Several natural products isolated from a red alga Gracilaria lemaneiformis and its evaluation of antialgal activity against six common red tide microalgae.

The ethanol extracts of Gracilaria lemaneiformis that have inhibitory effects on Karenia mikimotoi and Skeletonema costatum were separated by liquid-liquid extraction using different polar solvents into five fractions with antialgal activities (petroleum ether, chloroform, ethyl acetate, n-butanol, and water-soluble fractions). These fractions were chromatographed on silica gel to give, after repeated preparative thin-layer chromatography (PTLC) purification processes, 1-ß-D-ribofuranosyluracil (1), 3-hydroxymethyl-pyrrolopiperazine-2,5-dione (2), benzene-1,2-propanoic acid (3), 1-O-palmitoyl-2-O-palmitoleoyl-3-O-ß-D-galactopyranosyl glycerol (4), 7-oxabicyclo[4.1.0]-heptan-3-ol (5), linoleic acid (6), 3,4-dimethoxy-6-(methoxymethyl)-tetrahydro-2H-pyran-2,5-diol (7), and 3,7,11,16-tetramethyl -2-heptadecen-1-ol (8). Five of them, natural products 1, 2, 5, 7, and 8, were isolated from Gracilaria lemaneiformis for the first time, and three natural products (3, 5, and 8) were isolated from marine macroalgae for the first time. Among them, natural products (1, 2, 3, 4, and 6) showed the most obvious inhibition activities to the growth of Karenia mikimotoi and Skeletonema costatum at the concentration of 80 µg/mL. Therefore, antialgal activities of these five natural products against Amphidinium carterae, Heterosigma akashiwo, Karenia mikimotoi, Phaeocystis globosa, Prorocentrum donghaiense, and Skeletonema costatum were further tested at different concentrations (0.4, 2, 10, and 50 µg/mL). This was the first report of antialgal activities of five natural products (1, 2, 3, 4, and 6) to these six red tide microalgae. They showed significantly selective antialgal activities against all tested red tide microalgae. At the concentration of 50 µg/mL, the growth of Amphidinium carterae, Heterosigma akashiwo, Karenia mikimotoi, and Phaeocystis globosa was obviously inhibited; for Karenia mikimotoi, natural products 1, 2, and 6 have significant antialgal activities; the growth inhibition of Skeletonema costatum that was exposed to natural products 1, 3, and 4 was remarkable. Furthermore, by analyzing and comparing EC50-96 h values, it has been determined that natural product 3 (natural product 4) showed the superior application potential than potassium dichromate and some reported natural products (such as gossonorol isolated from Porphyra yezoensis, trehalose purified from Ulva pertusa) as a characteristic antialgal agent against Amphidinium carterae (Phaeocystis globosa). In addition, natural products 1 and 3 also showed good superiority than some reported natural products in inhibiting Skeletonema costatum; however, it was a pity that they were inferior to potassium dichromate in the inhibiting this red tide microalgae. Taken together, it is not hard to conclude that Gracilaria lemaneiformis was a good source of natural products with antialgal activities against some red tide microalgae.

Benthic invertebrate and microbial biodiversity in sub-tropical urban rivers: Correlations with environmental variables and emerging chemicals.

Urban rivers often function as sinks for various contaminants potentially placing the benthic communities at risk of exposure. We performed a comprehensive biological survey of the benthic macroinvertebrate and bacterial community compositions in six rivers from the suburb to the central urban area of Guangzhou city (South China), and evaluated their correlations with emerging organic contaminants, heavy metals and nutrients. Overall, the benthic macroinvertebrate community shifted from molluscs to oligochaete from the suburban to the central urban rivers that receive treated and untreated sewage. An exception was the site in the Sha River where chironomids were most abundant. The differences in macroinvertebrate community assemblages were significantly associated with chromium, total phosphorus, galaxolide, triclosan and sand content in the sediment. There was no significant difference in benthic macroinvertebrate composition between the dry and wet season. As assessed by double constrained ordination, sexual reproduction was the only trait of benthic macroinvertebrates that showed a significant correlation with pollution variables, as it was significantly positively correlated with chromium and total phosphorus. This suggests that r-strategist occurs in polluted sampling sites. The benthic bacterial community composition showed a significant difference between seasons and among the Liuxi River, Zhujiang River and central urban rivers. The differences in community composition of the benthic bacteria were significantly correlated with galaxolide, total phosphorus, lead and triclosan. These results suggest that input of treated and untreated sewage significantly altered the benthic macroinvertebrate and bacterial community compositions in urban rivers.

