Expression of nitric oxide synthase in the developing eye of Ze-brafish Danio rerio

1 INTRODUCTION Nitric oxide (NO), a highly free radical gas, is recently discovered as messenger molecule involved in different cellular and developmental events including cell proliferation, differentiation and apoptosis as well as neural pathfinding and synapse formation. NO is synthesized from amino acid L-arginine by catalytic action of a family of isoen-zymes called nitric oxide synthases (NOS) (Palmer, 1988). NOS require nicotinamide adenine dinucleo-tide phosphate (NADPH) as a…     

Antiangiogenic activity of low-temperature lysozyme from a marine bacterium <Emphasis Type="Italic">in vivo</Emphasis> and <Emphasis Type="Italic">in vitro</Emphasis>

We extracted marine low-temperature lysozyme (MLTL), a novel lysozyme, from a marine microorganism through fermentation. Our previous study suggested that a low molecular weight (16 kDa) may exert anti-tumor activity through antiangiogenesis. In this study, we extracted a high weight (39 kDa) and investigated its antiangiogenic activity in vivo and in vitro. Using zebrafish embryos as an in vivo study model, we found that treatment with MLTL significantly inhibited the growth of subintestinal vessels (SIVs) in a dose-dependent manner and that 400 μg/ml MLTL was sufficient to block the growth of SIVs. An in vitro study conducted using human umbilical vein endothelial cells (HUVECs) revealed that MLTL suppressed the proliferation, migration and tube formation of HUVECs in a dose-dependent manner. Interestingly, assays by flow cytometry and DNA electrophoresis indicated that MLTL was able to induce apoptosis of HUVECs. Moreover, further study demonstrated that the disruption of intracellular Ca²⁺ homeostasis may play an important role in MLTL induced apoptosis of HUVECs. Taken together, the results of this study demonstrate for the first time that MLTL inhibits angiogenesis through its pleiotropic effects on vascular endothelial cells and induces apoptosis through regulation of cellular Ca²⁺ levels. The results of this study also revealed a possible mechanism underlying the antiangiogenic effect of MLTL and suggested that MLTL may be a promising new antiangiogenic agent for use in cancer therapy.     

The antitumor effect of bromophenol derivatives <Emphasis Type="Italic">in vitro</Emphasis> and <Emphasis Type="Italic">Leathesia nana</Emphasis> extract <Emphasis Type="Italic">in vivo</Emphasis>

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 MTT 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 bromophenol 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. Supported by the National High Technology Research and Development Program of China (863 Program, No. 2007AA09Z410) and Knowledge Innovation Program of Chinese Academy of Sciences (No. KZCX2-YW-209)     

<Emphasis Type="Italic">GST</Emphasis> (<Emphasis Type="Italic">phi</Emphasis>) gene from Macrophyte <Emphasis Type="Italic">Lemna minor</Emphasis> is involved in cadmium exposure responses

Reactive oxygen species (ROS) scavengers, including ascorbate peroxidase, superoxide dismutase, catalase and peroxidase, are the most commonly used biomarkers in assessing an organisms’ response to many biotic and abiotic stresses. In this study, we cloned an 866 bp GST (phi) gene in Lemna minor and investigated its characteristics, expression and enzymatic activities under 75 μmol/L cadmium concentrations in comparison with other ROS scavengers. GST (phi) gene expression patterns were similar to those of other scavengers of ROS. This suggests that GST (phi) might be involved in responding to heavy metal (cadmium) stress and that its expression level could be used as a bio-indicator in monitoring cadmium pollution.     

Variations of ion-transport enzyme activities during early development of the shrimps <Emphasis Type="Italic">Fenneropenaeus chinensis</Emphasis> and <Emphasis Type="Italic">Marsupenaeus japonicus</Emphasis>

The ion-transport enzyme activities were studied in the nauplii 1–2, zoea 1–3, mysis 1–3, and postlarva 1–7 of the shrimps Marsupenaeus japonicus and Fenneropenaeus chinensis. The results showed that total ATPase, Na⁺-K⁺-ATPase, and V-ATPase (V-type H⁺-ATPase) activities increased during the early development for both the species, from zero in nauplii to stable levels after zoea stage. The enzyme activities of the latter species were significantly higher than those of the former species after zoea stage (F>F _0.05). The contributions of Na⁺-K⁺-ATPase, V-ATPase and HCO₃ ⁻-ATPase to the total ATPase activity of the two species varied in different developmental stages and accounted for 40%–70%, 22%–46% and 2%–13% in M. japonicus from zoea to postlarva stage, whilst the shares of them were 42%–69%, 28%–44% and 2.5%–22%, respectively in F.chinensis. These findings suggest the possible culture of the two species with varying water quality, especially the salinity and pH.     

