Biomedical subjects

Anchalee Tassanakajon

Publications and source records attributed to Anchalee Tassanakajon.

12 recordsLinked to original sources

PubMed2006

Molecular cloning, characterization and expression of a masquerade-like serine proteinase homologue from black tiger shrimp Penaeus monodon.

A full-length cDNA of a masquerade-like serine proteinase homologue (PmMasSPH) of Penaeus monodon was cloned and characterized by rapid amplification cDNA end (RACE) method. The complete cDNA sequence of 1958bp contains an open reading frame (ORF) of 1572bp, encoding a 523 amino acid protein including a 19 amino acid signal peptide. The calculated molecular mass of the mature protein (504 amino acids) is 51.58kDa with an estimated pI of 4.86. PmMasSPH has most of the structural characteristics of insect prophenoloxidase activating factors (PPAFs) (the N-terminal clip domain and the C-terminal serine proteinase-like domain) but in the N-terminal region there are extensive glycine-rich repeats (LGGQGGG). Sequence comparison showed that the deduced amino acid of PmMasSPH has an overall similarity of 69%, 68% and 61% to those of Apis mellifera PPAF, Callinectes sapidus PPAF and Tenebrio molitor PPAF, respectively. A neighbour-joining tree revealed a clear differentiation of each species and also indicated that PmMasSPH and C. sapidus PPAF are closely related phylogenetically. In situ hybridisation and real-time RT-PCR analyses showed that PmMasSPH transcript in haemocytes of P. monodon increased within 24h after Vibrio harveyi injection.

Animals↗

PubMed2006

Penaeus monodon gene discovery project: the generation of an EST collection and establishment of a database.

A large-scale expressed sequence tag (EST) sequencing project was undertaken for the purpose of gene discovery in the black tiger shrimp Penaeus monodon. Initially, 15 cDNA libraries were constructed from different tissues (eyestalk, hepatopancrease, haematopoietic tissue, haemocyte, lymphoid organ, and ovary) of shrimp, reared under normal or stress conditions, to identify tissue-specific genes and genes responding to infection and heat stress. A total of 10,100 clones were analyzed by single-pass sequencing from the 5' end. Clustering and assembling of these ESTs resulted in a total of 4845 unique sequences with 917 overlapping contigs and 3928 singletons. The redundancy of each cDNA library ranged from 13.4% to 61.3% with an overall redundancy of 61.1%. About half of these ESTs (2365 clones, 48.8%) showed significant homology (BLASTX, e-values <10(-4)) to known genes. A high proportion of P. monodon ESTs was most similar to the predicted protein sequences from various organisms, e.g. Homo sapiens (9%), Mus musculus (7%), Drosophila (6%), Gallus sp.(6%), and Anopheles (5%). Only 6% showed the highest similarity to other known genes from shrimp due to the limited sequence entries of the species in the public database. Several tissue-specific transcripts were identified as well as the candidate genes that may be implicated in the immune response. In addition, bioinformatic mining of microsatellites from the P. monodon ESTs identified 997 unique microsatellite containing ESTs in which 74 loci resided within the genes of known functions. Consequently, the P. monodon EST database was established. The EST sequence data and the BLAST results were stored and made available through a web-accessible database (). This EST database provides a useful resource for gene identification and functional genomic studies of shrimp.

Animals↗

PubMed2006

A five-domain Kazal-type serine proteinase inhibitor from black tiger shrimp Penaeus monodon and its inhibitory activities.

A novel five-domain Kazal-type serine proteinase inhibitor, SPIPm2, identified from the hemocyte cDNA library of black tiger shrimp Penaeus monodon was successfully expressed in the Escherichia coli expression system. The expressed recombinant SPIPm2 (rSPIPm2) as inclusion bodies was solubilized with a sodium carbonate buffer, pH10, and purified by gel filtration chromatography. The molecular mass of rSPIPm2 was determined using MALDI-TOF mass spectrometry to be 29.065 kDa. The inhibitory activities of rSPIPm2 were tested against trypsin, alpha-chymotrypsin, subtilisin and elastase. The inhibitor exhibited potent inhibitory activities against subtilisin and elastase, weak inhibitory activity against trypsin, and did not inhibit chymotrypsin. Tight-binding inhibition assay suggested that the molar ratios of SPIPm2 to subtilisin and elastase were 1:2 and 1:1, respectively. The inhibition against subtilisin and elastase was a competitive type with inhibition constants (Ki) of 0.52 and 3.27 nM, respectively. The inhibitory activity of SPIPm2 against subtilisin implies that, in shrimp, it may function as a defense component against proteinases from pathogenic bacteria but the elastase inhibitory function is not known.

