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Fulltext Characterisation of full-length cDNA sequences provides insights into the Eimeria tenella transcriptome

Amiruddin N, Lee XW, Blake DP, Suzuki Y, Tay YL, Lim LS, et al.

BMC Genomics, 2012 Jan 13;13:21.
PMID: 22244352 DOI: 10.1186/1471-2164-13-21

BACKGROUND: Eimeria tenella is an apicomplexan parasite that causes coccidiosis in the domestic fowl. Infection with this parasite is diagnosed frequently in intensively reared poultry and its control is usually accorded a high priority, especially in chickens raised for meat. Prophylactic chemotherapy has been the primary method used for the control of coccidiosis. However, drug efficacy can be compromised by drug-resistant parasites and the lack of new drugs highlights demands for alternative control strategies including vaccination. In the long term, sustainable control of coccidiosis will most likely be achieved through integrated drug and vaccination programmes. Characterisation of the E. tenella transcriptome may provide a better understanding of the biology of the parasite and aid in the development of a more effective control for coccidiosis.
RESULTS: More than 15,000 partial sequences were generated from the 5' and 3' ends of clones randomly selected from an E. tenella second generation merozoite full-length cDNA library. Clustering of these sequences produced 1,529 unique transcripts (UTs). Based on the transcript assembly and subsequently primer walking, 433 full-length cDNA sequences were successfully generated. These sequences varied in length, ranging from 441 bp to 3,083 bp, with an average size of 1,647 bp. Simple sequence repeat (SSR) analysis identified CAG as the most abundant trinucleotide motif, while codon usage analysis revealed that the ten most infrequently used codons in E. tenella are UAU, UGU, GUA, CAU, AUA, CGA, UUA, CUA, CGU and AGU. Subsequent analysis of the E. tenella complete coding sequences identified 25 putative secretory and 60 putative surface proteins, all of which are now rational candidates for development as recombinant vaccines or drug targets in the effort to control avian coccidiosis.
CONCLUSIONS: This paper describes the generation and characterisation of full-length cDNA sequences from E. tenella second generation merozoites and provides new insights into the E. tenella transcriptome. The data generated will be useful for the development and validation of diagnostic and control strategies for coccidiosis and will be of value in annotation of the E. tenella genome sequence.

Matched MeSH terms: Molecular Sequence Data
Varroa jacobsoni (Acari: Varroidae) is more than one species

Anderson DL, Trueman JW

Exp Appl Acarol, 2000 Mar;24(3):165-89.
PMID: 11108385

Varroa jacobsoni was first described as a natural ectoparasitic mite of the Eastern honeybee (Apis cerana) throughout Asia. It later switched host to the Western honeybee (A. mellifera) and has now become a serious pest of that bee worldwide. The studies reported here on genotypic, phenotypic and reproductive variation among V. jacobsoni infesting A. cerana throughout Asia demonstrate that V. jacobsoni is a complex of at least two different species. In a new classification V. jacobsoni is here redefined as encompassing nine haplotypes (mites with distinct mtDNA CO-I gene sequences) that infest A. cerana in the Malaysia Indonesia region. Included is a Java haplotype, specimens of which were used to first describe V. jacobsoni at the beginning of this century. A new name, V. destructor n. sp., is given to six haplotypes that infest A. cerana on mainland Asia. Adult females of V. destructor are significantly larger and less spherical in shape than females of V. jacobsoni and they are also reproductively isolated from females of V. jacobsoni. The taxonomic positions of a further three unique haplotypes that infest A. cerana in the Philippines is uncertain and requires further study. Other studies reported here also show that only two of the 18 different haplotypes concealed within the complex of mites infesting A. cerana have become pests of A. mellifera worldwide. Both belong to V. destructor, and they are not V. jacobsoni. The most common is a Korea haplotype, so-called because it was also found parasitizing A. cerana in South Korea. It was identified on A. mellifera in Europe, the Middle East, Africa, Asia, and the Americas. Less common is a Japan/Thailand haplotype, so-called because it was also found parasitizing A. cerana in Japan and Thailand. It was identified on A. mellifera in Japan, Thailand and the Americas. Our results imply that the findings of past research on V. jacobsoni are applicable mostly to V. destructor. Our results will also influence quarantine protocols for bee mites, and may present new strategies for mite control.

Matched MeSH terms: Molecular Sequence Data
Fulltext Isolation and characterization of microsatellite loci in an endangered palm, Johannesteijsmannia lanceolata (Arecaceae)

Ang CC, Lee SL, Lee CT, Tnah LH, Zakaria RM, Ng CC

Am J Bot, 2011 May;98(5):e117-9.
PMID: 21613176 DOI: 10.3732/ajb.1000494

Microsatellite markers were developed for Johannesteijsmannia lanceolata to assess the genetic diversity and mating system of this alarmingly endangered species.

