Molecular characterization of cu/Zn SOD gene in Asian clam Corbicula fluminea and mRNA expression and enzymatic activity modulation induced by metals.

The copper/zinc superoxide dismutase (Cu/Zn SOD) could effectively eliminate reactive oxygen species (ROS). In this study, the cDNA sequence of Cu/Zn SOD from Corbicula fluminea (designated as CfCu/Zn SOD) was cloned by the rapid amplification of cDNA ends (RACE) approaches. The full-length cDNA of CfCu/Zn SOD was of 1288 bp, including a 465 bp ORF encoding a protein of 154 amino acids. Two SOD family signatures were identified in CfCu/Zn SOD amino acids sequence. Multiple sequence alignments indicated that CfCu/Zn SOD amino acid sequences exhibited high similarities to those of other species. The tissue distribution of the CfCu/Zn SOD of C. fluminea was detected by fluorescent real-time quantitative PCR. The mRNA expression levels in digestive gland and gill were significantly higher than those of other four tissues. In response to metal ions (Cd2+, Cu2+, and Pb2+) challenge, the expression and enzymatic activities of CfCu/Zn SOD in the gills were measured after exposure for 0, 6, 12, 24, 48, and 72 h. The results showed that the mRNA expression and enzymatic activities of CfCu/Zn SOD could be induced by three metal ions. After Cd2+ and Cu2+ exposure, the mRNA expression levels increased gradually and reach the peak at 24 h, afterwards it slowed down. The change trends of enzymatic activities were similar to the mRNA expression. After Pb2+ exposure, the expression and activities CfCu/Zn SOD were raised up gradually and reach the highest value at 72 h. All these results indicated that the mRNA expression and enzymatic activity of CfCu/Zn SOD are sensitive to Cd2+, Cu2+ and Pb2+ ions and can be used as molecular biomarkers of metal pollution in water.

Molecular cloning and functional characterization of novel antimicrobial peptides from the skin of brown frog, Rana zhenhaiensis.

Rana zhenhaiensis, a species of brown frog, is widely distributed in central and south China. In the present study, a total of 14 cDNA sequences encoding eight novel antimicrobial peptides (AMPs) were cloned from the synthesized cDNAs of R. zhenhaiensis skin. The eight novel AMPs belong to four families: brevinin-1 (four peptides), brevinin-2 (one peptide), ranatuerin-2 (one peptide) and chensinin-1 (two peptides), five AMPs from the four families (brevinin-1ZHa, brevinin-1ZHb, brevinin-2ZHa, ranatuerin-2ZHa and chensinin-1ZHa) were chemically synthesized, their antimicrobial and hemolytic activities were examined. The results indicated that the five AMPs possess different antimicrobial and hemolytic activities. Of these, brevinin-2ZHa exhibited the strongest and most broad-spectrum antimicrobial activity. Furthermore, scanning electron microscopy (SEM) experiment was carried out to investigate the potential antimicrobial mechanism of chensinin-1ZHa. The result indicated that chensinin-1ZHa may exert its function through disruption of the bacterial membrane.

Effect of tetramethylpyrazine on primary afferent transmission mediated by P2X3 receptor in neuropathic pain states.

Neuropathic pain is the most difficult type of pain to treat. The P2X(3) receptors play a crucial role in facilitating pain transmission at peripheral and spinal sites. The present research investigated the effects of tetramethylpyrazine (TMP) on the primary afferent transmission induced by P2X(3) receptor in neuropathic pain states. Chronic constriction injury (CCI) model was adopted. Sprague-Dawley male rats (n=30) had been randomly divided into normal saline (sham+NS) group (I), TMP group (II), sham group (III), CCI+TMP group (IV), and CCI group (V). Mechanical withdrawal threshold (MWT) and thermal withdrawal latency (TWL) were measured and P2X(3) immunoreactivity in L4/L5 dorsal root ganglion (DRG) and spinal cord was detected by immunohistochemistry. The mechanical withdrawal threshold and thermal withdrawal latency in group V were lower than those in groups I-III or IV (p<0.05), while P2X(3) receptor expression of L4/L5 DRG and spinal cord in group V was higher than those in groups I-III (p<0.01) or group IV (p<0.05). The mechanical withdrawal threshold, thermal withdrawal latency and P2X(3) immunoreactivity of L4/L5 DRG and spinal cord in group IV showed no significant difference compared with those in groups I, II or III (p>0.05). The amplitudes of the currents in group V (CCI) were much larger than those obtained in other groups after application of same concentration adenosine 5'-triphosphate disodium (ATP) (p<0.01). alpha,beta-Methylene-ATP (alpha,beta-meATP)-activated currents in DRG neurons of CCI rats were more obvious than those obtained in other group rats (p<0.01). The results showed that TMP may inhibit the primary afferent transmission of neuropathic pain induced by P2X(3) receptor.