Development toxicity of functionalized single-walled carbon nanotubes on rare minnow embryos and larvae.

Carbon nanotubes (CNTs) are widely used in industrial and commercial applications, but few studies systematically evaluate their developmental toxicity on aquatic organism. Using rare minnow (Gobiocypris rarus) at early life stages as experimental models, developmental toxicity of functionalized single-walled CNTs (SWCNTs) was investigated following exposure to 0-320 mg/L for 144 h. Results revealed that significantly increased in mortality and malformation was only observed after hatching. Decreased body length, heart rate and swimming speed provide a concentration-dependent manner on larvae; values of 144 h LC50 and EC50 were 140.8 and 109.8 mg/L, respectively. Antioxidant enzyme activities (superoxide dismutase, catalase and glutathione S-transferase) and antioxidant enzyme related mRNA expressions were significant changed; cell apoptosis activities (caspase-3, -8, -9) and cell apoptosis related mRNA expressions were significant up-regulated; reactive oxygen species and DNA damage were significantly induced when the concentration of SWCNTs above 100 mg/L. Fluorescence and electron microscopy sliced observation show that SWCNTs were well dispersed in larvae within 0.5 h, eventually cleared from the larvae at 144 h. This is the first study to define uptake kinetics and to focus on behavioral consequences, physiological changes and mRNA expression following SWCNTs exposure in the early life stages of fish. The results obtained in the present study demonstrated that functionalized SWCNTs have the potential to affect aquatic life when released into the aquatic environment and reached high concentration. In the increasing economical context of SWCNTs, complementary studies must be undertaken, especially including mechanistic and environmental investigations.

Single-walled carbon nanotubes as candidate recombinant subunit vaccine carrier for immunization of grass carp against grass carp reovirus.

Grass carp reovirus (GCRV), the most pathogenic aquareovirus, can cause fatal hemorrhagic disease in fingerling and yearling grass carp. Vaccination by injection is by far the most effective method of combating disease. However it is labor intensive, costly and not feasible to vaccinate large numbers of the fish. Thus, an efficient and economic strategy for the prevention of GCRV infection becomes urgent. Here, functionalized single-walled carbon nanotubes (SWCNTs) as carrier were used to manufacture SWCNTs-VP7 subunit vaccine with chemical modification. Different developmental stages of grass carps were immunized by VP7/SWCNTs-VP7 subunit vaccine against GCRV by intramuscular injection and bath immunization. The results indicate that better immune responses of grass carp immunized with the SWCNTs-VP7 subunit vaccine were induced in comparison with VP7 subunit vaccine alone. Immunization doses/concentrations are significantly reduced (about 5-8 times) to prevent GCRV infection in different developmental stages of grass carp with injection or bath treatment when SWCNTs carrier was used. A good immune protective effect (relative percentage survival greater than 95%) is observed in smaller size fish (0.2 g) with SWCNTs-VP7 bath immunization. In addition, serum respiratory burst activity, complement activity, lysozyme activity, superoxide dismutase activity, alkaline phosphatase activity, immune-related genes and antibody levels were significantly enhanced in fish immunized with vaccine. This study suggested that functionalized SWCNTs was the promising carrier for recombinant subunit vaccine and might be used to vaccinate fish by bath approach.

Cyclic AMP concentration and protein kinase A (PKA) gene expression at different developmental stages of the polychaete Hydroides elegans.

The phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (IBMX) showed inductive effect on larval settlement of the polychaete Hydroides elegans. It has been suggested that IBMX triggers larval settlement by elevating the cellular adenosine 3',5'-cyclic monophosphate (cAMP) level in this species. To test this hypothesis, we first examined cAMP-level changes in both the competent (CL) and attached larvae (AL) and then characterized the cAMP-dependent protein kinase in H. elegans, which is the major mediator of cAMP action. Tissue extracts of the larvae were assayed for cAMP by enzyme immunoassay; the results showed that IBMX increased cAMP production up to approximately two-folds in the CL. However, there was no significant difference in the cAMP concentration between the CL and AL that were not treated with IBMX. The catalytic subunit of protein kinase A gene from H. elegans (designated HePKAc) was cloned, and its expression in different developmental stages of H. elegans was examined using quantitative real-time polymerase chain reaction. The gene expression level in the pre-competent trochophore larvae was the lowest, increased in the CL, reached the highest in the larvae undergoing normal and IBMX-induced metamorphosis, and then decreased in the adult stage. In situ hybridization results showed that HePKAc expressed mainly around eye regions and along body fragments of the CL and AL. Our results indicated that the IBMX-induced cAMP changes and the cAMP-dependent protein kinase gene may mediate larval development and settlement of H. elegans.