Axenic culture of free-living conchocelis of<Emphasis Type="Italic">Porphyra yezoensis</Emphasis> and<Emphasis Type="Italic">Porphyra haitanensis</Emphasis>

After discarding marine microorganisms from conchocelis of Porphyra yezoensis and Porphyra ha/tanens/s, their axenic cultures were obtained through treatment with antibiotics. Antibiotic disc tests were carried out to determine the effectiveness of each antibiotic in eliminating contaminating microorganisms. Five of 12 antibiotics tested were selected and used to produce the axenic cultures in this study, which showed that 200 μg/mL streptomycin, 250 μg/mL penicillin, 252 μg/mL kanamycin, 30 μg/mL neomycin, 200 μg/mL chloramphenicol were effective concentrations for eliminating microorganisms from conchocelis when antibiotics were added singly step by step; whereas simultaneous combination of 150 μg/mL streptomycin, 250 (or 350) μg/mL penicillin, 150 (or 250) μg/mL kanamycin, 70 μg/mL neomycin and 200 μg/mL chloramphenicol was also effective for producing the axenic cultures. However,it seemed that the treatments with antibiotics applied individually were more feasible than those with all antibiotics added at the same time. This may be due to the combined inhibiting effect of antibiotics on the growth and development of conchocelis.     

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.     

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)     

Fatty acid composition of <Emphasis Type="Italic">Euphausia superba</Emphasis>, <Emphasis Type="Italic">Thysanoessa macrura</Emphasis> and <Emphasis Type="Italic">Euphausia crystallorophias</Emphasis> collected from Prydz Bay,Antarctica

The information of trophic relationship is important for studying the Southern Ocean ecosystems. In this study, three dominant krill species, Euphausia superba, Thysanoessa macrura and Euphausia crystallorophias, were collected from Prydz Bay, Antarctica, during austral summer of 2009/2010. The composition of fatty acids in these species was studied. E. superba and T. macrura showed a similar fatty acid composition which was dominated by C14:0, C16:0, EPA (eicosapentenoic acid) and DHA (decosahexenoic acid) while E. crystallorophias showed higher contents of C18:1(n-9), C18:1(n-7), DHA and EPA than the former two. Higher fatty acid ratios of C18:1(n-9)/18:1(n-7), PUFA (polyunsaturated fatty acid)/SFA (saturated fatty acid), and 18PUFA/16PUFA indicated that E. crystallorophias should be classified as a typical omnivore with a higher trophic position compared with E. superba and T. macrura.     

Biodegradation of <Emphasis Type="Italic">Enteromorpha</Emphasis> polysaccharides by intestinal micro-community from <Emphasis Type="Italic">Siganus oramin</Emphasis>

Micro-communities are supposed to have more potential functions of biodegradation of polysaccharides than single strain; however, the intestinal micro-communities involved in the biodegradation of Enteromorpha polysaccharides (EP) were seldom reported. In order to obtain the EP-degrading micro-community, the intestines of Siganus oramin was obtained to isolate the micro-communities, which were enriched by 0.3% of EP as the sole carbon source. A stable micro-community with EP degradative capability was achieved after seven generations of subculture, named H1. Results showed that H1 was able to degrade 75% of EP within 24 hours, and the activity of EP lyases reached 500 U mL^?1 in 32 hours. With denaturing gradient gel electrophoresis (DGGE) and 16S rRNA gene clone library analysis, ten bacteria closely related to Marinomonas pontica, Microbacterium sp., Leucobacter chironomi, Cyclobacterium sp., Algoriphagus winogradskyi, Pseudoalteromonas sp. and Vibrio sp. were determined. Furthermore, compared with the DGGE bands sequence and the clone library analysis, the dominant bacteria of the EP-biodegrading micro- community were Pseudoalteromonas sp. and Vibrio sp., with the respective proportion of 38% and 46%, and they should play an important role in EP degradation together with other degrading bacteria in the micro-community H1.     

Genome size of <Emphasis Type="Italic">Alexandrium catenella</Emphasis> and <Emphasis Type="Italic">Gracilariopsis lemaneiformis</Emphasis> estimated by flow cytometry

Flow cytometry(FCM) technique has been widely applied to estimating the genome size of various higher plants. However, there is few report about its application in algae. In this study, an optimized procedure of FCM was exploited to estimate the genome size of two eukaryotic algae. For analyzing Alexandrium catenella, an important red tide species, the whole cell instead of isolated nucleus was studied, and chicken erythrocytes were used as an internal reference. The genome size of A. catenella was estimated to be 56.48 ± 4.14 Gb(1C), approximately nineteen times larger than that of human genome. For analyzing Gracilariopsis lemaneiformis, an important economical red alga, the purified nucleus was employed, and Arabidopsis thaliana and Chondrus crispus were used as internal references, respectively. The genome size of Gp. lemaneiformis was 97.35 ± 2.58 Mb(1C) and 112.73 ± 14.00 Mb(1C), respectively, depending on the different internal references. The results of this research will promote the related studies on the genomics and evolution of these two species.     

Tyrosinase, a new innate humoral immune parameter in large yellow croaker （Pseudosciaena crocea R）

We evaluated the immune response to infection with a pathogen in large yellow croaker(Pseudosciaena crocea Richardson).The fish were given an intraperitoneal(i.p.) injection of Vibrio parahaemolyticus or sterile sea water(control).We collected blood sera from the fish 0.17,1,2,4,8,12,or 16 d after injection(dpi).We measured tyrosinase activity and the concentrations of lysozyme,NOS,and antibodies.Serum tyrosinase activity was significantly higher at 0.17 and 4 dpi than in the control group,and peaked at 8 d...     

