Construction and characterization of bacterial artificial chromosome library of black-handed spider monkey (Ateles geoffroyi)

The large-insert genomic DNA library is a critical resource for genome-wide genetic dissection of target species. We constructed a high-redundancy bacterial artificial chromosome (BAC) library of a New World monkey species, the black-handed spider monkey

Construction, characterization and chromosomal mapping of bacterial artificial chromosome (BAC) library of Yunnan snub-nosed monkey (Rhinopithecus bieti)

We constructed a high redundancy bacterial artificial chromosome library of a seriously endangered Old World Monkey, the Yunnan snub-nosed monkey (Rhinopithecus bieti) from China. This library contains a total of 136 320 BAC clones. The average insert size of BAC clones was estimated to be 148 kb. The percentage of small inserts (50-100 kb) is 2.74%, and only 2.67% non-recombinant clones were observed. Assuming a similar genome size with closely related primate species, the Yunnan snub-nosed monkey BAC library has at least six times the genome coverage. By end sequencing of randomly selected BAC clones, we generated 201 sequence tags for the library. A total of 139 end-sequenced BAC clones were mapped onto the chromosomes of Yunnan snub-nosed monkey by fluorescence in-situ hybridization, demonstrating a high degree of synteny conservation between humans and Yunnan snub-nosed monkeys. Blast search against human genome showed a good correlation between the number of hit clones and the size of the chromosomes, an indication of unbiased chromosomal distribution of the BAC library. This library and the mapped BAC clones will serve as a valuable resource in comparative genomics studies and large-scale genome sequencing of nonhuman primates. The DNA sequence data reported in this paper were deposited in GenBank and assigned the accession number CG891489-CG891703.

Blood cells as targets of snake toxins

Snake venoms are mixtures of enzymes and peptides which exert toxicological effects by targeting their substrates or receptors upon envenomation. Snake venom proteins widely affect vascular system including circulating blood cells, coagulation factors, an

Molecular characterization and expression profiles in response to bacterial infection of Chinese soft-shelled turtle interleukin-8 (IL-8), the first reptilian chemokine gene

In this study, an IL-8 homologue has been cloned and identified from a reptile, Chinese soft-shelled turtle for the first time. The full-length cDNA of turtle IL-8 was 1188 bp and contained a 312 bp open reading frame (ORF) coding for a protein of 104 amino acids. The chemokine CXC domain, which contained Glu-Leu-Arg (ELR) motif and four cysteine residues, was well conserved in turtle IL-8. The 4924 bp genomic DNA of turtle IL-8 contained four exons and three introns. Phylogenetic analysis showed that the amino acid sequence of turtle IL-8 clustered together with birds. RT-PCR analysis showed that turtle IL-8 mRNA was constitutively expressed liver, spleen, kidney, heart, blood and intestine tissues of control turtles. Real-time quantitative PCR analysis further indicated that the turtle IL-8 mRNA expression was apparent in various tissues at 8 h and up-regulated significantly during 8 h-7 d after Aeromonas hydrophila infection. The present studies will help us to understand the evolution of IL-8 molecule and the inflammatory response mechanism in reptiles. (C) 2009 Elsevier Ltd. All rights reserved.

In vivo study on the effects of microcystin extracts on the expression profiles of proto-oncogenes (c-fos, c-jun and c-myc) in liver, kidney and testis of male Wistar rats injected i.v. with toxins

Microcystins (MCs) are a potent liver tumor promoter, possessing potent tumor-promoting activity and weak initiating activity. Proto-oncogenes are known to be involved in the tumor-promoting mechanisms of microcystin-LR. However, few data are available on the effects of MCs oil proto-oncogenes in the whole animal. To investigate the effects of MCs on the expression profile of the proto-oncogenes in different organs, male Wistar rats were injected intravenously with microcystin extracts at a dose of 86.7 mu g MC-LR eq/kg bw (MC-LR eq, MC-LR equivalents). mRNA levels of three proto-oncogenes c-fos, c-jun and c-myc in liver, kidney and testis were analyzed using quantitative real-time PCR at several time points post-injection. Significant induction of these genes at transcriptional level was observed in the three organs. In addition, the increase of mRNA expression of all three genes was much higher in liver than in kidney and testis. Meanwhile, the protein levels of c-Fos and c-Jun were investigated by western blotting. Both proteins were induced in the three organs. However, elevations of protein levels were Much lower than those of mRNA levels. These findings suggest that the expression of c-fos, c-jun and c-myc might be one possible mechanism for the tumor-promoting activity and initiating activity of microcystins. (c) 2008 Published by Elsevier Ltd.

