The antitumor effect of bromophenol derivatives in vitro and Leathesia nana extract in vivo

To investigate the antitumor effect of bromophenol derivatives in vitro and Leathesia nana extract in vivo, six bromophenol derivatives 6-(2,3-dibromo-4,5-dihydroxybenzyl)-2,3-dibromo-4,5-dihydroxy benzyl methyl ether (1), (+)-3-(2,3-dibromo-4,5-dihydroxyphenyl)-4-bromo-5,6-dihydroxy-1,3-dihydroisobenzofuran (2), 3-bromo-4-(2,3-dibromo-4,5-dihydroxybenzyl)-5-methoxymethyl-pyrocatechol (3),2,2',3,3'-tetrabromo-4,4',5,5'-tetrahydroxy-diphenylmethane (4), bis(2,3-dibromo-4,5-dihydroxybenzyl) ether (5), 2,2',3-tribromo-3',4,4',5-tetrahydroxy-6'-ethyloxymethyldiphenylmethane (6) were isolated from brown alga Leathesia nana, and their cytotoxicity were tested by MTr assays in human cancer cell lines A549, BGC-823, MCF-7, B16-BL6, HT-1080, A2780, Bel7402 and HCT-8. Their inhibitory activity against protein tyrosine kinase (PTK) with over-expression of c-kit was analyzed also by ELISA. The antitumor activity of ethanolic extraction of Leathesia nana (EELN) was evaluated on S180-bearing mice.All compounds showed very potent cytotoxicity against all of the eight cancer cell lines with IC50 below 10 μg/mL. In PTK inhibition study, all bromophenol derivatives showed moderate inhibitory activity and compounds 2, 5 and 6 showed significant bioactivity with the inhibition ratio of 77.5%, 80.1% and 71.4%,respectively. Pharmacological studies reveal that EELN could inhibit the growth of Sarcoma 180 tumor and increase the indices of thymus and spleen to improve the immune system remarkably in vivo. Results indicated that the bromophenoi derivatives and EELN can be used as potent antitumor agents for PTK over-expression of c-kit and considered in a new therapeutic strategy for treatment of cancer.     

Synthesis and protein tyrosine phosphatase 1B inhibition activities of two new synthetic bromophenols and their methoxy derivatives

3-bromo-4,5-bis(2,3-dibromo-4,5-dihydroxybenzyl)-1,2-benzenediol (1) is a natural bromophenol isolated from the red algae Rhodomela confervoides that exhibits significant inhibition against protein tyrosine phosphatase 1B (PTP1B). Based on its activity, we synthesized two new synthetic bromophenols and their methoxy derivatives from vanillin using the structure of natural bromophenol 1 as a scaffold. The structures of these bromophenols were elucidated from 1H NMR, 13C NMR, and high resolution electron ionization mass spectrometry as 2,3-dibromo-1-(2p-bromo-6p-(3q,4q-dimethoxybenzyl)- 3p,4p-dimethoxybenzyl)-4,5-dimethoxybenzene(2),2,3-dibromo-1-(2p-bromo-6p-(2q-bromo-4q,5q-dimethoxy-benzyl)-3p,4p-dimethoxybenzyl)-4,5-dimethoxybenzene(3),3,4-dibromo-5-(2p-bromo-6p-(2q-bromo-4q,5q-dihydroxybenzyl)-3p,4p-dihydroxybenzyl)pyrocatechol(4)and 3,4-dibromo-5-(2p-bromo-6p-(3q,4q-dihydroxybenzyl)-3p,4p-dihydroxybenzyl)pyrocatechol (5).PTP1B inhibition activities of these compounds were evaluated using a colorimetric assay,and compounds 3 and 4 demonstrated interesting activity against PTP1B.     

