Chromone and chromanone glucosides from Hypericumsikokumontanum and their anti-Helicobacter pylori activities

Chromone glucosides, takanechromones A-C (1, 2 and 5) and chromanone glucosides, named takanechromanones A and B (3 and 4), were isolated from the methanolic extracts of Hypericumsikokumontanum together with 27 known compounds. Their structures were established based on spectroscopic evidence. The isolated compounds and some chromone derivatives were assayed for antimicrobial activity against Helicobacter pylori and cytotoxicity against human cancer cell lines.     

Terpenoids from Dysoxylum densiflorum

Three degraded limonoids, dysodensiols A-C (1-3), and three sesquiterpenoids, dysodensiols D-F (4-6), along with 17 known compounds, were isolated from the twigs and leaves of Dysoxylum densiflorum. The structures of compounds 1-6 were established on the basis of extensive spectroscopic analysis.     

Secreted Opisthorchis viverrini glutathione S-transferase regulates cell proliferation through AKT and ERK pathways in cholangiocarcinoma

Opisthorchis viverrini can develop mitogenic substances into the excretory/secretory product (ESP) that may play an important role in promoting the genesis of cholangiocarcinoma (CCA). In the present study, glutathione S-transferase (GST) is identified as being secreted into Ov-ESP and acting as one of the parasitic mitogens. Its proliferative effect and possible mechanism were explored and its association with the tumor development is proposed. Ov-ESP was concentrated and purified by gel filtration chromatography. SDS-PAGE, 2-DE, and LC-MS/MS identified GST predominantly expressed in the proliferative ESP fraction. The recombinant OvGST (rOvGST) was produced by wheat germ cell-free expression and confirmed by an MTS assay to have a proliferative function on NIH-3T3 murine fibroblasts and MMNK1 non-tumorigenic human bile duct epithelial cells in a dose dependent manner with different optimal doses. The cell surface binding of rOvGST was confirmed in vitro and the activation of both pAKT and pERK was revealed as the mechanism of OvGST-mediated cell proliferation. With support from the observation of secreted OvGST on the biliary cells surrounding the parasites, it is suggested that OvGST can promote cell proliferation that consequently may accelerate the genesis of CCA.     

C-Glucosidic ellagitannin oligomers from Melaleuca squarrosa Donn ex Sm., Myrtaceae

C-Glucosidic ellagitannin dimers were classified as types A-C according to a putative biogenetic oligomerization mode. They were characterized by different positions of the C-C bond between the phenolic acyl unit in one monomer and the benzylic C-1 of the open-chain glucose core in the other monomer. In recent years, four C-glucosidic tannins, melasquanins A-D (18-21), have been found in the leaves of Melaleuca squarrosa Donn ex Sm. (Myrtaceae). These are characterized as a dimer (melasquanin A) of a dimerization mode (type D), and trimers (melasquanins B-D) based on spectroscopic analysis including various two-dimensional nuclear magnetic resonance (2D NMR) experiments. Melasquanins B (19) and D (21) are C-glucosidic tannin trimers with a structure containing, non-repeating condensation modes, which was hitherto unknown.     

In Vitro Repression of Brca1-associated RING Domain Gene,Bard1, Induces Phenotypic Changes in Mammary Epithelial Cells

BRCA1-associated RING domain (BARD1) was identified as a protein interacting with the breast cancer gene product BRCA1. The identification of tumorigenic missense mutations within BRCA1 that impair the formation of BARD1–BRCA1 complexes, and of BARD1 mutations in breast carcinomas, sustain the view that BARD1 is involved in BRCA1-mediated tumor suppression. We have cloned the murine Bard1 gene and determined that its expression in different tissues correlates with the expression profile of Brca1. To investigate the function of Bard1, we have reduced Bard1 gene expression in TAC-2 cells, a murine mammary epithelial cell line that retains morphogenetic properties characteristic of normal breast epithelium. Partial repression of Bard1, achieved by the transfection of TAC-2 cells with plasmids constitutively expressing ribozymes or antisense RNAs, resulted in marked phenotypic changes, consisting of altered cell shape, increased cell size, high frequency of multinucleated cells, and aberrant cell cycle progression. Furthermore, Bard1-repressed cell clones overcame contact inhibition of cell proliferation when grown in monolayer cultures and lost the capacity to form luminal structures in three-dimensional collagen gels. These results demonstrate that Bard1 repression induces complex changes in mammary epithelial cell properties which are suggestive of a premalignant phenotype.     