Matched MeSH terms: Molecular Sequence Data
Ambient temperature detection of PCR amplicons with a novel sequence-specific nucleic acid lateral flow biosensor

Ang GY, Yu CY, Yean CY

Biosens Bioelectron, 2012 Oct-Dec;38(1):151-6.
PMID: 22705404 DOI: 10.1016/j.bios.2012.05.019

In the field of diagnostics, molecular amplification targeting unique genetic signature sequences has been widely used for rapid identification of infectious agents, which significantly aids physicians in determining the choice of treatment as well as providing important epidemiological data for surveillance and disease control assessment. We report the development of a rapid nucleic acid lateral flow biosensor (NALFB) in a dry-reagent strip format for the sequence-specific detection of single-stranded polymerase chain reaction (PCR) amplicons at ambient temperature (22-25°C). The NALFB was developed in combination with a linear-after-the-exponential PCR assay and the applicability of this biosensor was demonstrated through detection of the cholera toxin gene from diarrheal-causing toxigenic Vibrio cholerae. Amplification using the advanced asymmetric PCR boosts the production of fluorescein-labeled single-stranded amplicons, allowing capture probes immobilized on the NALFB to hybridize specifically with complementary targets in situ on the strip. Subsequent visual formation of red lines is achieved through the binding of conjugated gold nanoparticles to the fluorescein label of the captured amplicons. The visual detection limit observed with synthetic target DNA was 0.3 ng and 1 pg with pure genomic DNA. Evaluation of the NALFB with 164 strains of V. cholerae and non-V. cholerae bacteria recorded 100% for both sensitivity and specificity. The whole procedure of the low-cost NALFB, which is performed at ambient temperature, eliminates the need for preheated buffers or additional equipment, greatly simplifying the protocol for sequence-specific PCR amplicon analysis.

Matched MeSH terms: Molecular Sequence Data
Fulltext Molecular evidence of cholera outbreak caused by a toxigenic Vibrio cholerae O1 El tor variant strain in Kelantan, Malaysia

Ang GY, Yu CY, Balqis K, Elina HT, Azura H, Hani MH, et al.

J Clin Microbiol, 2010 Nov;48(11):3963-9.
PMID: 20826646 DOI: 10.1128/JCM.01086-10

A total of 20 Vibrio cholerae isolates were recovered for investigation from a cholera outbreak in Kelantan, Malaysia, that occurred between November and December 2009. All isolates were biochemically characterized as V. cholerae serogroup O1 Ogawa of the El Tor biotype. They were found to be resistant to multiple antibiotics, including tetracycline, erythromycin, sulfamethoxazole-trimethoprim, streptomycin, penicillin G, and polymyxin B, with 35% of the isolates being resistant to ampicillin. All isolates were sensitive to ciprofloxacin, norfloxacin, chloramphenicol, gentamicin, and kanamycin. Multiplex PCR analysis confirmed the biochemical identification and revealed the presence of virulence genes, viz., ace, zot, and ctxA, in all of the isolates. Interestingly, the sequencing of the ctxB gene showed that the outbreak strain harbored the classical cholera toxin gene and therefore belongs to the newly assigned El Tor variant biotype. Clonal analysis by pulsed-field gel electrophoresis demonstrated that a single clone of a V. cholerae strain was responsible for this outbreak. Thus, we present the first molecular evidence that the toxigenic V. cholerae O1 El Tor variant has invaded Malaysia, highlighting the need for continuous monitoring to facilitate early interventions against any potential epidemic by this biotype.

Matched MeSH terms: Molecular Sequence Data
Molecular characterization, modeling and docking of CYP107CB2 from Bacillus lehensis G1, an alkaliphile

Ang SS, Salleh AB, Chor AL, Normi YM, Tejo BA, Rahman MB

Comput Biol Chem, 2015 Jun;56:19-29.
PMID: 25766878 DOI: 10.1016/j.compbiolchem.2015.02.015