Chemical constituents of marine medicinal mangrove plant <Emphasis Type="Italic">Sonneratia caseolaris</Emphasis>

Twenty-four compounds including eight steroids (1–8), nine triterpenoids (9–16, 24), three flavonoids (20–22), and four benzenecarboxylic derivatives (17–19, 23) were isolated and identified from stems and twigs of medicinal mangrove plant Sonneratia caseolaris. The structures of the isolated compounds were determined by extensive analysis of their spectroscopic data. Among these metabolites, compounds 1, 4–20 and 22–24 were isolated and identified for the first time from S. caseolaris. In the in vitro cytotoxic assay against SMMC-7721 human hepatoma cells, compound 21 (3′,4′,5,7-tetrahydroxyflavone) exhibited significant activity with IC₅₀ 2.8 μg/mL, while oleanolic acid (14), 3,3′-di-O-methyl ether ellagic acid (18), and 3,3′,4-O-tri-O-methyl ether ellagic acid (19) showed weak activity. None of these compounds displayed significant antibacterial activites. Supported by the National Natural Science Foundation of China (No. 30770234); Knowledge Innovation Program of Chinese Academy of Sciences (KZCX2-YW-211-04); Department of Science and Technology of Shandong Province (No.2006GG2205023)     

Bioaccumulation of Pb<Superscript>2+</Superscript> and its effects on growth,morphology and pigment contents of <Emphasis Type="Italic">Spirulina</Emphasis> (<Emphasis Type="Italic">Arthrospira</Emphasis>) <Emphasis Type="Italic">platensis</Emphasis>

A laboratory experiment was conducted to assess the bioaccumulation of Pb²⁺ and its effects on growth, morphology and pigment contents of Spirulina (Arthrospira) platensis. The specimen cultured in Zarrouk liquid medium was treated with various initial metal concentrations (0, 5, 10, 30, 50 and 100 μg mL⁻¹). The growth of S. platensis was adversely affected by Pb²⁺ at high concentrations (30, 50 and 100 μg mL⁻¹). However, at low concentrations (5 μg mL⁻¹), Pb²⁺ could stimulate its growth slightly. The pigment contents (chlorophyll α and β carotene) were decreased in a dose-dependent manner. The highest reductions (67% and 53% respectively in chlorophyll α and β carotene) were observed in 100 μg mL⁻¹ treatment group. The LC₅₀ (96 h) of Pb²⁺ was measured as 75.34 μg mL⁻¹. Apart from a few cases of filament breakages at elevated concentrations (50 and 100 μg mL⁻¹), morphological abnormalities are not specific. Metal bioaccumulation increased with Pb²⁺ concentrations, but decreased with exposure time. The maximum accumulated amount was 188 mg g⁻¹ dry weight. The bioconcentration factor (BCF) reached to a peak at day 2, followed by a gradual reduction for all the exposure concentrations. S. platensis is able to tolerate considerably high Pb²⁺ concentrations. Consequently it can be used as a potential species to remove heavy metal from contaminated waters.     

Synthesis of three bromophenols from red algae as PTP1B inhibitors

Bromophenols are a set of natural products widely distributed in seaweed, most of which exhibit interesting and useful biological activities. To develop a reliable and efficient synthetic route to these natural bromophenols, three of them, 3,4-dibromo-5-(2′-bromo-3′,4′-dihydroxy-6′-methoxymethyl-benzyl)-benzene-1,2-diol (compound 9), 3,4-dibromo-5-(2′-bromo-6′-ethoxy methyl-3′,4′-dihydroxybenzyl)-benzene-1,2-diol (compound 10), and 3-bromo-4-(3′-bromo-4′,5′-dihydroxy benzyl)-5-(ethoxymethyl)-benzene-1,2-diol (compound 14), isolated from red marine algae, have been synthesized in eight steps with an overall yield of 14.4%, 14.4%, and 18.2% respectively, via a practical approach employing bromination, Wolff-Kishner-Huang reduction and a Friedel-Crafts reaction as key steps. The protein tyrosine phosphatase 1B (PTP1B) inhibitory activities of the synthetic compounds were evaluated by the colorimetric assay. The results show that these compounds are moderate PTP1B inhibitors. The synthesis of these bromophenol derivatives makes in vivo studies of their structure-activity relationships and inhibition activity against PTP1B possible.     