The role of Wnt5a in prostate gland development

The Wnt genes encode a large family of secreted glycoproteins that play important roles in controlling tissue patterning, cell fate and proliferation during development. Currently, little is known regarding the role(s) of Wnt genes during prostate gland development. The present study examines the role of the noncanonical Wnt5a during prostate gland development in rat and murine models. In the rat prostate, Wnt5a mRNA is expressed by distal mesenchyme during the budding stage and localizes to periductal mesenchymal cells with an increasing proximal-to-distal gradient during branching morphogenesis. Wnt5a protein is secreted and localizes to periductal stroma, extracellular matrix and epithelial cells in the distal ducts. While Wnt5a expression is high during active morphogenesis in all prostate lobes, ventral prostate (VP) expression declines rapidly following morphogenesis while dorsal (DP) and lateral lobe (LP) expression remains high into adulthood. Steroids modulate prostatic Wnt5a expression during early development with testosterone suppressing Wnt5a and neonatal estrogen increasing expression. In vivo and ex vivo analyses of developing mouse and rat prostates were used to assess the functional roles of Wnt5a. Wnt5a^−/− murine prostates rescued by organ culture exhibit disturbances in bud position and directed outgrowth leading to large bulbous sacs in place of elongating ducts. In contrast, epithelial cell proliferation, ductal elongation and branchpoint formation are suppressed in newborn rat prostates cultured with exogenous Wnt5a protein. While renal grafts of Wnt5a^−/− murine prostates revealed that Wnt5a is not essential for cyto- and functional differentiation, a role in luminal cell polarity and lumenization of the ducts was indicated. Wnt5a suppresses prostatic Shh expression while Shh stimulates Wnt5a expression in a lobe-specific manner during early development indicating that Wnt5a participates in cross-talk with other members of the gene regulatory network that control prostate development. Although Wnt5a does not influence prostatic expression of other Wnt morphogens, it suppresses Wif-1 expression and can thus indirectly modulate Wnt signaling. In summary, the present finds demonstrate that Wnt5a is essential for normal prostate development where it regulates bud outgrowth, ductal elongation, branching, cell polarity and lumenization. These findings contribute to the growing body of knowledge on regulatory mechanisms involved in prostate gland development which are key to understanding abnormal growth processes associated with aging.     

Potentially human pathogenic Acanthamoeba isolated from a heated indoor swimming pool in Switzerland

Some free-living amoebae, including some species of the genus Acanthamoeba, can cause infections in humans and animals. These organisms are known to cause granulomatous amebic encephalitis (GAE) in predominantly immune-deficient persons. In the present study, we isolated a potentially human pathogenic Acanthamoeba isolate originating from a public heated indoor swimming pool in Switzerland. The amoebae, thermophilically preselected by culture at 37 °C, subsequently displayed a high thermotolerance, being able to grow at 42 °C, and a marked cytotoxicity, based on a co-culture system using the murine cell line L929. Intranasal infection of Rag2-immunodeficient mice resulted in the death of all animals within 24 days. Histopathology of brains and lungs revealed marked tissue necrosis and hemorrhagic lesions going along with massive proliferation of amoebae. PCR and sequence analysis, based on 18S rDNA, identified the agent as Acanthamoeba lenticulata. In summary, the present study reports on an Acanthamoeba isolate from a heated swimming pool suggestive of being potentially pathogenic to immunocompromised persons.     

CrxOS maintains the self-renewal capacity of murine embryonic stem cells

Embryonic stem (ES) cells maintain pluripotency by self-renewal. Several homeoproteins, including Oct3/4 and Nanog, are known to be key factors in maintaining the self-renewal capacity of ES cells. However, other genes required for the mechanisms underlying this process are still unclear. Here we report the identification by in silico analysis of a homeobox-containing gene, CrxOS, that is specifically expressed in murine ES cells and is essential for their self-renewal. ES cells mainly express the short isoform of endogenous CrxOS. Using a polyoma-based episomal expression system, we demonstrate that overexpression of the CrxOS short isoform is sufficient for maintaining the undifferentiated morphology of ES cells and stimulating their proliferation. Finally, using RNA interference, we show that CrxOS is essential for the self-renewal of ES cells, and provisionally identify foxD3 as a downstream target gene of CrxOS. To our knowledge, ours is the first delineation of the physiological role of CrxOS in ES cells.     

Cytotoxic dihydrobenzophenanthridine alkaloids from the roots of Macleaya microcarpa

Five dihydrobenzophenanthridine alkaloids, named as maclekarpine A-E, were isolated from the roots of Macleaya microcarpa, together with 10 known benzophenanthridine/dihydrobenzophenanthridine derivatives and a known amide. Their structures were determined on the basis of spectroscopic methods. The relative configuration of maclekarpine A was solved by X-ray single-crystal diffraction, which also contributed to the establishment of the relative configuration of maclekarpine B and C, as well as the assignment of the absolute configuration of maclekarpine A-C with aid of their CD curves. Cytotoxicity of the isolated compounds against five human tumor cell-lines was evaluated, with some having activity with IC₅₀ values ranging from 0.1 to 3.5 μM.     