Amino Acid Sequence↗

PubMed2006

Differentially expressed genes in hemocytes of Vibrio harveyi-challenged shrimp Penaeus monodon.

Differential Display PCR technique (DD-PCR) was used for the analysis of altered gene expression in hemocytes of Vibrio harveyi-infected Penaeus monodon. Forty-four combinations of arbitrary and oligo(dT) primers were used to screen for differentially expressed genes. A total of 79 differentially expressed bands could be identified from 33 primer combinations. These included 48 bands (61%) whose expression level increased and 31 bands (39%) decreased after V. harveyi challenge. Subsequently, forty-eight differential display fragments were successfully reamplified and cloned. A total of 267 clones were randomly selected and sequenced. The sequence analysis showed that 85 (31%) out of 267 clones were matched with sequences in the GenBank database which represented 24 different genes with known functions. Among the known genes, glucose transporter 1, interferon-related developmental regulator 1, lysozyme, profilin, SERPINB3, were selected for further confirmation of their differentially expression patterns by real-time PCR. The results showed increasing in expression level of the selected genes in shrimp hemocytes after microbial challenge suggesting the involvement of such genes in bacterial response in shrimp. The anti-lipopolysaccharide factor type 3 (ALFPm3) gene, previously reported in P. monodon (Supungul et al., 2002) was found among the up-regulated genes but diversity due to amino acid changes was observed. Increase in ALFPm3 transcripts upon V. harveyi injection is in accordance with that found in the previous study.

Amino Acid Sequence↗

PubMed2006

PenBase, the shrimp antimicrobial peptide penaeidin database: sequence-based classification and recommended nomenclature.

Antimicrobial peptides play a major role in innate immunity. The penaeidins, initially characterized from the shrimp Litopenaeus vannamei, are a family of antimicrobial peptides that appear to be expressed in all penaeid shrimps. As of recent, a large number of penaeid nucleotide sequences have been identified from a variety of penaeid shrimp species and these sequences currently reside in several databases under unique identifiers with no nomenclatural continuity. To facilitate research in this field and avoid potential confusion due to a diverse number of nomenclatural designations, we have made a systematic effort to collect, analyse, and classify all the penaeidin sequences available in every database. We have identified a common penaeidin signature and subsequently established a classification based on amino acid sequences. In order to clarify the naming process, we have introduced a 'penaeidin nomenclature' that can be applied to all extant and future penaeidins. A specialized database, PenBase, which is freely available at , has been developed for the penaeidin family of antimicrobial peptides, to provide comprehensive information about their properties, diversity and nomenclature.

Amino Acid Sequence↗

PubMed2005

Development of gene transfer technology for black tiger shrimp, Penaeus monodon.

An effective foreign gene transfer method for shrimp would have several potential uses in the shrimp culture industry, such as in preventing infectious diseases. We evaluated two gene transfer methods and used black tiger shrimp, Penaeus monodon, as a model target species. For a promoter, we used the 1,592-bp promoter region of the EF-1alpha gene, a house-keeping gene, of kuruma shrimp Marsupenaeus japonicus. The promoter region was linked to either the gene for green fluorescence protein (GFP) or the gene for chloramphenicol acetyl transferase (CAT). The fusion genes were designated pJEF-GFP and pJEF-CAT, respectively. The pJEF-GFP gene was introduced into fertilized eggs of black tiger shrimp by microinjection and particle gun bombardment. The survival rate of the microinjected eggs was 17.6%, and 1.0% of the treated embryos were found to be GFP-positive. However, the GFP-positive embryos were damaged and embryogenesis did not progress. The survival rate of the particle-bombarded eggs was 60.6%, and 0.42% of the treated embryos were found to be GFP-positive. Ubiquitous GFP expression was observed from 8 hr post-fertilization and these embryos developed and hatched normally. The pJEF-CAT gene was introduced into fertilized eggs of black tiger shrimp using the optimized conditions of the particle gun bombardment. CAT activity was observed from 1 to 7 days post-fertilization, with the highest activities being observed at 5 and 7 days post-hatching.