[Study on mitochondrial DNA genetic diversity of some cattle breeds in China].

The complete mitochondrial D-loop sequences, 910 bp in length, in 22 individuals from 8 cattle breeds in China were analyzed. The results showed that A% + T% was about 61.65%. Comparisons of these 22 sequences revealed 66 polymorphic sites, and 5 types of mutation, transition, transversion, insertion, deletion and coexistence site of transition and transversion were observed,with the percentage of 81.82%, 6.06%, 7.57%, 3.03%, 1.52%, respectively. In reference to complete mtDNA D-loop of the European cattle as a control, eight Chinese cattle breeds were classified into 3 groups according to the average percentage of D-loop nucleotide variations. The lowest average percentage of mtDNA D-loop nucleotide variation was in Xizhen cattle, Mongolian cattle, Holstein, Qinchuan cattle with 0.37%, 0.44%, 0.52%, 0.66%, the mediate in Nanyang cattle and Jiaxian cattle with 1.91%, 2.02% and the highest in Jinnan cattle and Yueyang cattle with 4.47%, 4.73%, respectively. The average sequence divergence estimated from D-loop region within breeds and among breeds in China varied from 0.55% - 5.39% and 1.21% - 6.59%, respectively. Comparisons of these 22 sequences revealed 19 mitochondrial haplotypes, the percentage of haplotype was 86.36%, showing that abundant mitochondrial genetic diversity exists in Chinese cattle. The molecular phylogenetic tree of mtDNA D-loop of 8 Chinese cattle breeds was constructed by Neighbor-Joining method. The NJ tree indicated that these mtDNA sequences fell into 3 distinct haplotype groups, it also suggested in molecular level that there were probably 3 maternal origins, of which the main origins of Chinese cattle were from Bos taurus and Bos indicus.

[Cloning and expression of tumor necrosis factor (TNFalpha) cDNA from red seabream pagrus major].

A fragment of TNFalpha cDNA sequence from red seabream was cloned by homology cloning approach with two degenerated primers which were designed based on the conserved regions of other animals' TNF sequences. The sequence was elongated by 3' and 5' RACE to get the full length CDS sequence. This sequence contained 1264 nucleotides that included a 5' UTR of 85 bp, a 3' UTR of 514 bp and an open reading frame (ORF) of 666 bp which could encode 222 amino acids propeptide. In 3' UTR, there were several mRNA instability motifs and three endotoxin-responsive sequences, but the sequence lacked the polyadenylation signal. The deduced peptide had a clear transmembrane domain, a TNFalpha family signature and a TNF2 family profile. The cell attachment sequence and the glycosaminoglycan attachment sites were also found in the sequence. The red seabream TNF sequence shared relatively high similarity with both mammalian TNFalpha and TNFbeta by multiple sequence alignments. Phylogenetic analysis showed that the piscine TNFalpha were located independently in a different branch compared with mammalian TNFalpha and TNFbeta. Based on the primary and secondary structure analysis and gene expression study, we could concluded that the red seabream TNF should be a TNFalpha, not TNFbeta. RT-PCR was used to study TNFalpha transcript expression. 24 h after the red seabream was challenged by Vibrio anguillarum, the RS TNFalpha transcript expression were detected in blood, brain, gill, heart, head kidney, kidney, liver, muscle and spleen. Results showed that TNFalpha mRNA was constitutively expressed in parts of the tissues both in stimulated and unstimulated fish and the expression could be enhanced after the pathogen infection.