Cho-Fat Hui

Epinecidin-1, an antimicrobial peptide from fish (Epinephelus coioides) which has an antitumor effect like lytic peptides in human fibrosarcoma cells

Epinecidin-1, a synthetic 21-mer antimicrobial peptide originally identified from grouper (Epinephelus coioides), specifically exhibited high antimicrobial activities against both Gram-negative and Gram-positive bacteria. In the current study we report on the in vitro cytotoxicity of the peptide, an important factor before it can be considered for further applications in cancer therapy. The cytotoxicity of epinecidin-1 was investigated against several cancer cells (A549, HA59T/VGH, HeLa, HepG2, HT1080, RAW264.7, and U937) and normal cells (AML-12, NIH3T3, and WS-1) with the MTT assay, and the inhibition of cancer cell growth was confirmed by a soft agar assay and scanning electron microscopy. However, cell variations were detected with AO/EtBr staining, while apoptosis and necrosis gene expressions in HT1080 cells after treatment with the epinecidin-1 peptide and Nec-1 showed that epinecidin-1 had an anti-necrosis function in HT1080 cells. The data presented here indicate that epinecidin-1 has in vitro antitumor activity against the HT1080 cell line, and functions like lytic peptides. In addition, our results suggest that epinecidin-1 may prove to be an effective chemotherapeutic agent for human fibrosarcoma cells in the future.

Variation in mitochondrial DNA and population structure of the Taipei treefrog Rhacophorus taipeianus in Taiwan.

Mitochondrial DNA (mtDNA) from 40 samples of the Taipei treefrog Rhacophorus taipeianus collected from seven populations in Taiwan were sequenced to document the DNA sequence variation in anuran mtDNA and to elucidate the phylogeographic population structure in the Taipei treefrog. Sequences of 722–764 bases in length, including a 108‐bp segment of the cytochrome b gene and a 614–656‐bp D‐loop segment, were obtained by direct sequencing using polymerase chain reaction (PCR). The variation in length was due to a 40‐bp region that tandemly repeated four to five times in the D‐loop region. The first repeat is the most conserved one among the five repeats because there are no variable sites in this repeat. Besides the 40‐bp length variation, 28 positions in the 764‐bp sequences are variable and distributed evenly in the cytochrome b gene fragment and D‐loop region. Variation in the D‐loop of the Taipei treefrog is comparable to those of other vertebrates. Two well‐differentiated lineages (northern and central) differing by mean sequence divergence of 1.7% are identified and concordant with their geographic distributions. The two lineages are inferred to have split from a common ancestral population in the early Pleistocene. However, the interpopulation divergence of the northern lineage (< 0.33%) is apparently lower than that of the central lineage (1.11%), implying that the two lineages evolved independently and had different demographic histories after divergence. This study reveals that anuran D‐loop has potential as a genetic marker in phylogenetic and population genetic analyses of anurans.

Insights into the antibacterial and immunomodulatory functions of the antimicrobial peptide, epinecidin-1, against Vibrio vulnificus infection in zebrafish.

In the present study, we used Vibrio vulnificus and a zebrafish model system to investigate the inhibitory effect of epinecidin-1 on acute bacterial infection and studied the impacts of pretreatment, co-treatment, and post-treatment with epinecidin-1 on its protective efficacy. In vivo experiments showed that co-treatment with epinecidin-1 and V. vulnificus achieved 78%-97% survival rates after 30 days. When epinecidin-1 and V. vulnificus were co-injected into zebrafish and zebrafish were re-challenged with V. vulnificus after 30 days, zebrafish had survival rates of 22%-47%. Pretreatment and post-treatment with epinecidin-1 obtained respective survival rates of 57% and 60%. In addition, epinecidin-1 modulated the expressions of immune-responsive genes like interleukin (IL)-10, IL-1b, tumor necrosis factor-α, and interferon-γ as analyzed by a microarray and qPCR approach. This study demonstrates the use of epinecidin-1 to develop inactivated material for fish bacterial infections which can provide guidelines for the future design of epinecidin-1-bacterial formulations for various in vivo applications.

