Background: Kraits (genus Bungarus) and cobras (genus Naja) are two representative toxic genera of elapids in the old world. Although they are closely related genera and both of their venoms are very toxic, the compositions of their venoms are very different. To unveil their detailed venoms and their evolutionary patterns, we constructed venom gland cDNA libraries and genomic bacterial artificial chromosome (BAC) libraries for Bungarus multicinctus and Naja atra, respectively. We sequenced about 1500 cDNA clones for each of the venom cDNA libraries and screened BAC libraries of the two snakes by blot analysis using four kinds of toxin probes; i.e., three-finger toxin (3FTx), phospholipase A2 (PLA2), kunitz-type protease inhibitor (Kunitz), and natriuretic peptide (NP). Results: In total, 1092 valid expressed sequences tags (ESTs) for B. multicinctus and 1166 ESTs for N. atra were generated. About 70% of these ESTs can be annotated as snake toxin transcripts. 3FTx (64.5%) and β bungarotoxin (25.1%) comprise the main toxin classes in B. multicinctus, while 3FTx (95.8%) is the dominant toxin in N. atra. We also observed several less abundant venom families in B. multicinctus and N. atra, such as PLA2, C-type lectins, and Kunitz. Peculiarly a cluster of NP precursors with tandem NPs was detected in B. multicinctus. A total of 71 positive toxin BAC clones in B. multicinctus and N. atra were identified using four kinds of toxin probes (3FTx, PLA2, Kunitz, and NP), among which 39 3FTx-postive BACs were sequenced to reveal gene structures of 3FTx toxin genes. Conclusions: Based on the toxin ESTs and 3FTx gene sequences, the major components of B. multicinctus venom transcriptome are neurotoxins, including long chain alpha neurotoxins (α-ntx) and the recently originated β bungarotoxin, whereas the N. atra venom transcriptome mainly contains 3FTxs with cytotoxicity and neurotoxicity (short chain α-ntx). The data also revealed that tandem duplications contributed the most to the expansion of toxin multigene families. Analysis of nonsynonymous to synonymous nucleotide substitution rate ratios (dN/dS) indicates that not only multigene toxin families but also other less abundant toxins might have been under rapid diversifying evolution.

Antimicrobial peptides (AMPs) have been considered alternatives to conventional antibiotics for drug-resistant bacterial infections. However, their comparatively high toxicity toward eukaryotic cells and poor efficacy in vivo hamper their clinical application. OH-CATH30, a novel cathelicidin peptide deduced from the king cobra, possesses potent antibacterial activity in vitro . The objective of this study is to evaluate the efficacy of OH-CATH30 and its analog OH-CM6 against drug-resistant bacteria in vitro and in vivo . The MICs of OH-CATH30 and OH-CM6 ranged from 1.56 to 12.5 μg/ml against drug-resistant clinical isolates of several pathogenic species, including Escherichia coli , Pseudomonas aeruginosa , and methicillin-resistant Staphylococcus aureus . The MICs of OH-CATH30 and OH-CM6 were slightly altered in the presence of 25% human serum. OH-CATH30 and OH-CM6 killed E. coli quickly (within 60 min) by disrupting the bacterial cytoplasmic membrane. Importantly, the 50% lethal doses (LD ₅₀ ) of OH-CATH30 and OH-CM6 in mice following intraperitoneal (i.p.) injection were 120 mg/kg of body weight and 100 mg/kg, respectively, and no death was observed at any dose up to 160 mg/kg following subcutaneous (s.c.) injection. Moreover, 10 mg/kg OH-CATH30 or OH-CM6 significantly decreased the bacterial counts as well as the inflammatory response in a mouse thigh infection model and rescued infected mice in a bacteremia model induced by drug-resistant E. coli . Taken together, our findings demonstrate that the natural cathelicidin peptide OH-CATH30 and its analogs exhibit relatively low toxicity and potent efficacy in mouse models, indicating that they may have therapeutic potential against the systemic infections caused by drug-resistant bacteria.

