Publications by authors named "Chin-I Chang"

8 Publications

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Determining the cleavage site for the mature antimicrobial peptide of Nile tilapia β-defensin using 2D electrophoresis, western blot, and mass spectrometry analysis.

Fish Shellfish Immunol 2017 Mar 9;62:41-46. Epub 2017 Jan 9.

Department of Marine Biotechnology, National Kaohsiung Marine University, 142 Hai-Chuan Road, Kaohsiung 81157, Taiwan.

Several proteomic techniques were used to determine the cleavage site of the mature antimicrobial peptide of Nile tilapia β-defensin. The computer-predicted Nile tilapia β-defensin (ASFPWSCLSLSGVCRKVCLPTELFFGPLGCGKGSLCCVSHFL) composed of 42 amino acids was chemically synthesized and prepared to produce an antibody for Western blotting. Total proteins from the skin of the Nile tilapia were separated on two-dimensional electrophoresis, and the spot of Nile tilapia β-defensin was recognized using Western blot analysis. It was then excised and extracted from the gel. The precise molecular mass of this spot was determined by LC-MS/MS spectrometry. Four major peptides were discovered, with molecular weights of 4293.2 Da, 4306.5 Da, 4678.9 Da, and 4715.0 Da. The calculated mass of the 40-amino-acid sequence (FPWSCLSLSGVCRKVCLPTELFFGPLGCGKGSLCCVSHFL) of Nile tilapia β-defensin starting from Phe27 and ending with Leu66 was 4293.18 Da, which completely matched the 4293.2 Da peptide that was obtained from the mass spectrometry analysis. This result confirmed that the cleavage site for the mature C-terminal Nile tilapia β-defensin is at residue Ser26-Phe27, not at Ala24-25 as predicted by computer analysis. This study provides a simple but reliable model to determine the cleavage site for a mature antimicrobial peptide.
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http://dx.doi.org/10.1016/j.fsi.2017.01.010DOI Listing
March 2017

Comparative proteomic analysis of Litopenaeus vannamei gills after vaccination with two WSSV structural proteins.

Fish Shellfish Immunol 2016 Feb 4;49:306-14. Epub 2016 Jan 4.

Department of Marine Biotechnology, National Kaohsiung Marine University, Kaohsiung, 81157, Taiwan, ROC. Electronic address:

White spot syndrome virus (WSSV) is one of the most devastating viral pathogens of cultured shrimp worldwide. Recently published papers show the ability of WSSV structural protein VP28 to vaccinate shrimp and raise protection against the virus. This study attempted to identify the joining proteins of the aforementioned shrimp quasi-immune response by proteomic analysis. The other envelope protein, VP36B, was used as the non-protective subunit vaccine control. Shrimp were intramuscularly injected with rVPs or PBS on day 1 and day 4 and then on day 7 their gill tissues were sampled. The two-dimensional electrophoresis (2-DE) patterns of gill proteins between vaccinated and PBS groups were compared and 20 differentially expressed proteins identified by mass spectrometry, some of which were validated in gill and hemocyte tissues using real-time quantitative RT-PCR. Many of identified proteins and their expression levels also linked with the shrimp response during WSSV infection. The list of up-regulated protein spots found exclusively in rVP28-vaccinated shrimp include calreticulin and heat shock protein 70 with chaperone properties, ubiquitin, and others. The two serine proteases, chymotrypsin and trypsin, were significantly increased in shrimp of both vaccinated groups compared to PBS controls. The information presented here should be useful for gaining insight into invertebrate immunity.
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http://dx.doi.org/10.1016/j.fsi.2015.12.044DOI Listing
February 2016

Immune responses of prophenoloxidase and cytosolic manganese superoxide dismutase in the freshwater crayfish Cherax quadricarinatus against a virus and bacterium.

Mol Immunol 2013 Nov 10;56(1-2):72-80. Epub 2013 May 10.

Department of Marine Biotechnology, National Kaohsiung Marine University, Kaohsiung 81157, Taiwan, ROC.