Bacterial diversity in activated sludge from a consecutively aerated submerged membrane bioreactor treating domestic wastewater

The bacterial diversity of activated sludge from submerged membrane bioreactor (SMBR) was investigated. A 16S rDNA clone library was generated, and 150 clones were screened using restriction fragment length polymorphism (RFLP). Of the screened clones, almost full-length 16S rDNA sequences of 64 clones were sequenced. Phylogenetic tree was constructed with a database containing clone sequences from this study and bacterial rDNA sequences from NCB1 for identification purposes. The 90.6% of the clones were affiliated with the two phyla Bacteroidetes (50%) and Proteobacteria (40%), and beta-, -gamma-, and delta-Proteobacteria accounted for 7.8%, 28.1%, and 4.7%, respectively. Minor portions were affiliated with the Actinobacteria and Firmicutes (both 3.1%). Only 6 out of 64 16S rDNA sequences exhibited similarities of more than 97% to classified bacterial species, which indicated that a substantial fraction of the clone sequences were derived from unknown taxa. Rarefaction analysis of operational taxonomic units (orrUs) clusters demonstrated that 150 clones screened were still insufficient to describe the whole bacterial diversity. Measurement of water quality parameter demonstrated that performance of the SMBR maintained high level, and the SMBR system remained stable during this study.

Distribution of toxins in various tissues of crucian carp intraperitoneally injected with hepatotoxic microcystins

An acute toxicity experiment was conducted to examine the distribution and depuration of microcystins (MCS) in crucian carp (Carassius aurutus) tissues. Fish were injected intraperitoneally with extracted MCs at a dose of 200 mu g MC-LR (where L = leucine and R = arginine) equivalent/kg body weight. Microcystin concentrations in various tissues and aquaria water were analyzed at 1, 3, 12, 24, and 48 h postinjection using liquid chromatography coupled with mass spectrometry. Microcystins were detected mainly in blood (3.99% of injected dose at 1 h), liver (1.60% at I h), gonad (1.49% at 3 h), and kidney (0.14% at 48 h). Other tissues, such as the heart, gill, gallbladder, intestine, spleen, brain, and muscle, contained less than 0.1% of the injected MCs. The highest concentration of MCs was found in blood (526-3,753 ng/g dry wt), followed by liver (103-1,656 ng/g dry wt) and kidney (279-1,592 ng/g dry wt). No MC-LR was detectable in intestine, spleen, kidney, brain, and muscle, whereas MC-RR was found in all examined fish tissues, which might result from organ specificity of different MCs. Clearance of MC-RR in brain tissue was slow. In kidney, the MC-RR content was negatively correlated with that in blood, suggesting that blood was important in the transportation of MC-RR to kidney for excretion.

Reduction in microcystin concentrations in large and shallow lakes: Water and sediment-interface contributions

Blooms of cyanobacteria, or blue-greens, are known to produce chemicals, such as microcystins, which can be toxic to aquatic and terrestrial organisms. Although previous studies have examined the fate of microcystins in freshwater lakes, primary elimination pathways and factors affecting degradation and loss have not been fully explained. The goal of the present study was to explore sources of algal toxins and investigate the distribution and biodegradation of microcystins in water and sediment through laboratory and field analyses. Water and sediment samples were collected monthly from several locations in Lake Taihu from February 2005 to January 2006. Samples were analyzed for the presence of microcystin. Water and sediment were also used in laboratory studies to determine microcystin degradation rates by spiking environmental samples with known concentrations of the chemical and observing concentration changes over time. Some water samples were found to efficiently degrade microcystins. Microcystin concentrations dropped faster in water collected immediately above lake sediment (overlying water). Degradation in sediments was higher than in water. Based on spatial distribution analyses of microcystin in Lake Taihu, higher concentrations (relative to water concentrations) of the chemical were found in lake sediments. These data suggest that sediments play a critical role in microcystin degradation in aquatic systems. The relatively low levels of microcystins found in the environment are most likely due to bacterial biodegradation. Sediments play a crucial role as a source (to the water column) of bio-degrading bacteria and as a carbon-rich environment for bacteria to proliferate and metabolize microcystin and other biogenic toxins produced by cyanobacteria. These, and other, data provide important information that may be applied to management strategies for improvement of water quality in lakes, reservoirs and other water bodies. (C) 2007 Elsevier Ltd. All rights reserved.

A screening study on persistent bioaccumulation toxins (PBT) in fuel combustion soot

Persistent bioaccumulative toxins (PBTs) are organic substances that are persistent, bioaccumulative and can cause severe toxic effects (e.g. potential oncogens, mutagenic, endocrine disrupters) to human health or environment which are the ones that need special attention. PBT chemicals could be released to the environment from several types of sources and are ubiquitous in environment. However, fast and efficiency monitoring and assessment methods to investigate PBTs in environment are still lacking. In this study, a cleaning-up procedure of analyzing PBTs in fuels combustion soot was developed and its performance was assessed through comparing the chromatograms of crude extracts with their cleaned extracts after the cleaning-up procedure. The results showed that polycyclic aromatic compounds (PACs) were the main components in fuel combustion soot and the clean-up procedure developed in this paper can be well used as the method of analyzing PBTs in fuels combustion soot.