Synedra ulna var. repanda,a new variety of Synedra （Bacillariophyta） from Xinjiang,China

Synedra ulna var, repanda Q. X. Wang ＆ Q. M. You, a new variety of Synedra （Bacillariophyta） from Xinjiang, China, is described and illustrated, and the characteristic of the variety： includes undulate-linear valves and straight pseudoraphe, differs from other species of Synedra.     

Competition Among Dinoflagellate Alexandrium Tam—arense, Raphidophyte Heterosigma Carterae and Diatom Skeletonema Costatum under Combinations ofTwo Temperatures and Five Salinities

Competition among HAB (Harmful Algal Bloom) species Dinoflagellate Alexandrium tamarense, Raphidophyte Heterosigma carterae, and Diatom Skeletonema costatum was studied in the laboratory. Experiments with these three major HAB species under combinations of different salinities (10, 18, 25, 30, 35) and temperatures (19℃, 25℃) were carried out. The results showed that S. costatum successfully competed with the other two species at salinities of 18, 25,30, and 35 at temperatures of 19℃ and 25℃. However, H. carterae showed its advantage at low salinity of 10 and became the single dominant species at salinity 10 and 25℃. A. tamarense could not compete successfully with the other two species especially at low salinities. However, it could remain at low density in the presence of higher densities of other algae.     

Steroids from green alga <Emphasis Type="Italic">Chaetomorpha basiretorsa</Emphasis> Setchell

Six steroids have been isolated from ethanolic extract of green alga Chaetomorpha basiretorsa Setchell by a combination of repeated normal phase silica gel and Sephadex LH-20 gel column chromatography as well as recrystallization. Using spectroscopic methods including MS and NMR, their structures were determined as β-1awsaritol （1）, saringosterol （2）, 24-hydroperoxy-24-vinyl- cholesterol （3）, β-stigmasterol （4）, stigmast-4-en-3α, 6β-diol （5）, 29-hydroxystigmasta-5, 24 （28）-dien-3β-ol （6）. All these compounds were obtained from this genus for the first time and they were inactive （IC50〉10μg/ml） against KB, Bel-7402, PC-3M, Ketr 3 and MCF-7 cell lines.     

In vitro acute cytotoxicity of neonicotinoid insecticide imidacloprid to gill cell line of flounder Paralichthy olivaceus

In vitro acute cytotoxicity of neonicotinoid insecticide imidacloprid (IMI) to the gill cell line of flounder (FG) that collected in the gill of Paralichthys olivaceus, was examined by 3 widely used endpoint bioassays: NR (neutral red), MTT (3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide) and TCP (total cell protein). The result shows that the IMI increased at concentrations ≥0.5 μg/ml. The IC50 value of NR, MTT, and TCP was 41.86, 38.46, and 39.08 μg/ml, respectively. The ultrastructural observation revealed that the mitochondria of the cells exposed to 60 μg/ml IMI for 48 h were severely damaged, swollen or disrupted, while their nuclei and rough endoplasmic reticulum (RER) remained normal. This would suggest that the mitochondria are probably the primary target of IMI.     

Effect of lead on cytoskeletal protein stability in crucian carp <Emphasis Type="Italic">Carassius auratus</Emphasis>

Inorganic lead （Pb） is one of the most common environmental pollutants. Much evidence indicates that Pb exposure could directly affect fish growth and development. In this study, we investigated the cytotoxic effects of Pb on cytoskeletal protein stability at both protein and mRNA level in crucian carp Carassius auratus. Pb（NO3）2 treatment in concentration of 100 μmol/L resulted in decreased expression of both α- and β-tubulin but γ-tubulin as assayed with SDS-PAGE, Western Blot, and ELISA. In vivo and in vitro analyses on protein expression of tubulins are consistent. The effect of Pb on mRNA expression varied among different tissues. Our results suggest that cytotoxicity of Pb at protein translation level is stronger than at mRNA expression level.     