Animals↗

PubMed2005

Genetic heterogeneity of the tropical abalone (Haliotis asinina) revealed by RAPD and microsatellite analyses.

Genetic heterogeneity of the tropical abalone, Haliotis asinina was examined using randomly amplified polymorphic DNA (RAPD) and microsatellite analyses. One hundred and thirteen polymorphic RAPD fragments were generated. The percentage of polymorphic bands of H. asinina across overall primers was 85.20%. The average genetic distance of natural samples within the Gulf of Thailand (HACAME and HASAME) was 0.0219. Larger distance was observed when those samples were compared with HATRAW from the Andaman Sea (0.2309 and 0.2314). Geographic heterogeneity and F(ST) analyses revealed population differentiation between H. asinina from the Gulf of Thailand and the Andaman Sea (p < 0.0001). Three microsatellite loci (CUHas1, CUHas4 and CUHas5) indicated relatively high genetic diversity in H. asinina (total number of alleles = 26, 5, 23 and observed heterozygosity = 0.84, 0.42 and 0.33, respectively). Significant population differentiation was also found between samples from different coastal regions (p < 0.0001). Therefore, the gene pool of natural H. asinina in coastal Thai waters can be genetically divided to 2 different populations; the Gulf of Thailand (A) and the Andaman Sea (B).

Animals↗

PubMed2005

Recombinant expression and anti-microbial activity of anti-lipopolysaccharide factor (ALF) from the black tiger shrimp Penaeus monodon.

Anti-lipopolysaccharide factors (ALFs), originally characterized from horseshoe crabs, have been recently identified from hemocytes of the black tiger shrimp, Penaeus monodon, by a genomic approach. In order to characterize the properties and biological activities of this immune effector in shrimp, ALFPm3, the most abundant isoform found in P. monodon, was expressed in the yeast Pichia pastoris. Large-scale production in fermentor provided 262 mg/l of recombinant ALFPm3 which was purified to homogeneity by single chromatography step on expanded-bed Streamline SP6XL. The rALFPm3 was further characterized in terms of N-terminal sequencing and mass spectrometry. Anti-microbial assays demonstrated that rALFPm3 has a broad spectrum of anti-fungal properties against filamentous fungi, and anti-bacterial activities against both Gram-positive and Gram-negative bacteria, associated with a bactericidal effect. Interestingly, rALFPm3 is highly efficient against various Vibrio species including strains pathogenic for shrimp. Finally, a synthetic peptide corresponding to a part of the putative LPS-binding site of ALFPm3 was shown to display activities mainly directed against Gram-positive bacteria indicating the involvement of the full molecule to the anti-microbial activity for Gram-negative bacteria.

Amino Acid Sequence↗

PubMed2005

Recombinant expression and characterization of five-domain Kazal-type serine proteinase inhibitor of black tiger shrimp (Penaeus monodon).

Four full-length complementary DNAs of Kazal-Type serine proteinase inhibitors (SPIs) were identified from hemocyte cDNA libraries of the black tiger shrimp Penaeus monodon and recognized as having 4 (SPIPm1) and 5 (SPIPm2, SPIPm3, and SPIPm4) Kazal domains. SPIPm2 encoding a complete 5-Kazal-domain inhibitor was expressed in the Escherichia coli system. Inhibitory activity of crude proteins against various serine proteases was tested using SPI activity gelatin with sodium dodecylsulfate polyacrylamide gel electrophoresis and inhibitory spectrum assays. A 32-kDa recombinant protein (rSPIPm2) showed inhibitory activity against trypsin, chymotrypsin, and subtilisin, but not elastase. Concordantly, inhibitory spectrum assays showed that crude rSPIPm2 strongly inhibited trypsin (89%) and chymotrypsin (70%), but less effectively inhibited subtilisin (8%), and did not inhibit elastase activity. Northern blot analysis of hemocyte total RNA showed 2 SPI transcripts of 1.6 and 1.7 kb in size. Tissue-specific expression using reverse transcriptase polymerase chain reaction suggests that SPIPm2 is exclusively expressed in hemocytes of P. monodon.

