Structure-function relationship of three neurotoxins from the venom of Naja kaouthia: a comparison between the NMR-derived structure of NT2 with its homologues, NT1 and NT3

Three homologous short-chain neurotoxins, named NT1, NT2 and NT3, were purified from the venom of Naja kaouthia. NT1 has an identical amino acid sequence to cobrotoxin from Naja naja atra [Biochemistry 32 (1993) 2131]. NT3 shares the same sequence with cobrotoxin b [J. Biochem. (Tokyo) 122 (1997) 1252], whereas NT2 is a novel 6 1 -residue neurotoxin. Tests of their physiological functions indicate that NT1 shows a greater inhibition of muscle contraction induced by electrical stimulation of the nerve than do NT2 and NT3. Homonuclear proton two-dimensional NMR methods were utilized to study the solution tertiary structure of NT2. A homology model-building method was employed to predict the structure of NT3. Comparison of the structures of these three toxins shows that the surface conformation of NT1 facilitates the substituted base residues, Arg28, Arg30, and Arg36, to occupy the favorable spatial location in the central region of loop 11, and the cation groups of all three arginines face out of the molecular surface of NT1 This may contribute greatly to the higher binding of NT1 with AchR compared to NT2 and NT3. (C) 2002 Elsevier Science B,V. All rights reserved.

Fish-Specific Duplicated dmrt2b Contributes to a Divergent Function through Hedgehog Pathway and Maintains Left-Right Asymmetry Establishment Function

Gene duplication is thought to provide raw material for functional divergence and innovation. Fish-specific dmrt2b has been identified as a duplicated gene of the dmrt2a/terra in fish genomes, but its function has remained unclear. Here we reveal that Dmrt2b knockdown zebrafish embryos display a downward tail curvature and have U-shaped somites. Then, we demonstrate that Dmrt2b contributes to a divergent function in somitogenesis through Hedgehog pathway, because Dmrt2b knockdown reduces target gene expression of Hedgehog signaling, and also impairs slow muscle development and neural tube patterning through Hedgehog signaling. Moreover, the Dmrt2b morphants display defects in heart and visceral organ asymmetry, and, some lateral-plate mesoderm (LPM) markers expressed in left side are randomized. Together, these data indicate that fish-specific duplicated dmrt2b contributes to a divergent function in somitogenesis through Hedgehog pathway and maintains the common function for left-right asymmetry establishment.

The profound effects of microcystin on cardiac antioxidant enzymes, mitochondrial function and cardiac toxicity in rat

Deaths from microcystin toxication have widely been attributed to hypovolemic shock due to hepatic interstitial hemorrhage, while some recent studies suggest that cardiogenic complication is also involved. So far, information on cardiotoxic effects of MC has been rare and the underlying mechanism is still puzzling. The present study examined toxic effects of microcystins on heart muscle of rats intravenously injected with extracted MC at two doses, 0.16LD(50) (14 mu g MC-LReq kg(-1) body weight) and 1LD(50) (87 mu g MC-LReq kg(-1) body weight). In the dead rats, both TTC staining and maximum elevations of troponin I levels confirmed myocardial infarction after MC exposure, besides a serious interstitial hemorrhage in liver. In the 1LD(50) dose group, the coincident falls in heart rate and blood pressure were related to mitochondria dysfunction in heart, while increases in creatine kinase and troponin I levels indicated cardiac cell injury. The corresponding pathological alterations were mainly characterized as loss of adherence between cardiac myocytes and swollen or ruptured mitochondria at the ultrastructural level. MC administration at a dose of 1LD(50) not only enhanced activities and up-regulated mRNA transcription levels of antioxidant enzymes, but also increased GSH content. At both doses, level of lipid peroxides increased obviously, suggesting serious oxidative stress in mitochondria. Simultaneously. complex I and III were significantly inhibited, indicating blocks in electron flow along the mitochondrial respiratory chain in heart. In conclusion, the findings of this study implicate a role for MC-induced cardiotoxicity as a potential factor that should be considered when evaluating the mechanisms of death associated with microcystin intoxication in Brazil. (C) 2009 Elsevier Ireland Ltd. All rights reserved.