Cytochrome P450s are a superfamily of heme monooxygenases which catalyze a wide range of biochemical reactions. The reactions involve the introduction of an oxygen atom into an inactivated carbon of a compound which is essential to produce an intermediate of a hydroxylated product. The diversity of chemical reactions catalyzed by cytochrome P450s has led to their increased demand in numerous industrial and biotechnology applications. A recent study showed that a gene sequence encoding a CYP was found in the genome of Bacillus lehensis G1, and this gene shared structural similarity with the bacterial vitamin D hydroxylase (Vdh) from Pseudonocardia autotrophica. The objectives of present study was to mine, for a novel CYP from a new isolate B. lehensis G1 alkaliphile and determine the biological properties and functionalities of CYP in this bacterium. Our study employed the usage of computational methods to search for the novel CYP from CYP structural databases to identify the conserved pattern, functional domain and sequence properties of the uncharacterized CYP from B. lehensis G1. A computational homology model of the protein's structure was generated and a docking analysis was performed to provide useful structural knowledge on the enzyme's possible substrate and their interaction. Sequence analysis indicated that the newly identified CYP, termed CYP107CB2, contained the fingerprint heme binding sequence motif FxxGxxxCxG at position 336-345 as well as other highly conserved motifs characteristic of cytochrome P450 proteins. Using docking studies, we identified Ser-79, Leu-81, Val-231, Val-279, Val-383, Ala-232, Thr-236 and Thr-283 as important active site residues capable of stabilizing interactions with several potential substrates, including vitamin D3, 25-hydroxyvitamin D3 and 1α-hydroxyvitamin D3, in which all substrates docked proximally to the enzyme's heme center. Biochemical analysis indicated that CYP107CB2 is a biologically active protein to produce 1α,25-dihydroxyvitamin D3 from 1α-hydroxyvitamin D3. Based on these results, we conclude that the novel CYP107CB2 identified from B. lehensis G1 is a putative vitamin D hydroxylase which is possibly capable of catalyzing the bioconversion of parental vitamin D3 to calcitriol, or related metabolic products.

Matched MeSH terms: Molecular Sequence Data
Fulltext Occurrence and molecular identification of Anisakis Dujardin, 1845 from marine fish in southern Makassar Strait, Indonesia

Anshary H, Sriwulan, Freeman MA, Ogawa K

Korean J Parasitol, 2014 Feb;52(1):9-19.
PMID: 24623876 DOI: 10.3347/kjp.2014.52.1.9

Anisakis spp. (Nematoda: Anisakidae) parasitize a wide range of marine animals, mammals serving as the definitive host and different fish species as intermediate or paratenic hosts. In this study, 18 fish species were investigated for Anisakis infection. Katsuwonus pelamis, Euthynnus affinis, Caranx sp., and Auxis thazard were infected with high prevalence of Anisakis type I, while Cephalopholis cyanostigma and Rastrelliger kanagurta revealed low prevalence. The mean intensity of Anisakis larvae in K. pelamis and A. thazard was 49.7 and 5.6, respectively. A total of 73 Anisakis type I larvae collected from K. pelamis and A. thazard were all identified as Anisakis typica by PCR-RFLP analysis. Five specimens of Anisakis from K. pelamis and 15 specimens from A. thazard were sequenced using ITS1-5.8S-ITS2 region and 6 specimens from A. thazard and 4 specimens from K. pelamis were sequenced in mtDNA cox2 region. Alignments of the samples in the ITS region showed 2 patterns of nucleotides. The first pattern (genotype) of Anisakis from A. thazard had 100% similarity with adult A. typica from dolphins from USA, whereas the second genotype from A. thazard and K. pelamis had 4 base pairs different in ITS1 region with adult A. typica from USA. In the mtDNA cox2 regions, Anisakis type I specimens from A. thazard and K. pelamis showed similarity range from 94% to 99% with A. typica AB517571/DQ116427. The difference of 4 bp nucleotides in ITS1 regions and divergence into 2 subgroups in mtDNA cox2 indicating the existence of A. typica sibling species in the Makassar Strait.

Matched MeSH terms: Molecular Sequence Data
Microwave modified non-crosslinked pectin films with modulated drug release

Anuar NK, Wong TW, Taib MN

Pharm Dev Technol, 2012 Jan-Feb;17(1):110-7.
PMID: 20958167 DOI: 10.3109/10837450.2010.522584

The effects of microwave on drug release properties of pectin films carrying sulfanilamide (SN-P), sulfathiazole (ST-P) and sulfamerazine (SM-P) of high to low aqueous solubilities were investigated. These films were prepared by solvent evaporation technique and treated by microwave at 80 W for 5-40 min. Their profiles of drug dissolution, drug content, matrix interaction and matrix crystallinity were determined by drug dissolution testing, drug content assay, differential scanning calorimetry, X-ray diffractometry and scanning electron microscopy techniques. Microwave induced an increase in matrix amorphousness but lower drug release propensity with a greater retardation extent in SN-P films, following a rise in strength of matrix interaction. A gain in amorphous structure does not necessarily increase the drug release of film. Microwave can possibly retard drug release of pectin film carrying water-soluble drug through modulating its state of matrix interaction.