Trefoil factor 3 (TFF3) is a small peptide that plays an important role in mucosal protection, cell proliferation, and cell migration. The aberrant expression of TFF3 is correlated with gastrointestinal inflammation, solid tumors, and other clinical diseases. The objective of this study was to identify the distribution characteristics of serum TFF3 in common clinical diseases. A large prospective randomized study of 1,072 Chinese patients was performed using an enzyme-linked immunosorbent assay (ELISA) to examine the serum TFF3 concentrations in patients with different diseases. A matched case-control study was conducted on patients with chronic kidney disease (CKD) stages 1–5. Immunohistochemistry (IHC) was performed using renal tissues to determine the relationship between the severity of CKD and the serum and urine concentrations of TFF3 peptides. The mean serum concentrations of TFF3 in patients with CKD, metastatic and secondary carcinoma (MC) and acute gastroenteritis (AG) (200.9 ng/ml, 95.7 ng/ml and 71.7 ng/ml, respectively) were significantly higher than those in patients with other common clinical diseases. A positive correlation tendency was observed between the serum TFF3 concentrations and the severity of CKD. The mean serum TFF3 values for CKD stages 1–5 were 23.6 ng/ml, 29.9 ng/ml, 54.9 ng/ml, 85.0 ng/ml and 176.6 ng/ml, respectively. The same trend was observed in the urine TFF3 concentrations and the CKD stages. The creatinine(Cr)-corrected concentrations of TFF3 in urine were 367.1 ng/mg·Cr, 910.6 ng/mg·Cr, 1,149.0 ng/mg·Cr, 1,610.0 ng/mg·Cr and 3,475.0 ng/mg·Cr for CKD stages 1–5, respectively. IHC revealed that TFF3 expression was concentrated in tubular epithelial cells. The influence of kidney injuries must be fully considered when performing clinical TFF3 research. Further studies on TFF3 in CKD will contribute to our understanding of its pathological roles and mechanisms in other diseases.

In vertebrates, non-lens βγ-crystallins are widely expressed in various tissues, but their functions are unknown. The molecular mechanisms of trefoil factors, initiators of mucosal healing and being greatly involved in tumorigenesis, have remained elusive. A naturally existing 72-kDa complex of non-lens βγ-crystallin (α-subunit) and trefoil factor (β-subunit), named βγ-CAT, was identified from frog Bombina maxima skin secretions. Its α-subunit and β-subunit (containing three trefoil factor domains), with a non-covalently linked form of αβ₂, show significant sequence homology to ep37 proteins, a group of non-lens βγ-crystallins identified in newt Cynops pyrrhogaster and mammalian trefoil factors, respectively. βγ-CAT showed potent hemolytic activity on mammalian erythrocytes. The specific antiserum against each subunit was able to neutralize its hemolytic activity, indicating that the two subunits are functionally associated. βγ-CAT formed membrane pores with a functional diameter about 2.0 nm, leading to K⁺ efflux and colloid-osmotic hemolysis. High molecular weight SDS-stable oligomers (>240-kDa) were detected by antibodies against the α-subunit with Western blotting. Furthermore, βγ-CAT showed multiple cellular effects on human umbilical vein endothelial cells. Low dosages of βγ-CAT (25–50 pM) were able to stimulate cell migration and wound healing. At high concentrations, it induced cell detachment (EC₅₀ 10 nM) and apoptosis. βγ-CAT was rapidly endocytosed via intracellular vacuole formation. Under confocal microscope, some of the vacuoles were translocated to nucleus and partially fused with nuclear membrane. Bafilomycin A1 (a specific inhibitor of the vacuolar-type ATPase) and nocodazole (an agent of microtuble depolymerizing), while inhibited βγ-CAT induced vacuole formation, significantly inhibited βγ-CAT induced cell detachment, suggesting that βγ-CAT endocytosis is important for its activities. These findings illustrate novel cellular functions of non-lens βγ-cyrstallins and action mechanism via association with trefoil factors, serving as clues for investigating the possible occurrence of similar molecules and action mechanisms in mammals.