Prophenoloxidase (proPO) and cytosolic manganese superoxide dismutase (cytMnSOD) play crucial roles in crustacean innate immunity. In the present study, both of the above genes were cloned from hemocytes of the red claw crayfish Cherax quadricarinatus. A phylogenetic analysis of the amino acid sequences showed that C. quadricarinatus proPO and cytMnSOD were more closely related to the proPO and cytMnSOD of other crayfish than to those of penaeids, crabs, lobsters, or freshwater prawns. A tissue distribution analysis revealed that proPO was primarily expressed in hemocytes, gills, and the heart, while cytMnSOD was detected in all tissues examined. All of the crayfish artificially infected with white spot syndrome virus (WSSV) died within 4 days. According to a non-lethal dose, there was no mortality in crayfish when infected deliberately with Aeromonas hydrophila. Total hemocyte counts (THCs) had significantly decreased in crayfish at 48 and 72 h after infection with WSSV compared to the control group. In contrast, THCs of crayfish after A. hydrophila challenge had recovered by 48 and 72 h from a lower level at 24 h. There were similar responses in enzyme activities toward WSSV and A. hydrophila infection. Phenoloxidase (PO) and superoxide dismutase (SOD) activities per hemocyte significantly increased from 48 to 72 h compared to the control group. After WSSV challenge, expressions of proPO and cytMnSOD transcripts in hemocytes significantly decreased at 12h, then had respectively recovered and increased at 24 h. At 48-72 h, transcript levels were finally downregulated. No significant differences in the expression profiles of proPO and cytMnSOD were observed between the A. hydrophila-infected and control groups, besides the significant upregulation at 24h post-infection. These results implicate proPO and cytMnSOD in the immune response, and they presented similar expression patterns, although different defense mechanisms may exist for crayfish induced by WSSV and A. hydrophila.
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http://dx.doi.org/10.1016/j.molimm.2013.03.023DOI Listing
November 2013

Viral resistance and immune responses of the shrimp Litopenaeus vannamei vaccinated by two WSSV structural proteins.

Immunol Lett 2012 Nov-Dec;148(1):41-8. Epub 2012 Aug 13.

Department of Marine Biotechnology, National Kaohsiung Marine University, Kaohsiung 81157, Taiwan, ROC.

Although adaptive immunity is believed to exist only in higher vertebrates, recent studies showed the ability to vaccinate shrimp and other crayfish against white spot syndrome virus (WSSV). This study attempted to establish parameters of vaccination coordinated with subsequent viral challenge to gain insights into the mechanisms of the protective response of penaeid shrimp. Two WSSV envelope proteins, VP28 and VP36B, were used as subunit vaccines expressed in Escherichia coli followed by histidine-tag affinity chromatographic purification. Shrimp vaccinated with the recombinant WSSV proteins and challenged with diluted WSSV inocula were intramuscularly injected in order to give a precise load. Results of the viral challenge trials showed complete survival in the rVP28 group in contrast to the rVP36B and PBS groups which exhibited 100% mortality. But this effective protection was exclusively induced from a combination of a higher dosage of rVP28 and a lower viral challenge pressure. The innate immune parameters analyzed among the three groups revealed that rVP28-treated shrimp showed the highest activity level (p<0.05) of phenoloxidase and superoxide dismutase during the entire period of 7 days post-vaccination. But there were no significant differences (p>0.05) in mRNA abundances of the Down syndrome cell adhesion molecule (Dscam) among all groups. In addition, total hemocyte counts significantly decreased in shrimp treated with the recombinant viral proteins compared to the PBS group. These results indicated that the existence of structure- and dose-dependent protective responses and the elevated innate immunity in shrimp following a protein-based vaccination might be responsible for conferring resistance against WSSV.
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http://dx.doi.org/10.1016/j.imlet.2012.08.004DOI Listing
April 2013

Simultaneous detection of multiple fish pathogens using a naked-eye readable DNA microarray.

Sensors (Basel) 2012 29;12(3):2710-28. Epub 2012 Feb 29.

Aquaculture Division, Fisheries Research Institute, Ministry of Agriculture, Keelung 20246, Taiwan.

We coupled 16S rDNA PCR and DNA hybridization technology to construct a microarray for simultaneous detection and discrimination of eight fish pathogens (Aeromonas hydrophila, Edwardsiella tarda, Flavobacterium columnare, Lactococcus garvieae, Photobacterium damselae, Pseudomonas anguilliseptica, Streptococcus iniae and Vibrio anguillarum) commonly encountered in aquaculture. The array comprised short oligonucleotide probes (30 mer) complementary to the polymorphic regions of 16S rRNA genes for the target pathogens. Targets annealed to the microarray probes were reacted with streptavidin-conjugated alkaline phosphatase and nitro blue tetrazolium/5-bromo-4-chloro-3'-indolylphosphate, p-toluidine salt (NBT/BCIP), resulting in blue spots that are easily visualized by the naked eye. Testing was performed against a total of 168 bacterial strains, i.e., 26 representative collection strains, 81 isolates of target fish pathogens, and 61 ecologically or phylogenetically related strains. The results showed that each probe consistently identified its corresponding target strain with 100% specificity. The detection limit of the microarray was estimated to be in the range of 1 pg for genomic DNA and 10(3) CFU/mL for pure pathogen cultures. These high specificity and sensitivity results demonstrate the feasibility of using DNA microarrays in the diagnostic detection of fish pathogens.
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http://dx.doi.org/10.3390/s120302710DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3376613PMC
October 2012

Two cathelicidin genes are present in both rainbow trout (Oncorhynchus mykiss) and atlantic salmon (Salmo salar).