Molecular Cloning and Tissue-Specific, Developmental-Stage-Specific, and Hormonal Regulation of IGFBP3 Gene in Zebrafish

The biological functions of insulin-like growth factor (IGF) I and II are modulated by a family of IGF-binding proteins (IGFBPs) in complex IGF-dependent and IGF-independent pathways. For further understanding of the actions of IGFs, some of these binding proteins have been cloned and characterized. We report the molecular cloning of IGFBP-3 cDNA for zebrafish. The tissue-specific and developmental stage-specific expression of IGFBP-3 and the hormonal regulation of its expression have also been determined by comparative reverse transcription polymerase chain reaction. Zebrafish IGFBP-3 cDNA contains an open reading frame of 879 bp, encoding a polypeptide of 293 amino acid residues. Results of this analysis revealed high levels of IGFBP-3 messenger RNA in ovary and fin tissue. Expression of IGFBP-3 mRNA was throughout the entire embryonic development, with the highest level of expression observed at 36 hours after the onset of development. Elevated levels of expression of IGFBP-3 were observed 24 hours after injection with IGF-I and 48 hours with IGF-II or insulin. These results suggest that the expression of IGFBP3 gene might be modulated by IGF-I, IGF-II, and insulin.

Epinecidin-1 peptide induces apoptosis which enhances antitumor effects in human leukemia U937 cells.

Epinecidin-1 is an antimicrobial peptide present in the grouper (Epinephelus coioides). In this study, the antitumor activity of a synthetic epinecidin-1 peptide was tested. The in vitro results showed that epinecidin-1 inhibited the proliferation of human leukemia U937 cells and increased the ADP/ATP ratio after 24h of treatment. The DNA fragmentation assay, flow cytometric assay, and caspases-3, -8, and -9 assays indicated that epinecidin-1 could induce apoptosis in U937 cells. Real-time RT-PCR results showed regular increases in tumor necrosis factor (TNF)-alpha after treatment with 4 microg/ml epinecidin-1 from 4 to 24h; interleukin (IL)-10, interferon (INF)-r, p53, IL-15, and IL-6 increased after treatment with 2 microg/ml epinecidin-1 for 4-12h. These results suggest that the epinecidn-1 inhibited U937 cells, induced apoptosis in response to cytokine production, and may have pleiotropic effects on different cells.

Molecular phylogeny of 48 species of damselfishes (Perciformes: Pomacentridae) using 12S mtDNA sequences

Phylogenetic relastionships within the family pomacentridae teleostei: Perciformes) were inferred by analyzing a portion of the 12S mitochondrial ribosomal DNA gene. Thirty-four pomacentrid species were sequenced for this study and the resulting data were combined with previously published pomacentrid sequence data to form a combined matrix of 53 pomacentrids representing 48 different species in 18 genera. Four outgroup species were also drawn from published data; these taxa were taken from the other three putative families of the suborder Labroidei, as well as a single representative of the family Moronidae. The data set contained 1053 data columns after alignment according to ribosomal secondary structure and the removal of all ambiguously aligned positions. The resulting strict consensus tree topology generally agreed with the previous molecular hypothesis, and recovers a monophyletic Pomacentridae and subfamily Amphiprioninae. The two other subfamilies included, Chrominae and Pomacentrinae, were found to be polyphyletic. A monophyletic group consisting of the Amphiprioninae, Pomacentrus, Acanthochromis, Amblyglyphidodon, Neoglyphidodon, Chrysiptera, Neopomacentrus, and Teixeirichthys was found. This group was recovered as the sister group to a clade consisting of a paraphyletic Chromis and a monophyletic Dascyllus. A sister-group relationship between the genus Pomacentrus and the subfamily Amphiprioninae was observed.

Truncated antimicrobial peptides from marine organisms retain anticancer activity and antibacterial activity against multidrug-resistant Staphylococcus aureus

Antimicrobial peptides (AMPs) were recently determined to be potential candidates for treating drug-resistant bacterial infections. The aim of this study was to develop shorter AMP fragments that combine maximal bactericidal effect with minimal synthesis cost. We first synthesized a series of truncated forms of AMPs (anti-lipopolysaccharide factor from shrimp, epinecidin from grouper, and pardaxin from Pardachirus marmoratus). The minimum inhibitory concentrations (MICs) of modified AMPs against ten bacterial species were determined. We also examined the synergy between peptide and non-peptide antibiotics. In addition, we measured the inhibitory rate of cancer cells treated with AMPs by MTS assay. We found that two modified antibacterial peptides (epinecidin-8 and pardaxin-6) had a broad range of action against both gram-positive and gram-negative bacteria. Furthermore, epinecidin and pardaxin were demonstrated to have high antibacterial and anticancer activities, and both AMPs resulted in a significant synergistic improvement in the potencies of streptomycin and kanamycin against methicillin-resistant Staphylococcus aureus. Neither AMP induced significant hemolysis at their MICs. In addition, both AMPs inhibited human epithelial carcinoma (HeLa) and fibrosarcoma (HT-1080) cell growth. The functions of these truncated AMPs were similar to those of their full-length equivalents. In conclusion, we have successfully identified shorter, inexpensive fragments with maximal bactericidal activity. This study also provides an excellent basis for the investigation of potential synergies between peptide and non-peptide antibiotics, for a broad range of antimicrobial and anticancer activities.