Occurrence and ecological risk assessment of emerging organic chemicals in urban rivers: Guangzhou as a case study in China.

Urban rivers may receive contamination from various sources including point sources like domestic sewage and nonpoint sources (e.g., runoff), resulting in contamination with various chemicals. This study investigated the occurrence of emerging organic contaminants (3 endocrine disrupting compounds (EDCs), and 17 pharmaceuticals and personal care products (PPCPs)) in six urban rivers of a representative subtropical city, Guangzhou (southern China). Our results showed that EDCs and personal care products were frequently detected in the water phase and sediment phase. 4-nonylphenol (4-NP) was the most predominant compound with the highest concentration of 5050ng/L in the water phase and 14,400ng/g dry weight (dw) in the sediment. Generally, higher total concentrations of EDCs and PPCPs were detected in the four urban streams compared to the main stream Zhujiang River and the Liuxi River at the suburb area. A screening-level risk assessment showed that 4-nonylphenol and triclosan (TCS) pose potential risks to aquatic organisms in most sampling sites. For individual taxa, 4-NP may pose risks to various groups of aquatic organisms, while TCS only might pose high risks to algae. CAPSULE: Higher contamination of EDCs and PPCPs was observed in rivers in urban area; 4-nonylphenol and triclosan showed RQs>1 in >70% of the reported area.

Ecological risks of home and personal care products in the riverine environment of a rural region in South China without domestic wastewater treatment facilities.

Home and personal care products (HPCPs) including biocides, benzotriazoles (BTs) and ultraviolet (UV) filters are widely used in our daily life. After use, they are discharged with domestic wastewater into the receiving environment. This study investigated the occurrence of 29 representative HPCPs, including biocides, BTs and UV filters, in the riverine environment of a rural region of South China where no wastewater treatment plants were present, and assessed their potential ecological risks to aquatic organisms. The results showed the detection of 11 biocides and 4 BTs in surface water, and 9 biocides, 3 BTs and 4 UV filters in sediment. In surface water, methylparaben (MeP), triclocarban (TCC), and triclosan (TCS) were detected at all sites with median concentrations of 9.23 ng/L, 2.64 ng/L and 5.39 ng/L, respectively. However, the highest median concentrations were found for clotrimazole (CLOT), 5-methyl-1H-benzotriazole (MBT) and carbendazim (CARB) at 55.6 ng/L, 33.7 ng/L and 13.8 ng/L, respectively. In sediment, TCC, TCS, and UV-326 were detected with their maximum concentrations up to 353 ng/g, 155 ng/g, and 133 ng/g, respectively. The concentrations for those detected HPCPs in surface water and sediment were generally lower in the upper reach (rural area) of Sha River than in the lower reach of Sha River with close proximity to Dongjiang River (Pt-test<0.05), indicating other input sources of HPCPs in the lower reach. Biocides showed significantly higher levels in surface water in the wet season than in the dry and intermediate seasons. Preliminary risk assessment demonstrated that the majority of HPCPs monitored represented low risk in surface waters. There are potentially greater risks to aquatic organisms from the use of TCS and TCC in the wet season than in dry and intermediate seasons in surface waters. This preliminary assessment also indicates potential concerns associated with TCC, TCS, DEET, CARB, and CLOT in sediments, although additional data should be generated to assess this fully. Thus future research is needed to investigate ecological effects of these HPCPs on benthic organisms in sediment of rural rivers receiving untreated wastewater discharge.

Brazilin plays an anti-inflammatory role with regulating Toll-like receptor 2 and TLR 2 downstream pathways in Staphylococcus aureus-induced mastitis in mice.

