The structure of Aquifex aeolicus sulfide:quinone oxidoreductase, a basis to understand sulfide detoxification and respiration

Sulfide: quinone oxidoreductase (SQR) is a flavoprotein with homologues in all domains of life except plants. It plays a physiological role both in sulfide detoxification and in energy transduction. We isolated the protein from native membranes of the hyperthermophilic bacterium Aquifex aeolicus, and we determined its X-ray structure in the "as-purified,'' substrate-bound, and inhibitor-bound forms at resolutions of 2.3, 2.0, and 2.9 angstrom, respectively. The structure is composed of 2 Rossmann domains and 1 attachment domain, with an overall monomeric architecture typical of disulfide oxidoreductase flavoproteins. A. aeolicus SQR is a surprisingly trimeric, periplasmic integral monotopic membrane protein that inserts about 12 angstrom into the lipidic bilayer through an amphipathic helix-turn-helix tripodal motif. The quinone is located in a channel that extends from the si side of the FAD to the membrane. The quinone ring is sandwiched between the conserved amino acids Phe-385 and Ile-346, and it is possibly protonated upon reduction via Glu-318 and/or neighboring water molecules. Sulfide polymerization occurs on the re side of FAD, where the invariant Cys-156 and Cys-347 appear to be covalently bound to polysulfur fragments. The structure suggests that FAD is covalently linked to the polypeptide in an unusual way, via a disulfide bridge between the 8-methyl group and Cys-124. The applicability of this disulfide bridge for transferring electrons from sulfide to FAD, 2 mechanisms for sulfide polymerization and channeling of the substrate, S2-, and of the product, S-n, in and out of the active site are discussed.

Enriched accumulation and biotransformation of selenium in the edible seaweed Laminaria japonica

Accumulations of selenium in kelp Laminaria japonica cultured in seawater was achieved by adding selenite (Na2SeO3) with or without N-P (NaNO3 + NaH2PO4) nutrients at different concentrations. Biotransformation of selenium in the kelp was investigated through measuring the selenium of biological samples and different biochemical fractionations. The results showed that the optimal selenite-enrichment concentration is 200 mg L-1, which can allow the kelp to accumulate a total selenium content from 0.51 +/- 0.15 to 26.23 +/- 3.12 mug g(-1) of fresh weight (fw). Selenium composition analysis of kelp (control group) showed that selenium is present as organic selenium, which is up to 86.22% of the total selenium, whereas inorganic selenium is barely 4.85%. When L. japonica was exposed for 56 h in seawater containing 200 mg L-1 Na2SeO3, the organic selenium was 16.70 mug g(-1) of fw (68.23%) and inorganic selenium was 4.71 mug g(-1) of fw (19.26%). The capability of accumulation of selenium was further enhanced by adding N-P nutrients to the selenite-enriched medium. Total selenium is increased to be 33.65 mug g(-1) of fw at optimal concentration of N-P nutrient (150 mg L-1 NaNO3 and 25 mg L-1 NaH2PO4), whereas the inorganic selenium was not increased and remained at 4.597 mug g(-1) of fw (13.36%), and the increased part of selenium was organic selenium. This implied that kelp L. japonica could effectively transform inorganic selenium into organic selenium through metabolism.

High-throughput synergy screening identifies microbial metabolites as combination agents for the treatment of fungal infections

The high mortality rate of immunocompromised patients with fungal infections and the limited availability of highly efficacious and safe agents demand the development of new antifungal therapeutics. To rapidly discover such agents, we developed a high-throughput synergy screening (HTSS) strategy for novel microbial natural products. Specifically, a microbial natural product library was screened for hits that synergize the effect of a low dosage of ketoconazole (KTC) that alone shows little detectable fungicidal activity. Through screening of approximate to 20,000 microbial extracts, 12 hits were identified with broadspectrum antifungal activity. Seven of them showed little cytotoxicity against human hepatoma cells. Fractionation of the active extracts revealed beauvericin (BEA) as the most potent component, because it dramatically synergized KTC activity against diverse fungal pathogens by a checkerboard assay. Significantly, in our immunocompromised mouse model, combinations of BEA (0.5 mg/kg) and KTC (0.5 mg/kg) prolonged survival of the host infected with Candida parapsilosis and reduced fungal colony counts in animal organs including kidneys, lungs, and brains. Such an effect was not achieved even with the high dose of 50 mg/kg KTC. These data support synergism between BEA and KTC and thereby a prospective strategy for antifungal therapy.