Generation of recombinant monoclonal antibodies to study structure-function of envelope protein VP28 of white spot syndrome virus from shrimp

White spot syndrome virus (WSSV) is a major pathogen in shrimp aquaculture. VP28 is one of the most important envelope proteins of WSSV. In this study, a recombinant antibody library, as single-chain fragment variable (scFv) format, displayed on phage was constructed using mRNA from spleen cells of mice immunized with-full-length VP28 expressed in Escherichia coli. After several rounds of panning, six scFv antibodies specifically binding to the epitopes in the N-terminal, middle, and C-terminal regions of VP28, respectively, were isolated from the library. Using these scFv antibodies as tools, the epitopes in VP28 were located on the envelope of the virion by immuno-electron Microscopy, Neutralization assay with these antibodies in vitro suggested that these epitopes may not be the attachment site of WSSV to host cell receptor. This study provides a new way to investigate the structure and function of the envelope proteins of WSSV. (c) 2008 Published by Elsevier Inc.

Carbon source/sink function of a subtropical, eutrophic lake determined from an overall mass balance and a gas exchange and carbon burial balance

Although studies on carbon burial in lake sediments have shown that lakes are disproportionately important carbon sinks, many studies on gaseous carbon exchange across the water-air interface have demonstrated that lakes are supersaturated with CO2 and CH4 causing a net release of CO2 and CH4 to the atmosphere. In order to more accurately estimate the net carbon source/sink function of lake ecosystems, a more comprehensive carbon budget is needed, especially for gaseous carbon exchange across the water-air interface. Using two methods, overall mass balance and gas exchange and carbon burial balance, we assessed the carbon source/sink function of Lake Donghu, a subtropical, eutrophic take, from April 2003 to March 2004. With the overall mass balance calculations, total carbon input was 14 905 t, total carbon output was 4950 1, and net carbon budget was +9955 t, suggesting that Lake Donghu was a great carbon sink. For the gas exchange and carbon burial balance, gaseous carbon (CO2 and CH4) emission across the water-air interface totaled 752 t while carbon burial in the lake sediment was 9477 t. The ratio of carbon emission into the atmosphere to carbon burial into the sediment was only 0.08. This low ratio indicates that Lake Donghu is a great carbon sink. Results showed good agreement between the two methods with both showing Lake Donghu to be a great carbon sink. This results from the high primary production of Lake Donghu, substantive allochthonous carbon inputs and intensive anthropogenic activity. Gaseous carbon emission accounted for about 15% of the total carbon output, indicating that the total output would be underestimated without including gaseous carbon exchange. (C) 2007 Elsevier Ltd. All rights reserved.

Determinations of residual furazolidone and its metabolite, 3-amino-2-oxazolidinone (AOZ), in fish feeds by HPLC-UV and LC-MS/MS, respectively

The antibacterial drug furazolidone belonging to the group of nitrofuran antibacterial agents has been widely used as an antibacterial and antiprotozoal feed additive for poultry, cattle, and farmed fish in China. During application a large proportion of the administered drug may reach the environment directly or via feces. Although the use of furazolidone is prohibited in numerous countries, there are indications of its illegal use. It is known that furazolidone can be rapidly metabolized to 3-amino-2-oxazolidinone (AOZ) in the body of the target organism. In this study, a total of 21 fish feed samples, including 17 commercial fish feeds from local markets in China (representing 15 different formulations) and 4 fish feeds obtained from Germany and Turkey, respectively, are analyzed to determine whether the drug is still illegally used or commercially available feeds are contaminated by this drug. High-performance liquid chromatography (HPLC) and liquid chromatography-electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS) methods have been implemented to determine furazolidone and its metabolite AOZ in fish feeds containing animal protein, respectively. An efficient and convenient cleanup method for the determination of furazolidone in fish feeds is developed, and a simple cleanup method for the determination of AOZ is used. Method recoveries for samples used were determined as 87.7-98.3% for furazolidone at two spike levels of 2.0 and 5.0 ng g(-1) and as 95.6-102.8% for AOZ at spike levels of 0.4 and 0.8 ng g(-1). Limits of detections were 0.4 ng g(-1) for furazolidone and 0.05 ng g(-1) for AOZ. The established methods are therefore suitable for the determination of furazolidone and its metabolite AOZ in fish feeds at trace contamination levels. Using the established methods, all fish feed samples have been proved to be furazolidone negative; however, AOZ is tested in 16 of 17 fish feeds obtained from local markets in the Hubei province of China, with a positive rate as high as 94.1%.