Mastitis, which commonly occurs during the postpartum period, is caused by the infection of the mammary glands. The most common infectious bacterial pathogen of mastitis is Staphylococcus aureus (S. aureus) in both human and animals. Brazilin, a compound isolated from the traditional herbal medicine Caesalpinia sappan L., has been shown to exhibit multiple biological properties. The present study was performed to determine the effect of brazilin on the inflammatory response in the mouse model of S. aureus mastitis and to confirm the mechanism of action involved. Brazilin treatment was applied in both a mouse model and cells. After brazilin treatment of cells, Western blotting and qPCR were performed to detect the protein levels and mRNA levels, respectively. Brazilin treatment significantly attenuated inflammatory cell infiltration and inhibited the expressions of TNF-α, IL-1ß and IL-6 in a dose-dependent manner. Administration of brazilin in mice suppressed S. aureus-induced inflammatory injury and the production of proinflammatory mediators. This suppression was achieved by reducing the increased expression of TLR2 and regulating the NF-κB and MAPK signaling pathways in the mammary gland tissues and cells with S. aureus-induced mastitis. These results suggest that brazilin appears to be an effective drug for the treatment of mastitis and may be applied as a clinical therapy.

Bergenin Plays an Anti-Inflammatory Role via the Modulation of MAPK and NF-κB Signaling Pathways in a Mouse Model of LPS-Induced Mastitis.

Mastitis is a major disease in humans and other animals and is characterized by mammary gland inflammation. It is a major disease of the dairy industry. Bergenin is an active constituent of the plants of genus Bergenia. Research indicates that bergenin has multiple biological activities, including anti-inflammatory and immunomodulatory properties. The objective of this study was to evaluate the protective effects and mechanism of bergenin on the mammary glands during lipopolysaccharide (LPS)-induced mastitis. In this study, mice were treated with LPS to induce mammary gland mastitis as a model for the disease. Bergenin treatment was initiated after LPS stimulation for 24 h. The results indicated that bergenin attenuated inflammatory cell infiltration and decreased the concentration of NO, TNF-α, IL-1ß, and IL-6, which were increased in LPS-induced mouse mastitis. Furthermore, bergenin downregulated the phosphorylation of nuclear factor-kappaB (NF-κB) and mitogen-activated protein kinases (MAPK) signaling pathway proteins in mammary glands with mastitis. In conclusion, bergenin reduced the expression of NO, TNF-α, IL-1ß, and IL-6 proinflammatory cytokines by inhibiting the activation of the NF-κB and MAPKs signaling pathways, and it may represent a novel treatment strategy for mastitis.

hARIP2 is a putative growth-promoting factor involved in human colon tumorigenesis.

Activin is a multifunctional growth and differentiation factor of the growth factor-beta (TGF-ß) superfamily, which inhibits the proliferation of colon cancer cells. It induces phosphorylation of intracellular signaling molecules (Smads) by interacting with its type I and type II receptors. Previous studies showed that human activin receptor-interacting protein 2 (hARIP2) can reduce activin signaling by interacting with activin type II receptors; however, the activity of hARIP2 in colon cancer has yet to be detailed. In vitro, overexpression of hARIP2 reduced activin-induced transcriptional activity and enhanced cell proliferation and colony formation in human colon cancer HCT8 cells and SW620 cells. Also, hARIP2 promoted colon cancer cell apoptosis, suggesting that a vital role in the initial stage of colon carcinogenesis. In vivo, immunohistochemistry revealed that hARIP2 was expressed more frequently and much more intensely in malignant colon tissues than in controls. These results indicate that hARIP2 is involved in human colon tumorigenesis and could be a predictive maker for colon carcinoma aggressiveness.

Construction of novel chloroplast expression vector and development of an efficient transformation system for the diatom Phaeodactylum tricornutum.

Plastids are ideal subcellular hosts for the expression of transgenes and have been successfully used for the production of different biopolymers, therapeutic proteins and industrial enzymes. Phaeodactylum tricornutum is a widely used aquatic feed species. In this study, we focused on developing a high-efficiency plastid expression system for P. tricornutum. In the plastid transformation vector, the site selected for integration was the transcriptionally active intergenic region present between the trnI and trnA genes, located in the IR (inverted repeat) regions of the plastid genome. Initially, a CAT reporter gene (encoding chloramphenicol acetyltransferase) was integrated at this site in the plastid genome. The expression of CAT in the transformed microalgae conferred resistance to the antibiotic chloramphenicol, which enabled growth in the selection media. Overall, the plastid transformation efficiency was found to be approximately one transplastomic colony per 1,000 microalgae cells. Subsequently, a heterologous gene expression cassette for high-level expression of the target gene was created and cloned between the homologous recombination elements. A TA cloning strategy based on the designed XcmI-XcmI sites could conveniently clone the heterologous gene. An eGFP (green fluorescent protein) reporter gene was used to test the expression level in the plastid system. The relatively high-level expression of eGFP without codon optimisation in stably transformed microalgae was determined to account for 0.12 % of the total soluble protein. Thus, this study presents the first and convenient plastid gene expression system for diatoms and represents an interesting tool to study diatom plastids.