Interaction between Thymidylate Synthase and Its Cognate mRNA in Zebrafish Embryos

Thymidylate synthase (TS), which catalyzes the de novo synthesis of dUMP, is an important target for cancer therapy. In this report, the effects of 5-fluorouracil (5-FU) and ZD1694 on the regulation of TS gene expression were evaluated in zebrafish embryos. Our results revealed that the expression of TS was increased by about six-fold when embryos were treated with 1.0 mu M 5-FU and there was a greater than 10-fold increase in the TS protein level after treatment with 0.4 mu M ZD1694. Northern blot analysis confirmed that expression of TS mRNA was identical in treated or untreated embryos. Gel shift and immunoprecipitation assays revealed that zebrafish TS was specifically bound with its cognate mRNA in vitro and in vivo. We identified a 20 nt RNA sequence, TS:N20, localized to the 5'-UTR of TS mRNA, which corresponded to nt 13-32; TS:N20 bound to the TS protein with an affinity similar to that of the full-length TS mRNA. The MFold program predicted that TS:N20 formed a stable stem-loop structure similar to that of the cis-acting element found in human TS mRNA. Variant RNAs with either a deletion or mutation in the core motif of TS:N20 were unable to bind to the TS protein. In vitro translation experiments, using the rabbit lysate system, confirmed that zebrafish TS mRNA translation was significantly repressed when an excess amount of TS protein was included in the system. Additionally, a TS stability experiment confirmed that treatment of zebrafish embryos with 5-FU could increase the TS stability significantly, and the half life of TS protein was about 2.7 times longer than in untreated embryos. Our study revealed a structural requirement for the interaction of TS RNA with TS protein. These findings also demonstrated that the increase in TS protein induced by 5-FU occurs at the post-transcriptional level and that increased stability and translation efficiency both contributed to the increase in TS protein levels induced by TS inhibitors.

Interaction between Thymidylate Synthase and Its Cognate mRNA in Zebrafish Embryos

Thymidylate synthase (TS), which catalyzes the de novo synthesis of dUMP, is an important target for cancer therapy. In this report, the effects of 5-fluorouracil (5-FU) and ZD1694 on the regulation of TS gene expression were evaluated in zebrafish embryos. Our results revealed that the expression of TS was increased by about six-fold when embryos were treated with 1.0 mu M 5-FU and there was a greater than 10-fold increase in the TS protein level after treatment with 0.4 mu M ZD1694. Northern blot analysis confirmed that expression of TS mRNA was identical in treated or untreated embryos. Gel shift and immunoprecipitation assays revealed that zebrafish TS was specifically bound with its cognate mRNA in vitro and in vivo. We identified a 20 nt RNA sequence, TS:N20, localized to the 5'-UTR of TS mRNA, which corresponded to nt 13-32; TS:N20 bound to the TS protein with an affinity similar to that of the full-length TS mRNA. The MFold program predicted that TS:N20 formed a stable stem-loop structure similar to that of the cis-acting element found in human TS mRNA. Variant RNAs with either a deletion or mutation in the core motif of TS:N20 were unable to bind to the TS protein. In vitro translation experiments, using the rabbit lysate system, confirmed that zebrafish TS mRNA translation was significantly repressed when an excess amount of TS protein was included in the system. Additionally, a TS stability experiment confirmed that treatment of zebrafish embryos with 5-FU could increase the TS stability significantly, and the half life of TS protein was about 2.7 times longer than in untreated embryos. Our study revealed a structural requirement for the interaction of TS RNA with TS protein. These findings also demonstrated that the increase in TS protein induced by 5-FU occurs at the post-transcriptional level and that increased stability and translation efficiency both contributed to the increase in TS protein levels induced by TS inhibitors.

Enrichment and hydrogen production by marine anaerobic hydrogen-producing microflora

Acid, alkali, heat-shock, KNO3 and control pretreatment methods applied to anaerobic sludge were evaluated for their ability to selectively enrich the marine hydrogen-producing mixed microflora. Seawater culture medium was used as the substrate. The hydrogen yield of pretreated microflora was higher than that of the un-pretreated control (P < 0.05). Among the pretreatment methods studied, heat-shock pretreatment yielded the greatest hydrogen production, which was 14.6 times that of the control. When the effect of initial pH on hydrogen production of heat-shock pretreated samples was studied, hydrogen was produced over the entire pH range (pH 4-10). The hydrogen yield peaked at initial pH 8 (79 mL/g sucrose) and then steadily decreased as the initial pH increased. Sucrose consumption was high at neutral initial pH. During the process of hydrogen production, pH decreased gradually, which indicated that the acquired microflora consisted of acidogenic bacteria.