Modeling phytoplankton dynamics in the River Darling (Australia) using the radial basis function neural network

A radial basis function neural network was employed to model the abundance of cyanobacteria. The trained network could predict the populations of two bloom forming algal taxa with high accuracy, Nostocales spp. and Anabaena spp., in the River Darling, Australia. To elucidate the population dynamics for both Nostocales spp. and Anabaena spp., sensitivity analysis was performed with the following results. Total Kjeldahl nitrogen had a very strong influence on the abundance of the two algal taxa, electrical conductivity had a very strong negative relationship with the population of the two algal species, and flow was identified as one dominant factor influencing algal blooms after a scatter plot revealed that high flow could significantly reduce the algal biomass for both Nostocales spp. and Anabaena spp. Other variables such as turbidity, color, and pH were less important in determining the abundance and succession of the algal blooms.

Residual plastids of bleached mutants of Euglena gracilis and their effects on the expression of nucleus-encoded genes

Bleached mutants of Euglena gracilis were obtained by treatment with ofloxacin (Ofl) and streptomycin (Sm) respectively. As shown by electron microscopy, the residual plastids contain prothylakoids in an Ofl mutant, and the highly developed and tightly stacked membranous structure found in cells of two Sm, mutants. Nine genes of the plastid genome were examined with PCR, showing that ribosomal protein genes and most other plastid genes were lost in all but one Sm mutant. Using differential display and RT-PCR, it was shown that chloroplast degeneration could cause changes in transcription of certain nucleus-encoded genes during heterotrophic growth in darkness.

Photosynthetic physiology and growth as a function of colony size in the cyanobacterium Nostoc sphaeroides

Algal size can affect the rate of metabolism and of growth. Different sized colonies of Nostoc sphaeroides were used with the aim of determining the effects of colony size on photosynthetic physiology and growth. Small colonies showed higher maximum photosynthetic rates per unit chlorophyll, higher light saturation point, and higher photosynthetic efficiency (a) than large colonies. Furthermore, small colonies had a higher affinity for DIC and higher DIC-saturated photosynthetic rates. In addition, small colonies showed higher photosynthetic rates from 5-45degreesC than large colonies. There was a greater decrease in Fv/Fm after exposure to high irradiance and less recovery in darkness for large colonies than for small colonies. Relative growth rate decreased with increasing colony size. Small colonies had less chl a and mass per unit surface area. The results indicate that small colonies can harvest light and acquire DIC more efficiently and have higher maximum photosynthetic rates and growth rates than large colonies.

Structure-function analysis of the inverted terminal repeats of the Sleeping Beauty transposon

Translocation of Sleeping Beauty (SB) transposon requires specific binding of SB transposase to inverted terminal repeats (ITRs) of about 230 bp at each end of the transposon, which is followed by a cut-and-paste transfer of the transposon into a target DNA sequence. The ITRs contain two imperfect direct repeats (DRs) of about 32 bp. The outer DRs are at the extreme ends of the transposon whereas the inner DRs are located inside the transposon, 165-166 bp from the outer DRs. Here we investigated the roles of the DR elements in transposition. Although there is a core transposase-binding sequence common to all of the DRs, additional adjacent sequences are required for transposition and these sequences vary in the different DRs. As a result, SB transposase binds less tightly to the outer DRs than to the inner DRs. Two DRs are required in each ITR for transposition but they are not interchangeable for efficient transposition. Each DR appears to have a distinctive role in transposition. The spacing and sequence between the DR elements in an ITR affect transposition rates, suggesting a constrained geometry is involved in the interactions of SB transposase molecules in order to achieve precise mobilization. Transposons are flanked by TA dinucleotide base-pairs that are important for excision; elimination of the TA motif on one side of the transposon significantly reduces transposition while loss of TAs on both flanks of the transposon abolishes transposition. These findings have led to the construction of a more advanced transposon that should be useful in gene transfer and insertional mutagenesis in vertebrates. (C) 2002 Elsevier Science Ltd. All rights reserved.