<Emphasis Type="Italic">Skeletonema</Emphasis> cf. <Emphasis Type="Italic">costatum</Emphasis> biogenic silica production rate determinated by PDMPO method

Diatoms are the only ecological phytoplankton that require silicic acid for growth. They are also the dominant contributor of ocean’s total primary productivity. Generation and circulation with silica walls, which the siliceous organisms form, is an important component of the marine biological pump. It is crucial to the study of the operational mechanisms of biological pump with different sea areas. Moreover, it is the key link to the study of global silicon cycle. This paper introduces the basic mechanism of the formation of diatom silica walls and a new way of researching silicic acid metabolism, namely the 2-(4-pyridyl)-5-((4-(2-dimethylaminoethylaminocarbamoyl)- methoxy)phenyl)oxazole (PDMPO) dyeing method. Under a fluorescence microscope after excitation with bright green fluorescence, it can combine with silicic acid to form a complex into the Si deposition within diatom cells. The advantage of this method is that it can monitor the metabolism of silicate after adding PDMPO. For experimentation and sample collection in each of the specified time points, samples were determinated through the unutilized silicic acid, silica dissoluble intracellular and Si deposition within diatom cells, not only using hot alkaline digestions method but also PDMPO dyeing method. Results showed a good linear relationship between PDMPO fluorescent value and biogenic silica concentration. It was also indicated that PDMPO had no deleterious impact on Skeletonema cf. costatum growth for 34 h and was useful for tracking newly-deposited biogenic silica in diatoms’ frustules.     

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.     

Lipid and fatty acid compositions of cod (<Emphasis Type="Italic">Gadus morhua</Emphasis>), haddock (<Emphasis Type="Italic">Melanogrammus aeglefinus</Emphasis>) and halibut (<Emphasis Type="Italic">Hippoglossus hippoglossus</Emphasis>)

This study was conducted to compare lipid and fatty acid composition of cod, haddock and halibut. Three groups of cod (276 g ± 61 g), haddock (538 g ± 83 g) and halibut (3704 g ± 221 g) were maintained with commercial feeds mainly based on fish meal and marine fish oil for 12 weeks prior to sampling. The fatty acid compositions of muscle and liver were determined by GC/FID after derivatization of extracted lipids into fatty acid methyl esters (FAME). Lipids were also fractionated into neutral and polar lipids using Waters silica Sep-Pak?. The phospholipid fraction was further separated by high-performance thin-layer chromatography (HPTLC) and the FAME profile was obtained. Results of the present study showed that cod and haddock were lean fish and their total muscle lipid contents were 0.8% and 0.7%, respectively, with phospholipid constituting 83.6% and 87.5% of the total muscle lipid, respectively. Halibut was a medium-fat fish and its muscle lipid content was 8%, with 84% of the total muscle lipid being neutral lipid. Total liver lipid contents of cod, haddock and halibut were 36.9%, 67.2% and 30.7%, respectively, of which the neutral lipids accounted for the major fraction (88.1%–97.1%). Polyunsaturated fatty acids were the most abundant in cod and haddock muscle neutral lipid. Monounsaturated fatty acid level was the highest in halibut muscle neutral lipid. Fatty acid compositions of phospholipid were relatively constant. In summary, the liver of cod and haddock as lean fish was the main lipid reserve organ, and structural phospholipid is the major lipid form in flesh. However, as a medium-fat fish, halibut stored lipid in both their liver and muscle.     

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.     

Variation in the immunoglobulin levels in turbot （Scophthalmus maximus） after vaccination with Streptococcus iniae

A pathogenic bacterium(S636),identified as Streptococcus iniae,was isolated from turbot(Scophthalmus maximus) in 2005.We immunized turbot with formalin-killed S.iniae four times(on days 1,14,21,and 28) by intraperitoneal inoculation.After each vaccination,we obtained serum samples and isolated the lymphocytes from the peripheral blood,spleen,pronephros,and mesonephros.We measured surface Ig-positive(sIg+) lymphocytes and serum antibody levels from these organs using flow cytometry and enzyme-linked immunoso...     

Metabolism of pectenotoxins in brown crabs <Emphasis Type="Italic">Cancer pagurus</Emphasis> fed with blue mussels <Emphasis Type="Italic">Mytilus edulis</Emphasis>

We investigated the metabolism of pectenotoxins in brown crabs(Cancer pagurus).The crabs were fed with blue mussels(Mytilus edulis) for 21 d then depurated for 42 d.We extracted the toxins from the digestive glands of contaminated crabs,uncontaminated crabs(control group),and the meat of blue mussels using methanol.Extracts of the crab digestive glands were fractionated by liquid-liquid partitioning and solid phase extraction.The fractions were analyzed by liquid chromatography coupled with tandem mass spec...