Gene expression profiles in zebrafish (Danio rerio) liver after acute exposure to okadaic acid.

Okadaic acid (OA), a main component of diarrheic shellfish poisoning (DSP) toxins, is a strong and specific inhibitor of the serine/threonine protein phosphatases PP1 and PP2A. However, not all of the OA-induced effects can be explained by this phosphatase inhibition, and controversial results on OA are increasing. To provide clues on potential mechanisms of OA other than phosphatase inhibition, here, acute toxicity of OA was evaluated in zebrafish, and changes in gene expression in zebrafish liver tissues upon exposure to OA were observed by microarray. The i.p. ED50 (6 h) of OA on zebrafish was 1.54 µg OA/g body weight (bw). Among the genes analyzed on the zebrafish array, 55 genes were significantly up-regulated and 36 down-regulated in the fish liver tissue upon exposure to 0.176 µg OA/g bw (low-dose group, LD) compared with the low ethanol control (LE). However, there were no obvious functional clusters for them. On the contrary, fish exposure to 1.760 µg OA/g bw (high-dose group, HD) yielded a great number of differential expressed genes (700 up and 285 down) compared with high ethanol control (HE), which clustered in several functional terms such as p53 signaling pathway, Wnt signaling pathway, glutathione metabolism and protein processing in endoplasmic reticulum, etc. These genes were involved in protein phosphatase activity, translation factor activity, heat shock protein binding, as well as transmembrane transporter activity. Our findings may give some useful information on the pathways of OA-induced injury in fish.

Construction and immunogenicity of a DNA vaccine containing clumping factor A of Staphylococcus aureus and bovine IL18.

Selection of potent cytokine adjuvants is important for the development of Staphylococcus aureus DNA vaccines. Several potential cytokines have been proven to induce enhanced immune responses in animal models and clinical tests. There is still no reported use of IL18 as an adjuvant to design DNA vaccines against S. aureus. In this study, we cloned the main fibronectin binding protein gene (a fragment from clumping factor A, ClfA(221-550)) of S. aureus and bovine interleukin 18 (bIL18). Then recombinant plasmids were constructed based on the eukaryotic expression vector pVAX1 with or without bIL18. Indirect immunofluorescence assays in transfected HeLa cells indicated that the recombinant DNAs (rDNAs) could be expressed correctly and had antigenicity. BALB/c mice were used as experimental models to examine the immunogenicity of rDNAs in vivo. The ClfA(221-550) rDNA provoked antibody production. The bIL18 rDNA induced production of the Th1 type cytokines IL2 and IFNgamma, and ClfA(221-550) and bIL18 synergistically stimulated T-lymphocyte proliferation. The data demonstrated that bIL18 is a potent adjuvant that could be used to enhance cellular immunity.

[Cloning of alpha-bungarotoxin gene and its prokaryotic expression as a non-fusion protein].

On the basis of the reported amino acid sequence of alpha-bungarotoxin (alpha-BGT), DNA sequence of alpha-BGT was deduced and fourteen partially complementary oligonucleotides were designed and synthesized. A plasmid carrying the coding region of alpha-BGT was obtained by primer extension, PCR and ligation with pMD-18-T. The target fragment was digested with Xba I and EcoR I, recovered and ligated with pET28a(+). The resultant expression vector was transformed into BL21 (DE3), BL21 (DE3) Codon plus, and BL21 (DE3) plysS, respectively. Recombinant alpha-BGT was expressed in BL21 (DE3) and was analyzed by 15% Tris/tricine SDS-PAGE. The result showed that the recombinant protein, mostly found in inclusion bodies, accounted for 11.98% of the total bacterial lysate. The expression capacity could be increased to 16.28% by optimizing expression conditions. Western blotting results showed that the expressed protein had similar immunogenicity with the natural alpha-BGT protein purified from the venom of Krait Bungarus spp. In vivo toxicity assay of purified and renatured proteins in mice showed that LD50 was about 1.28 microg/g.