Methotrexate and aminopterin lack in vivo antimalarial activity against murine malaria species

The antifolate anticancer drug methotrexate (MTX) has potent activity against Plasmodium falciparum in vitro. Experience of its use in the treatment of rheumatoid arthritis indicates that it could be safe and efficacious for treating malaria. We sought to establish a murine malaria model to study the mechanism of action and resistance of MTX and its analogue aminopterin (AMP). We used Plasmodium berghei, Plasmodium yoelii yoelii, Plasmodium chabaudi and Plasmodium vinckei. None of these species were susceptible to either drug. We have also tested the efficacy of pyrimethamine in combination with folic acid in P. berghei, and data indicate that folic acid does not influence pyrimethamine efficacy, which suggests that P. berghei may not transport folate. Since MTX and AMP utilise folate receptor/transport to gain access to cells, their lack of efficacy against the four tested murine malaria species may be the result of inefficiency of drug transport.     

The aPKCι blocking agent ATM negatively regulates EMT and invasion of hepatocellular carcinoma

Epithelial-to-mesenchymal transition (EMT) has an important role in invasion and metastasis of hepatocellular carcinoma (HCC). To explore the regulatory mechanism of atypical protein kinase C ι (aPKCι) signaling pathways to HCC development, and find an agent for targeted therapy for HCC, immortalized murine hepatocytes were employed to establish an EMT cell model of HCC, MMH-RT cells. Our study showed that EMT took place in MMH-R cells under the effect of transforming growth factor-β1 (TGF-β1) overexpressing aPKCι. Furthermore, we showed that the aPKCι blocking agent aurothiomalate (ATM) inhibited EMT and decreased invasion of hepatocytes. Moreover, ATM selectively inhibited proliferation of mesenchymal cells and HepG2 cells and induced apoptosis. However, ATM increased proliferation of epithelial cells and had little effect on apoptosis and invasion of epithelial cells. In conclusion, our result suggested that aPKCι could be an important bio-marker of tumor EMT, and used as an indicator of invasion and malignancy. ATM might be a promising agent for targeted treatment of HCC.     

A study on the immune receptors for polysaccharides from the roots of Astragalus membranaceus, a Chinese medicinal herb

The immunopotentiating effect of the roots of Astragalus membranaceus, a medicinal herb, has been associated with its polysaccharide fractions (Astragalus polysaccharides, APS). We herein demonstrate that APS activates mouse B cells and macrophages, but not T cells, in terms of proliferation or cytokine production. Fluorescence-labeled APS (fl-APS) was able to selectively stain murine B cells, macrophages and a also human tumor cell line, THP-1, as determined in flow cytometric analysis and confocal laser scanning microscopy. The specific binding of APS to B cells and macrophages was competitively inhibited by bacterial lipopolysaccharides. Rabbit-anti-mouse immunoglobulin (Ig) antibody was able to inhibit APS-induced proliferation of, and APS binding to, mouse B cells. Additionally, APS effectively stimulated the proliferation of splenic B cells from C3H/HeJ mice that have a mutated TLR4 molecule incapable of signal transduction. These results indicate that APS activates B cells via membrane Ig in a TLR4-independent manner. Interestingly, macrophages from C3H/HeJ mice were unable to respond to APS stimulation, suggesting a positive involvement of the TLR4 molecule in APS-mediated macrophage activation. Monoclonal Ab against mouse TLR4 partially inhibited APS binding with macrophages, implying direct interaction between APS and TLR4 on cell surface. These results may have important implications for our understanding on the molecular mechanisms of immunopotentiating polysaccharides from medicinal herbs.     

Leishmania (Viannia) peruviana (MHOM/PE/LCA08): Comparison of THP-1 cell and murine macrophage susceptibility to axenic amastigotes for the screening of leishmanicidal compounds

This study, undertaken to compare the susceptibility of THP-1 cells and murine peritoneal macrophages to Leishmania peruviana amastigotes, obtained THP-1 infection with 10 parasites/cell compared to 2 parasites/murine macrophage. The parasite burden was maximal at 72 h post-infection (h.p.i.) for THP-1 cells, while it was still increasing at 120 h.p.i. for murine macrophages. Since in both cases the infection with L. peruviana affected cell viability, we recommend evaluating any leishmanicidal activity at 72 h.p.i. Amphotericin B reduced Leishmania infection by 50% at concentrations of 0.1 μM in THP-1 and murine macrophages at 72 h.p.i.Our results demonstrate that amastigotes of L. peruviana can infect THP-1 cells and murine macrophages and indicate the suitability of this model to screen compounds for leishmanicidal activity.     