Vaccination in three different ways against vibriosis of <Emphasis Type="Italic">Seriola dumerili</Emphasis> caused by <Emphasis Type="Italic">Vibrio hollisae</Emphasis>

Bacterin was prepared by formalin-inactivating the virulent strain of Vibrio hollisae isolated from diseased Seriola dumerili （amberjack） suffering from vibriosis. Healthy S. dumeriIi were vaccinated by respective procedures of intramuscular injection, immersion, and orally administration. Results of the three different vaccinations were compared. Blood was drawn from the vaccinated fish every 7 days, and the antibody titers and lysozyme activities of the sera were determined. The antibody titer of injected fish was 1：40 at 7 d, and reached its peak of 1：320 at 28 d, while the fish vaccinated by immersion and orally administration exhibited weak antibody responses, the antibody titres of 〈1：10, 1：20, 1：160 were observed at 7 d, 14 d, 35 d respectively. Compared with the control, the vaccinated fish exhibited significantly higher lysozyme activities （P〈0.05）. Upon challenge with virulent strain, the relative percent survival （RPS） of injected, immersed and oral administrated fish were 75%, 45%, and 40% respectively, and the injected fish showed significantly higher RPS than immersed and oral administrated fish. The results suggested that vaccination of S. dumerili by the injection would be the best strategy to prevent the vibriosis in S. dumerili farm.     

Assessment of nitrogen maintenance requirement in juvenile Black Porgy <Emphasis Type="Italic">Acanthopagrus schlegeli</Emphasis>

Nitrogen balance method and nitrogen-free diet were used in this study to determine nitrogen maintenance requirement (NM) and nitrogen maintenance requirement per unit metabolism body weight (NM′) of black porgy Acanthopagrus schlegeli. Fish with body weight (BW) of 50, 80, 120, 160 and 200 g were fed by the diets containing three graded levels of crude protein (380, 420 and 460 g/kg). The results from nitrogen balance experiment showed that the amount of nitrogen deposition varied from 0.15 to 0.31 mg/g BW per day, accounting for 12.2% to 21.1% of nitrogen intake. The amount of fecal nitrogen excretion varied from 0.21 to 0.32 mg/g BW per day, accounting for 16.3% to 21.6% of nitrogen intake. The endogenous nitrogen excretion, a main part of nitrogen consumption varied from 0.79 to 0.97 mg/g BW per day, accounting for 63.3 % to 68.0% of nitrogen intake. Positive correlation was found between NM and body weight, while a negative correlation was found between NM of unit body weight and the growth duration. No significant differences (p>0.05) were found in NM′ among different growth stages. The average of NM′ was 0.485 7mg/g per day. The results from nitrogen-free diet experiment showed that a negative correlation between NM and feed intake of nitrogen-free diet. NM increased with the decrease of feed intake of fish. The average of NM was 0.482 9 mg/g BW per day that was close to 0.483 8 mg/g BW obtained from fish with 120 g BW in nitrogen balance experiment. The nitrogen balance method is recommended to be a better method for determining NM in consideration of fish stress and result stability. This study also provides a calculated result of the protein content in diets, which is necessary for maintaining fish body protein at different growth stages. The calculation was based on the amount of nitrogen required for maintaining body protein per kg BW. Supported by Scientific Research Project Grant (No.2004C100059) from the city government of Ningbo, China.     