PCDD/F-s, PCBs, HCHs and HCB in sediments and soils of Ya-Er lake area in China: Results on residual levels and correlation to the organic carbon and particle size

Sediments and soils collected from the Ya-Er Lake area in China were analysed for the polychlorinated dibenzo-p-dioxins and dibenzofurans (PCDD/Fs), polychlorinated biphenyl (PCBs), hexachlorocyclohexane (HCHs) and hexachlorobenzene (HCB). The results indicated the main pollution problems in the Ya-Er Lake area, which was heavily polluted by HCHs and chlorobenzenes, now is dominantly polluted by PCDD/Fs, PCBs and HCB. The occurrence of PCDD/Fs and PCBs with relatively high levels of HpCDDs, OCDD and low chlorinated-substituted PCBs, is attributed to the discharge of waste water and biodegradation. The vertical distributions of HCH-residues are related with the content of organic carbon and particle size. Copyright (C) 1996 Elsevier Science Ltd

MOBILITY AND TRANSFER OF RESIDUAL HEXACHLOROCYCLOHEXANE (HCH) IN AQUATIC ENVIRONMENT

An investigation was carried out to study the mobility and transfer of HCH-residues in Yanjia lake, Hubei, China, from 1990-1993. By sampling at different locations, the horizontal distribution of HCH-residues was determined; by sampling at different depths in the same location, the vertical mobility of HCH-residues and its penetration were measured. The vertical profiles of HCH-residues in sediment showed a gradient distribution, which was reduced with the increase in depth. This was very similar in shape to the organic carbon and nitrogen content profiles. The penetration of HCH-residues was measured as 60cm approximately. Meanwhile, water, sediment (0-12cm), benthos and benthic fish collected from the lake were analyzed to determine the transfer of HCH-residues along aquatic food chain. The BCF values of HCH-residues indicated that the bioaccumulation of HCH-residues in freshwater food chain still exsisted in spite of the banning of HCH since 1983. The maxium BCF value of HCH-residues in the liver samples of 5-year-old common carp was up to 61966 (calculated by beta-HCH).

HORMONAL REPLACEMENT THERAPY IN FISH - HUMAN GROWTH-HORMONE GENE-FUNCTION IN HYPOPHYSECTOMIZED CARP

Transgenic common carp, Cyprinus carpio, produced by the microinjection of fertilized eggs with a linearized chimeric plasmid pMThGH, a human growth hormone (hGH) gene with a mouse metallothionein-I (MT) gene promoter in pBR322, were used to produce F1 and F2 transgenics. Following hypophysectomy of the transgenic F2 common carp, non-transgenic common carp and non-transgenic crucian carp, growth was monitored for up to 110 days. In addition, recombinant hGH was injected subcutaenously into a group of the non-transgenic crucian carp. Growth rate analyses indicated that (1) hypophysectomy of non-transgenic common carp and crucian carp results in the cessation of growth, (2) hGH administration can stimulate the growth of hypophysectomized crucian carp and (3) hypophysectomized hGH-transgenic common carp continue to grow in the absence of their own growth hormone, suggesting that the hGH-transgene is being expressed in tissues other than the pituitary.

Exact result for the Green's function of a two-dimensional interacting electron gas with spin-orbit interaction in a perpendicular magnetic field

An exact property is established for the Green's function of a uniform two-dimensional interacting electron gas in a perpendicular magnetic field with spin-orbit interaction. It is shown that the spin-diagonal Green's function is exactly diagonal in the Landau level index even in the presence of electron-electron interactions. For the Green's function with different spin indexes, only that with adjacent Landau level indexes is non-zero. This exact result should be helpful in calculating the Green's function approximately.

Injection locked Fabry-Perot laser diodes for WDM passive optical network spare function

In this paper, a protection scheme for transmitters in wavelength-division-multiplexing passive optical network (WDM-PON) has been proposed and demonstrated. If any downstream transmitter encounters problems at the central office (CO), the interrupted communication can be restored immediately by injecting a Fabry-Perot laser diode (FP-LD) with the upstream lightwave corresponding to the failure transmitter. Compared with the conventional methods, this proposed architecture provides a cost-effective and reliable protection scheme employing a common FP-LD. In the experiment, a 1 36 protection capability was implemented with a 2.5 Gbit/s downstream transmission capability. (C) 2009 Elsevier B.V. All rights reserved.