Benzodiazepine Alkaloids from Marine-Derived Endophytic Fungus Aspergillus ochraceus

A new fungus-derived benzodiazepine analogue, 2-hydroxycircumdatin C (1), and a compound which has been isolated from a natural resource for the first time, but has been previously synthesized, namely (11aS)-2,3-dihydro-7-methoxy-1H-pyrrolo[2,1-c][1,4]benzodiazepine-5,11(10H,11aH)-dione (2), along with five structurally related known alkaloids (3-7), were isolated from Aspergillus ochraceus, an endophytic fungus derived from the marine brown alga Sargassum kjellmanianum. Their structures were established on the basis of spectroscopic methods. The absolute configuration of I was determined through CD evidence. Compound 1 displayed significant DPPH radical-scavenging activity with an IC50 value of 9.9 mu M, which is 8.9-fold more potent than that of butylated hydroxytoluene (BHT), a well-known synthetic positive control.

Comparison of photobioreactors and optimal light regime for rapid vegetative propagation of transgenic Undaria pinnatifida gametophytes

BACKGROUND: Previously, tachyplesin gene (tac) has been successfully transferred into Undaria pinnatifida gametophytes using the method of microprojectile bombardment transformation. The objectives of this study were to compare and evaluate the performance of bubble-column and airlift bioreactors to determine a preferred configuration of bioreactor for vegetative propagation of transgenic U. pinnatifida gametophytes, and to then investigate the influence of light on vegetative propagation of these gametophytes, including incident light intensity, photoperiod and light quality to resolve the problems of rapid vegetative propagation within the selected bioreactor. RESULTS: Experimental results showed that final dry cell density in the airlift bioreactor was 12.7% higher than that in the bubble-column bioreactor under the optimal aeration rate of 1.2 L air min(-1) L-1 culture. And a maximum final dry cell density of 2830 mg L-1 was obtained within the airlift bioreactor using blue light at 40 mu mol m(-2) s(-1) with a light/dark cycle of 14/10 (h). Polymerase chain reaction (PCR) analysis indicated that genes (bar and tac) were not lost during rapid vegetative propagation within the airlift bioreactor. CONCLUSION: The airlift bioreactor was shown to be much more suitable for rapid vegetative propagation of transgenic U. pinnatifida gametophytes than the bubble-column bioreactor in the laboratory. The use of blue light allows improvement of vegetative propagation of transgenic U. pinnatifida gametophytes. (C) 2009 Society of Chemical Industry

Halogenated Sesquiterpenes from the Marine Red Alga Laurencia saitoi (Rhodomelaceae)

Four new halogenated sesquiterpenes, 10-bromo-3-chloro-2,7-epoxychamigr-9-en-8a-of (1), 2,10 beta-dibromochamigra-2,7-dien-9 alpha-ol (2), (9S)-2-bromo-3-chloro-6,9-epoxybisabola-7(14),10-diene (3), and (9R)-2-bromo-3-chloro-6,9-epoxybisabola-7(14),10-diene (4), were characterized from the marine red alga Laurencia saitoi. In addition, two known halosesquiterpenes, 2,10-dibromo-3-chlorochamigr-7-en-9 alpha-ol (5) and isolaurenisol (6), were also isolated and identified. Their structures were established on the basis of extensive analysis of spectroscopic data.

Alkaloids from the Leaves of Daphniphyllum longeracemosum ROSENTH.

Four new Daphniphyllum alkaloids. daphlongamines E-H (1-4, resp.), along with 19 known alkaloids, were isolated front the leaves of Daphniphyllum longeracemosum. Their structures were characterized by spectroscopic methods, especially 2D-NMR techniques. It is noteworthy that aconitine- and veatchine-type diterpenoid alkaloids were also isolated from this species.

A new rearranged chamigrane sesquiterpene from Laurencia okamurai

A new rearranged chamigrane sesquiterpene, named laurenokamurin, was isolated from the marine red alga Laurencia okamurai. Its structure was determined on the basis of spectroscopic methods. (C) 2008 Bin Gui Wang. Published by Elsevier B.V. on behalf of Chinese Chemical Society. All rights reserved.