Amino Acid Sequence↗

PubMed2004

Antimicrobial peptides discovered in the black tiger shrimp Penaeus monodon using the EST approach.

Two cDNA libraries were prepared from hemocytes of normal and Vibrio harveyi-challenged black tiger shrimp Penaeus monodon. A total of 1062 expressed sequence tag (EST) clones were sequenced unidirectionally. ESTs representing the antimicrobial peptide (AMP) homologues, antilipopolysaccharide factors (ALF), penaeidins and crustins were discovered. They predominated among immune-related genes, representing 29.2% and 64.0% of the normal and challenged libraries, respectively. Several types of each AMP homologue were found. Sequence alignments of ALF in P. monodon (ALFPm 1 to 5) implied possible alternative splicing of different exons at both NH2 and COOH-termini. Only one major type of penaeidin (penPm3) was found in P. monodon. In addition, crustin homologues (crusPms1 to 4) and a newly identified glycine-rich antibacterial peptide (GAMPPm1) were also isolated and characterized. Using RT-PCR analysis, expression of ALF, penaeidin and crustin transcripts was detected in various tissues but the main expression site was in hemocytes. Expression of these antimicrobial peptides in P. monodon subjected to V. harveyi challenge revealed a significant increase in expression of ALFPms (p < 0.05) but a decrease in expression of crustins and penaeidins.

Alternative Splicing↗

PubMed2004

Species identification of the tropical abalone (Haliotis asinina, Haliotis ovina, and Haliotis varia) in Thailand using RAPD and SCAR markers.

A randomly amplified polymorphic DNA (RAPD) analysis was used to identify the species- and population-specific markers of abalone; Haliotis asinina, H. ovina, and H. varia in Thai waters. Fifteen species-specific and six population-specific RAPD markers were identified. In addition, an 1650 bp band (UBC195) that was restricted to H. ovina from the Gulf of Thailand (east) was also found. All of the specific RAPD markers were cloned and sequenced. Twenty pairs of primers were designed and specificity-tested (N = 12 and 4 for target and non-target species, respectively). Seven primer pairs (CUHA1, 2, 4, 11, 12, 13, and 14) were specifically amplified by H. asinina DNA, whereas a single pair of primers showed specificity with H. ovina (CUHO3) and H. varia (CUHV1), respectively. Four primer pairs, including CUHA2, CUHA12, CUHO3, and CUHV1, were further examined against 216 individuals of abalone (N = 111, 73, and 32, respectively). Results indicated the species-specific nature of all of them, except CUHO3, with the sensitivity of detection of 100 pg and 20 pg of the target DNA template for CUHA2 and CUHA12 and CUHV1, respectively. The species-origin of the frozen, ethanol-preserved, dried, and boiled H. asinina specimens could also be successfully identified by CUHA2.

Animals↗

PubMed2002

Identification of immune-related genes in hemocytes of black tiger shrimp (Penaeus monodon).

An expressed sequence tag (EST) library was constructed from hemocytes of the black tiger shrimp (Penaeus monodon) to identify genes associated with immunity in this economically important species. The number of complementary DNA clones in the constructed library was approximately 4 x 10(5). Of these, 615 clones having inserts larger than 500 bp were unidirectionally sequenced and analyzed by homology searches against data in GenBank. Significant homology to known genes was found in 314 (51%) of the 615 clones, but the remaining 301 sequences (49%) did not match any sequence in GenBank. Approximately 35% of the matched ESTs were significantly identified by the BLASTN and BLASTX programs, while 65% were recognized only by the BLASTX program. Of the 615 clones, 55 (8.9%) were identified as putative immune-related genes. The isolated genes were composed of those coding for enzymes and proteins in the clotting system and the prophenoloxidase-activating system, antioxidative enzymes, antimicrobial peptides, and serine proteinase inhibitors. Three full-length ESTs encoding antimicrobial peptides (antilipopolysaccharide and penaeidin homologues) and a heat shock protein (cpn10 homologue) are reported.

Journal Article↗