Rare merosesquiterpenoids from basidiomycete Craterellus odoratus and their inhibition of 11β-hydroxysteroid dehydrogenases

Rare merosesquiterpenoids, craterellins A-C (1-3), were isolated from cultures of basidiomycete Craterellus odoratus together with the previously known massarinolin C (4). Structures of 1-3 were elucidated on the basis of extensive spectroscopic analysis. Compounds 1-3 possess a rare, epoxymethylenecyclohexanetriol-bicyclofarnesane sesquiterpene hybrid skeleton. Compounds 1-4 were evaluated for their inhibitory activities against two isozymes of 11β-hydroxysteroid dehydrogenases (11β-HSD1 and 11β-HSD2).     

Northward expansion of a marine parasite: Testing the role of temperature adaptation

The known range of the eastern oyster (Crassostrea virginica) parasite, Perkinsus marinus, expanded into the northeastern United States in the early 1990s. We used both in vitro and in vivo data to test the hypothesis that the northward expansion was associated with a low-temperature adapted strain of the parasite. In vitro proliferation of nine P. marinus isolates from three geographic sites, Massachusetts and New Jersey in the new range, and South Carolina in the historic southern range, was measured at seven temperatures (5 to 35 °C) using a tetrazolium blue dye assay. We wanted to determine if there were between- and within-geographic location differences in the P. marinus proliferation rate, and if so, whether they were associated with temperature. We found no evidence of low-temperature adaptation based on the fact that net proliferation rates for isolates from all three geographic locations were similar at temperatures from 5 to 20 °C. On the other hand, at temperatures of 25 to 35 °C, the South Carolina isolates exhibited higher proliferation rates than the northern isolates suggesting possible high-temperature adaptation of parasite strains that are routinely exposed to higher temperatures. Although there was significant within-location variation among isolates, the data tended to group together by geographic location supporting the hypothesis that there is an important regional component to the proliferation rate of P. marinus isolates. A survey of published data showed that the temperature at which in vivo proliferation was first observed in oysters at sites from the Gulf of Mexico to Massachusetts was typically between 20 and 23 °C with no evidence of a geographic cline. These results lend support to the hypothesis that the recent warming trend in the northeastern US is the most likely explanation for the P. marinus range extension.     

Abnormalities in cell proliferation and apico-basal cell polarity are separable in Drosophila lgl mutant clones in the developing eye

In homozygous mutants of Drosophila lethal-2-giant larvae (lgl), tissues lose apico-basal cell polarity and exhibit ectopic proliferation. Here, we use clonal analysis in the developing eye to investigate the effect of lgl null mutations in the context of surrounding wild-type tissue. lgl⁻ clones in the larval eye disc exhibit ectopic expression of the G1-S regulator, Cyclin E, and ectopic proliferation, but do not lose apico-basal cell polarity. Decreasing the perdurance of Lgl protein in larval eye disc clones, by forcing extra proliferation of lgl⁻ tissue (using a Minute background), leads to a loss in cell polarity and to more extreme ectopic cell proliferation. Later in development at the pupal stage, lgl mutant photoreceptor cells show aberrant apico-basal cell polarity, but this is not associated with ectopic proliferation, presumably because cells are differentiated. Thus in a clonal context, the ectopic proliferation and cell polarity defects of lgl⁻ mutants are separable. Furthermore, lgl⁻ mosaic eye discs have alterations in the normal patterns of apoptosis: in larval discs some lgl⁻ and wild-type cells at the clonal boundary undergo apoptosis and are excluded from the epithelia, but apoptosis is decreased elsewhere in the disc, and in pupal retinas lgl⁻ tissue shows less apoptosis.     

Evaluation of phenolic profile,antioxidant and anticancer potential of two main representants of Zingiberaceae family against B164A5 murine melanoma cells

Background

Curcuma longa Linnaeus and Zingiber officinale Roscoe are two main representatives of Zingiberaceae family studied for a wide range of therapeutic properties, including: antioxidant, anti-inflammatory, anti-angiogenic, antibacterial, analgesic, immunomodulatory, proapoptotic, anti-human immunodeficiency virus properties and anticancer effects. This study was aimed to analyse the ethanolic extracts of Curcuma rhizome (Curcuma longa Linnaeus) and Zingiber rhizome (Zingiber officinale Roscoe) in terms of polyphenols, antioxidant activity and anti-melanoma potential employing the B164A5 murine melanoma cell line.

Results

In order to evaluate the total content of polyphenols we used Folin-Ciocâlteu method. The antioxidant activity of the two ethanolic extracts was determined by DPPH assay, and for the control of antiproliferative effect it was used MTT proliferation assay, DAPI staining and Annexin-FITC-7AAD double staining test. Results showed increased polyphenols amount and antioxidant activity for Curcuma rhizome ethanolic extract. Moreover, 100 μg/ml of ethanolic plant extract from both vegetal products presented in a different manner an antiproliferative, respectively a proapoptotic effect on the selected cell line.

Conclusions

The study concludes that Curcuma rhizome may be a promising natural source for active compounds against malignant melanoma.