Antimicrob Agents Chemother 2006 Jan;50(1):185-95

Scottish Fish Immunology Research Centre, School of Biological Sciences, University of Aberdeen, Zoology Building, Tillydrone Avenue, Aberdeen AB24 2TZ, United Kingdom.

Further to the previous finding of the rainbow trout rtCATH_1 gene, this paper describes three more cathelicidin genes found in salmonids: two in Atlantic salmon, named asCATH_1 and asCATH_2, and one in rainbow trout, named rtCATH_2. All the three new salmonid cathelicidin genes share the common characteristics of mammalian cathelicidin genes, such as consisting of four exons and possessing a highly conserved preproregion and four invariant cysteines clustered in the C-terminal region of the cathelin-like domain. The asCATH_1 gene is homologous to the rainbow trout rtCATH_1 gene, in that it possesses three repeat motifs of TGGGGGTGGC in exon IV and two cysteine residues in the predicted mature peptide, while the asCATH_2 gene and rtCATH_2 gene are homologues of each other, with 96% nucleotide identity. Salmonid cathelicidins possess the same elastase-sensitive residue, threonine, as hagfish cathelicidins and the rabbit CAP18 molecule. The cleavage site of the four salmonid cathelicidins is within a conserved amino acid motif of QKIRTRR, which is at the beginning of the sequence encoded by exon IV. Two 36-residue peptides corresponding to the core part of rtCATH_1 and rtCATH_2 were chemically synthesized and shown to exhibit potent antimicrobial activity. rtCATH_2 was expressed constitutively in gill, head kidney, intestine, skin and spleen, while the expression of rtCATH_1 was inducible in gill, head kidney, and spleen after bacterial challenge. Four cathelicidin genes have now been characterized in salmonids and two were identified in hagfish, confirming that cathelicidin genes evolved early and are likely present in all vertebrates.
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http://dx.doi.org/10.1128/AAC.50.1.185-195.2006DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1346769PMC
January 2006

Identification of a novel cathelicidin gene in the rainbow trout, Oncorhynchus mykiss.

Infect Immun 2005 Aug;73(8):5053-64

Scottish Fish Immunology Research Centre, School of Biological Sciences, University of Aberdeen, Zoology Building, Tillydrone Avenue, Aberdeen, AB24 2TZ, United Kingdom.

We report the cloning of a novel antimicrobial peptide gene, termed rtCATH_1, found in the rainbow trout, Oncorhynchus mykiss. The predicted 216-residue rtCATH_1 prepropeptide consists of three domains: a 22-residue signal peptide, a 128-residue cathelin-like region containing two identifiable cathelicidin family signatures, and a predicted 66-residue C-terminal cationic antimicrobial peptide. This predicted mature peptide was unique in possessing features of different known (mammalian) cathelicidin subgroups, such as the cysteine-bridged family and the specific amino-acid-rich family. The rtCATH_1 gene comprises four exons, as seen in all known mammalian cathelicidin genes, and several transcription factor binding sites known to be of relevance to host defenses were identified in the 5' flanking region. By Northern blot analysis, the expression of rtCATH_1 was detected in gill, head kidney, and spleen of bacterially challenged fish. Primary cultures of head kidney leukocytes from rainbow trout stimulated with lipopolysaccharide or poly(I x C) also expressed rtCATH_1. A 36-residue peptide corresponding to the core part of the fish cathelicidin was chemically synthesized and shown to exhibit potent antimicrobial activity and a low hemolytic effect. Thus, rtCATH_1 represents a novel antimicrobial peptide gene belonging to the cathelicidin family and may play an important role in the innate immunity of rainbow trout.
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http://dx.doi.org/10.1128/IAI.73.8.5053-5064.2005DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1201231PMC
August 2005

Discovery and characterization of two types of liver-expressed antimicrobial peptide 2 (LEAP-2) genes in rainbow trout.

Vet Immunol Immunopathol 2004 Oct;101(3-4):259-69

Department of Zoology, Scottish Fish Immunology Research Centre, University of Aberdeen, Zoology Building, Tillydrone Avenue, Aberdeen AB24 2TZ, UK.

The sequences and gene organisation of two LEAP-2 molecules (LEAP-2A and LEAP-2B) from rainbow trout, Oncorhynchus mykiss are presented. Both genes consist of a 3 exon/2 intron structure, with exon sizes comparable to known mammalian genes. LEAP-2A notably differs from LEAP-2B in having larger introns and a larger 3' UTR. The predicted proteins contain a signal peptide and prodomain, followed by a mature peptide of 41 aa containing four conserved cysteines. The RXXR cleavage site to release the mature peptide was also conserved. Both genes were found to be constitutively expressed in the liver, with expression in the intestine, and to a lesser extent the skin, evident after bacterial challenge.
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http://dx.doi.org/10.1016/j.vetimm.2004.05.005DOI Listing
October 2004
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