Notes on Two Marine Ciliates from the Yellow Sea,China： Placus salinus and Strombidium apolatum（Protozoa, Ciliophora）

The living morphology and infraciliature of two rare marine ciliates, Placus salinus Dietz, 1964 and Strombidium apolatum Wilbert and Song, 2005, collected from the coastal waters near Qingdao, China, were investigated by in vivo observation and protargol impregnation technique. The improved diagnosis for Placus salinus is as follows: medium-sized marine Placus, in vivo (50–60)μm×(30–40)μm; cell elliptical to barrel-shaped; 28–31 somatic kineties; single macronucleus usually ellipsoid and one micronucleus located in the indention of the macronucleus; one contractile vacuole posteriorly positioned. Strombidium apolatum is characterized by: marine strombidium (40–60)μm×(30–45)μmm in vivo, cordiform in shape with somewhat pointed posterior end and conspicuous apical protrusion; extrusomes prominent, about 15μm in length and evenly arranged along the circle kinety; about 16 collar and 5–6 buccal membranelles; one elongate macronucleus and one micronucleus; circle and ventral kineties consisting of about 53 and 45 dikinetids respectively.     

Isolation and characterization of pathogenic <Emphasis Type="Italic">Listonella anguillarum</Emphasis> of diseased half-smooth tongue sole (<Emphasis Type="Italic">Cynoglossus semilaevis</Emphasis> Günther)

Studies were conducted to determine the cause of the acute mortality of half-smooth tongue sole Cynoglossus semilaevis Günther juveniles in a fish farm in Jimo, Shandong Province, China, in June 2006. Gross signs of the diseased tongue sole included several petechiae and ecchymoses on the body and fin necrosis and hemorrhagic lesion at the base of the fin. Bacteria were isolated from kidney, liver and hemorrhagic lesions of the diseased tongue sole. Among14 strains, SJ060621 was proved to be highly virulent to juvenile tongue sole with LD₅₀ value of <1.0×10⁵ colony forming units (CFU) mL⁻¹, while the remaining 13 were avirulent. Among the 16 antibiotics tested, SJ060621 was sensitive to gentamicin and nitrofurantoin. It was identified as Listonella anguillarum with conventional plate and tube tests in combination with API 20E analysis. 16S rRNA gene and partial HSP60 gene sequenceing analysis revealed that the strain was highly homologous with L. anguillarum. Examination of the infected musculature by electron microscopy indicated numerous bacteria and lots of macrophages containing phagocytosed bacteria. Histopathological investigations revealed severe necrotic degenerative changes in the infected organs. Indirect immunofluorescence assay (IFA) was employed to detect the location of occurrence of bacteria, and bacteria were found in aggregations in the inflammatory areas in musculature.     

Immune response of <Emphasis Type="Italic">Pseudosciaena crocea</Emphasis> to the injection of <Emphasis Type="Italic">Vibrio alginolyticus</Emphasis>

For the investigation of anti-infection immune response of Pseudosciaena crocea, 160 healthy fish samples were categorized into infected and control groups. Each individual fish in the infected group was injected intraperitoneally (i.p.) with 0.2 ml bacterial suspension of Vibrio alginolyticus in density of 2×10⁷ CFU/ml, while each individual in the control group was injected i.p. with 0.2 ml sterile saline solution (0.85%). It was observed that the artificial injection of V. alginolyticus significantly increased the number of erythrocytes, leucocytes, lymphocytes in peripheral blood as well as peripheral serum antibacterial activity and antibody titer of large yellow croaker, and significantly reduced the number of peripheral blood granulocytes as compared with those in the control group. No significant difference in acid phosphytase and superoxide dismutase activity of serum was detected between the two groups. It is suggested that non-specific immune factors including leucocytes and anti-bacteria substance in peripheral blood played important role at the initial stage of infection, and specific immune factors such as antibody then played important role in response to anti-infection at the latter stage. Supported by the National High Technology Research and Development Program of China (863 program, No. 2007AA09Z115) and Technology Program of Xiamen (No. 3502Z73019)     

CYTOLOGICAL COMPARISON OF GAMETOGENESIS OF SCALLOPS, ARGOPECTEN IRRADIANS AND CHLAMYS FARRERI

Histological characteristics of gametogenesis of two kinds of scallops, gonochoric Chinese scallop, Chlamys farreri and hermaphroditic bay scallop, Argopecten irradians were investigated in this study. Spermatogenesis in C. farreri has different developmental stages: spermatogonia, primary spermatocyte, second spermatocyte, spermatid and spermatozoon. A large number of same developmental stage spermatic cells converge at a definite area of the testis. Premeiotic, previtellogenic and vitellogenic oocytes can be found during oogenesis in C. farreri, where oocyte distribution is obviously irregular. The A. irradians gonad consists of two different parts in one individual: one part functions as testis, the other as ovary. Between these two parts is a special appearance area, where a large number of spermatic cells are bound with two layers of acellular substance with many oocytes in it.     