Study on the photosynthetic performances of Enteromorpha prolifera collected from the surface and bottom of the sea of Qingdao sea area

In this work, the photosynthetic performances of Enteromorpha prolifera thalli collected from the surface and bottom of the sea of Qingdao sea area were studied with chlorophyll fluorescence and oxygraph technology. The samples with the highest photosynthetic activity among their kinds, the floating thalli from the sea surface of the south of the Qingdao Olympic Sailing Center and the sedimentary thalli from the mud surface of the bottom Tuandao bay, were chosen as representatives of surface thalli and bottom thalli, respectively. The results showed that the maximal PSII quantum yield of the floating thalli was significantly lower than the normal level although their photosynthetic activities were relatively high; the photosynthetic potential of the thalli form the mud surface was extremely low. Thus, it is indicated that the floating thalli are seriously stressed by their environment and the thalli from the mud surface are already dead or are dying. On the other hand, the results of the laboratory cultivation showed that the sedimentary thalli cannot regain normal photosynthetic activity even under normal illumination conditions. Thus, the thalli from the mud surface cannot become reproductive source of the alga even if they can reach sea surface again. Therefore, a preliminary conclusion can be reached that, up to mid-July 2008, the environmental conditions of the Qingdao sea area are not suitable for the growth of the alga E. prolifera and for this reason the biomass of E. prolifera, in the area, could be declining.

Features of Late Cenozoic Deepwater Sedimentation in Southern Qiongdongnan Basin, Northwestern South China Sea

Based on high resolution 2D and 3D seismic data acquired in recent years, using sequence stratigraphy analysis and geophysical methods, we discuss the features of Late Cenozoic deepwater sedimentation in the southern Qiongdongnan (sic) basin. The study area entered a bathyal slope environment in the Miocene. The channel developed in the Sanya (sic) Formation was controlled by a fault break, and its shingled seismic characteristics represent multiple erosion and fill, which may indicate that turbidite current developed in the slope environment. The polygon faults found in mudstone of the Meishan (sic) Formation represent the deepwater hungry sedimentary environment. The large-scale channels developed on the top of Huangliu (sic) Formation could be the result of a big sea level drop and an increase of sediment supply. The fantastic turbidite channel developed in Late Quaternary in the slope environment has "fan-like" body and long frontal tiny avulsion channel. The analysis of these features suggests that the sediment supply of the study area in the post-rifting period was dominant from the Vietnam uplift in the southwest. These deepwater sedimentary features could be potential reservoirs or migration pathways for deepwater petroleum systems.

Analysis of tandem repeats in the genome of Chinese shrimp Fenneropenaeus chinensis

Through random sequencing, we found a total of 884000 base-pairs (bp) of random genomic sequences in the genome of Chinese shrimp (Fenneropenaeus chinensis). Using bio-soft Tandem Repeat Finder (TRF) software, 2159 tandem repeats were found, in which there were 1714 microsatellites and 445 minisatellites, accounting for 79.4% and 20.6% of repeat sequences, respectively. The cumulative length of repeat sequences was found to be 116685 bp, accounting for 13.2% of the total DNA sequence; the cumulative length of microsatellites occupied 9.78% of the total DNA sequence, and that of minisatellites occupied 3.42%. In decreasing order, the 20 most abundant repeat sequence classes were as follows: AT (557), AC (471), AG (274), AAT (92), A (56), AAG (28), ATC (27), ATAG (27), AGG (18), ACT (15), C (11), AAC (11), ACAT (11), CAGA (10), AGAA (9), AGGG (7), CAAA (7), CGCA (6), ATAA (6), AGAGAA (6). Dinucleotide repeats, not only in the aspect of the number, but also in cumulative length, were the preponderant repeat type. There were few classes and low copy numbers of repeat units of the pentanucleotide repeat type, which included only three classes: AGAGA, GAGGC and AAAGA. The classes and copy numbers of heptanucleotide, eleven-nucleotide and thirteen-nucleotide primer-number-composed repeats were distinctly less than that of repeat types beside them.

A novel hydroperoxyl substituted cembranolide diterpene from marine soft coral Lobophytum crassum

A new cembranolide diterpene with a hydroperoxyl substitution was isolated from the marine soft coral Lobophytum crassum. The structure was elucidated on the basis of chemical and spectral methods.