Analysis of paralytic shellfish toxins in Aphanizomenon DC-1 from Lake Dianchi, China

Lake Dianchi is in Yunnan Province in southwestern China. In recent years, significant cyanobacterial blooms have occurred in this lake nearly every year because of eutrophication. Monitoring data for the past 5 years acquired by our research group showed that phytoplankton composition alternated between species of Microcystis sp. during warm seasons and those of Aphanizomenon sp. during cool seasons. In March 2003, when phytoplankton composition was highly dominated by Aphanizomenon sp., samples were taken from the lake for toxin detection and immediate strain isolation. A mouse bioassay with extracts from the lyophilized field material showed obvious intoxication from paralytic shellfish poisons (PSPs), and all mice died within 30 min. Further analysis of both field and isolated algal strain Aphanizomenon DC-1 by the postcolumn HPLC-FLD method confirmed its PSP-producing ability The analogues found in the extracts from the field material were neoSTX, dcSTX, and dcGTX3, with contents of 2.279, 1.135, and 0.547 ng/mg DW, respectively. Under laboratory culture condition, toxin content in the Aphanizomenon strain DC-1 varied greatly during different growth phases, with two peaks: in the early-exponential and late-stationary growth phases. When the culture grew at a relatively high rate during the mid- to late-exponential growth phase, toxin content declined gradually. Moreover, the types of toxin in the DC-1 strain varied greatly during a single culture cycle. The HPLC results showed that dcSTX was the only toxin isomer detected throughout the culture period, and its level remained stable. On the other hand, dcGTX2 and GTX4 were the major toxins during the early-exponential and stationary phases, respectively. This article presents the first data on the identification and detection of paralytic shellfish toxins from cyanobacteria in Lake Dianchi. As far as we know, this is also the first report of this type of toxin in inland water bodies in China. Our study indicates the threat associated with PSP toxins in Lake Dianchi and suggests that necessary measures and programs for control are urgently needed to prevent the spread of toxic cyanobacterial blooms. (c) 2006 Wiley Periodicals, Inc.

Measurement of 5-day biochemical oxygen demand without sample dilution or bacterial and nutrient enhancement

A direct method for measuring the 5-day biochemical oxygen demand (BODS) of aquaculture samples that does not require sample dilution or bacterial and nutrient enrichment was evaluated. The regression coefficient (R-2) between the direct method and the standard method for the analyses of 32 samples from catfish ponds was 0.996. The slope of the regression line did not differ from 1.0 or the Y-intercept from 0.0 at P = 0.05. Thus, there was almost perfect agreement between the two methods. The control limits (three standard deviations of the mean) for a standard solution containing 15 mg/L each of glutamic acid and glucose were 17.4 and 20.4 mg/L. The precision of the two methods, based on eight replicate analyses of four pond water samples did not differ at P = 0.05. (c) 2005 Elsevier B.V All rights reserved.

Cloning of biologically active genomes from a Helicoverpa armigera single-nucleocapsid nucleopolyhedrovirus isolate by using a bacterial artificial chromosome

Purification of genotypes from baculovirus isolates provides understanding of the diversity of baculoviruses and may lead to the development of better pesticides. Here, we report the cloning of different genotypes from an isolate of Helicoverpa armigera single-nucleocapsid nucleopolyhedrovirus (HaSNPV) by using a bacterial artificial chromosome (BAC). A transfer vector (pHZB10) was constructed which contained an Escherichia coli mini-F replicon cassette within the upstream and downstream arms of HaSNPV polyhedrin gene. Hz2e5 cells were co-transfected with wild-type HaSNPV DNA and pHZB10 to generate recombinant viruses by homologous recombination. The DNA of budded viruses (BVs) was used to transform E. coli. One of the bacmid colonies, HaBacHZ8, has restriction enzyme digestion profiles similar to an in vivo cloned strain HaSNPV-G4, the genome of which has been completely sequenced. For testing the oral infectivity, the polyhedrin gene of HaSNPV was reintroduced into HaBacHZ8 to generate the recombinant bacmid HaBacDF6. The results of one-step growth curves, electron microscopic examination, protein expression analysis and bioassays indicated that HaBacDF6 replicated as well as HaSNPV-G4 in vitro and in vivo. The biologically functional HaSNPV bacmids obtained in this research will facilitate future studies on the function genomics and genetic modification of HaSNPV. (C) 2003 Elsevier B.V. All rights reserved.