Isolation and pharmacological activities of bromophenols from Rhodomela confervoides

Four bromophenols were isolated from the extract of marine red alga,Rhodomela confervoides by column chromatography and HPLC methods. By means of spectroscopic methods inclding IR, MS, 1D, and 2D-NMR techniques, their structures were elucidated as (1) 3-bromo-4,5-dihydroxy benzoic acid methyl ester; (2) bis (2,3-dibromo-4,5-dihydroxybenzyl) ether; (3) 3,4-dibromo-5-(methoxymethyl)-1,2-benzenediol and (4) 3-bromo-4,5-dihydroxy-benzaldehyde. Compound 1 was first isolated from the algae in nature, and 1, 4 were found to have selective cytotoxic activities against KB, Bel 7402 and A549 cells, 2 showed powerful antibacterial activities againstStaphylococcus aurens andPseudomonas aeruginosa. Supported by National “863” Program (No. 2004AA625030, 2001AA620503), Quingdao marine sciences project (No. 04-2-NN-26) and Key Knowledge Innovative Project of the Chinese Academy of Sciences (KZCX3-SW-215).     

Acute Toxicity of Formalin to the Red-tide Dinoflageilate Prorocentrum minimum Schiller （Protozoa, Mastigophora）

The acute toxic levels of formalin to a marine red-tide dinoflagellate Prorocentrum minimum Schiller (Mastigophora, Dinoflagellida) were determined by linear regression analysis. The data obtained revealed that there is a close regression relationship between logarithmic concentrations of formalin and mortality probit scales of the organism when exposure times are 1, 2, 6, 12, 24 h. The median lethal concentrations (LC50 values) obtained from correlation analysis for these exposure times (with 95% confidence intervals), were 11.83, 6.76, 4.37, 4.27 and 3.98mgL^-1 respectively. A toxicity curve was obtained to connect the exposure time and LC50 value from the correlation between them. The results indicated that P. minimum could be killed or fixed by formalin in the concentration range of 4-12mgL^-1 within 1-24h.     

Genetic diversity analysis with ISSR PCR on green algae <Emphasis Type="Italic">Chlorella vulgaris</Emphasis> and <Emphasis Type="Italic">Chlorella pyrenoidosa</Emphasis>

In the present study, genetic polymorphism and diversity in unicellular clones of Chlorella vulgaris Beijerinck and Chlorella pyrenoidosa Chick were studied with Inter Simple Sequence Repeats PCR （ISSR PCR）. Samples including four clones of C. vulgaris and three clones of C. pyrenoidosa were purified by single-clone-choice method. For four C. vulgaris unicellular clones, the total number of the bands scored for 18 primers was 298; and the number of the polymorphic bands was 118, of which 39.6% were polymorphic. The size of PCR products ranged from 200 to 2 500 bp. The total number of bands scored for 18 primers, the number of polymorphic bands and the percentage of three C. pyrenoidosa unicellular clones was 194.83 and 30.8%, respectively. POPGENE analysis show that the average Nei genetic diversity （h^＊） and Shannon index of diversity （I^＊） in the four C. vulgaris unicellular clones was 0.2181 and 0.3208, respectively, which is slightly higher than those of the three C. pyrenoidosa unicellular clones （0.190 3 and 0.274 8）, which agreed with the percentage of polymorphic bands in the mixed samples of the two species. The results suggest that ISSR is a useful method to Chlorella for intra-species genetic analysis.