Shrimp anti-lipopolysaccharide factor peptide enhances the antitumor activity of cisplatin in vitro and inhibits HeLa cells growth in nude mice

The antitumor activity of the shrimp anti-lipopolysaccharide factor (SALF), an antimicrobial peptide, was not previously examined. In this study, a synthetic SALF was tested for antitumor activity using HeLa cells as the study model. We show that the SALF inhibited the proliferation of HeLa cells and reduced colony formation in a soft agar assay. An enhanced effect was observed when the SALF and cisplatin were used in combination, which caused significant inhibition of HeLa cells. Scanning electron microscopy (SEM) and transmission electron microscopy (TEM) showed that the SALF altered the membrane structure similar to what a lytic peptide does. A flow cytometric analysis, qRT-PCR, and Western blotting showed that the SALF induced apoptosis, activated caspases-6, -7, and -9, and downregulated Bcl-2 and nuclear factor (NF)-kappaB suggesting that the SALF induces apoptosis through the death receptor/NF-kappaB signaling pathway. An in vivo analysis revealed that the SALF displayed significant tumor suppressive activity in mice with tumor xenografts. Overall, these results indicated that the SALF possesses the potential to be a novel therapeutic agent for treating cervical cancer.

Antimicrobial peptide of an anti-lipopolysaccharide factor modulates of the inflammatory response in RAW264.7 cells.

In this study, to clarify the protective mechanism of a peptide from shrimp anti-lipopolysaccharide (LPS) factor (SALF) against endotoxin shock, we evaluated the effects of the SALF and LPS on the production and release of tumor necrosis factor (TNF)-alphain vitro using the RAW264.7 murine macrophage cell line. Stimulation by LPS induced the production of inflammatory cytokines, and the SALF was able to modulate TNF-alpha production in LPS-stimulated RAW264.7 cells. Microarray studies revealed a transcriptional profile which was assessed in the presence or absence of the SALF by a quantitative real-time polymerase chain reaction. Pretreatment with the SALF significantly downregulated the expression of nuclear factor (NF)-kappaB in the presence of LPS. In contrast, pretreatment with the SALF significantly elevated the expressions of Anp32a, CLU, and SLPI, which are considered to be immune-related genes in the presence of LPS. Inhibitor studies suggested that the SALFs modulation of LPS-induced TNF-alpha production involved a complex mechanism with mitogen-activated protein kinase kinase, calcium, and protein kinase C. The data from this study, which imply that the SALF can suppress TNF-alpha production, suggest a role for the SALF in the defense mechanism which can potentially be applied to mammals for endotoxin treatment.

Recombinant protein composed of Pseudomonas exotoxin A, outer membrane proteins I and F as vaccine against P. aeruginosa infection

Abstract We have constructed a chimeric protein composed of the receptor binding and membrane translocation domains of Pseudomonas exotoxin A (PE) with the outer membrane proteins I and F, together designated as PEIF. The potential of PEIF as a vaccine against Pseudomonas infection was evaluated in BALB/c mice and New Zealand white rabbits. We examined titers of anti-PE and anti-OprF antibodies, and the ability both to neutralize PE cytotoxicity and to increase opsonophagocytic uptake of Pseudomonas aeruginosa strain PAO1, serogroups 2 and 6. The results showed that PEIF can induce antibodies not only to neutralize the PE cytotoxicity but also to promote the uptake of various strains of P. aeruginosa by murine peritoneal macrophages. In a burned mouse model, PEIF afforded significant protection against infection by the homologous P. aeruginosa strain PAO1, heterologous serogroup 2, and the PE hyperproducing strain PA103. These observations thus indicate that PEIF may be used as a novel vaccine against P. aeruginosa infection.