effect of Ipriflavone on mineralization of osteoblasts (MC3T3E1 cells, a preosteoblast cell line derived from calvaria of C57BL/6 mice). Results indicated the potency on enhancing mineralization in D1 cells (a bone marrow mesenchymal cell line derived from BALB/c mice) decreased in an order 4>Ipriflavone>5a. However, the potency on enhancing mineralization in human adipose tissue derived stem cells (hADSCs) decreased in an order 5a>4>Raloxifene>Ipriflavone. Compound 5a has been found to be non-cytotoxic and especially active in the enhancement of mineralization in human adipose tissue derived stem cells. Therefore, 5a was selected as a potential lead for further structural optimization.

New lignans and cytotoxic constituents from Wikstroemia lanceolata.

Two new lignans, (-)-aptosimon ( 1) and (-)-diasesamin-di-gamma-lactone ( 2), together with twenty-two known compounds, have been isolated from the stems and roots of Wikstroemia lanceolata. The structures of the new compounds were determined through spectral analyses. The proton and carbon assignments of the known sesquiterpenoid, 1alpha,7alpha,10alpha H-guaia-4,11-dien-3-one ( 24) were revised by 2D NMR techniques. Among the isolates, three compounds, (+)-hinokinin ( 6), 2,6-dimethoxy- p-benzoquinone ( 17), and 1alpha,7alpha,10alpha H-guaia-4,11-dien-3-one ( 24) exhibited effective cytotoxicities (ED50 values < 4.0 microg mL-1) against P-388 and HT-29 tumor cell lines.