Matched MeSH terms: Molecular Sequence Data
Fulltext Human giardiasis in Malaysia: correlation between the presence of clinical manifestation and Giardia intestinalis assemblage

Anuar TS, Moktar N, Salleh FM, Al-Mekhlafi HM

Southeast Asian J. Trop. Med. Public Health, 2015 Sep;46(5):835-43.
PMID: 26863854

Clinical manifestations of giardiasis vary from asymptomatic infection to chronic diarrhea. A total of 611 stool samples from Aboriginal participants residing in Jelebu, Gerik and Temerloh States, Malaysia, ages 2 to 74 years were screened for Giardia intestinalis using microscopic examination and sequence analysis of a fragment of nested-PCR amplified triosephosphate isomerase (tpi) gene. Demographic data was collected through a structured questionnaire. tpi was successfully amplified from 98/110 samples microscopically positive for G. intestinalis, with 62 and 36 belonging to assemblage A and B, respectively. There is a significant correlation between assemblage A and symptomatic infection only in participants of < 15 years of age. In the other age group, host factors may have more effects on the presence of clinical signs and symptoms than G. intestinalis assemblage types.

Matched MeSH terms: Molecular Sequence Data
Cross-reactive T-cell responses to the nonstructural regions of dengue viruses among dengue fever and dengue hemorrhagic fever patients in Malaysia

Appanna R, Huat TL, See LL, Tan PL, Vadivelu J, Devi S

Clin Vaccine Immunol, 2007 Aug;14(8):969-77.
PMID: 17567768

Dengue virus infections are a major cause of morbidity and mortality in tropical and subtropical areas in the world. Attempts to develop effective vaccines have been hampered by the lack of understanding of the pathogenesis of the disease and the absence of suitable experimental models for dengue viral infection. The magnitude of T-cell responses has been reported to correlate with dengue disease severity. Sixty Malaysian adults with dengue viral infections were investigated for their dengue virus-specific T-cell responses to 32 peptides antigens from the structural and nonstructural regions from a dengue virus isolate. Seventeen different peptides from the C, E, NS2B, NS3, NS4A, NS4B, and NS5 regions were found to evoke significant responses in a gamma interferon enzyme-linked immunospot (ELISPOT) assay of samples from 13 selected patients with dengue fever (DF) and dengue hemorrhagic fever (DHF). NS3 and predominantly NS3(422-431) were found to be important T-cell targets. The highest peaks of T-cell responses observed were in responses to NS3(422-431) and NS5(563-571) in DHF patients. We also found almost a sevenfold increase in T-cell response in three DHF patients compared to three DF patient responses to peptide NS3(422-431). A large number of patients' T cells also responded to the NS2B(97-106) region. The ELISPOT analyses also revealed high frequencies of T cells that recognize both serotype-specific and cross-reactive dengue virus antigens in patients with DHF.

Matched MeSH terms: Molecular Sequence Data
Fulltext Comparative sequence and structure analysis reveals the conservation and diversity of nucleotide positions and their associated tertiary interactions in the riboswitches

Appasamy SD, Ramlan EI, Firdaus-Raih M

PLoS One, 2013;8(9):e73984.
PMID: 24040136 DOI: 10.1371/journal.pone.0073984

The tertiary motifs in complex RNA molecules play vital roles to either stabilize the formation of RNA 3D structure or to provide important biological functionality to the molecule. In order to better understand the roles of these tertiary motifs in riboswitches, we examined 11 representative riboswitch PDB structures for potential agreement of both motif occurrences and conservations. A total of 61 unique tertiary interactions were found in the reference structures. In addition to the expected common A-minor motifs and base-triples mainly involved in linking distant regions the riboswitch structures three highly conserved variants of A-minor interactions called G-minors were found in the SAM-I and FMN riboswitches where they appear to be involved in the recognition of the respective ligand's functional groups. From our structural survey as well as corresponding structure and sequence alignments, the agreement between motif occurrences and conservations are very prominent across the representative riboswitches. Our analysis provide evidence that some of these tertiary interactions are essential components to form the structure where their sequence positions are conserved despite a high degree of diversity in other parts of the respective riboswitches sequences. This is indicative of a vital role for these tertiary interactions in determining the specific biological function of riboswitch.

Matched MeSH terms: Molecular Sequence Data
Nucleoprotein gene analysis of fixed and street rabies virus variants using RT-PCR

Arai YT, Yamada K, Kameoka Y, Horimoto T, Yamamoto K, Yabe S, et al.

Arch Virol, 1997;142(9):1787-96.
PMID: 9672637

A simple and rapid single-step reverse transcriptase-polymerase chain reaction (RT-PCR) was used to investigate the nucleoprotein (N) gene of 11 rabies viruses. A conserved set of RT-PCR primers was designed to amplify the most variable region in the N gene. N gene regions were amplified from 6 fixed laboratory viruses, 4 street viruses from dogs in Thailand, and a horse in Zambia. Sequences of the amplified products, together with the database of 91 additional sequences, were analyzed by using PILEUP program of the GCG package. The rabies viruses grouped into at least 9 distinct clusters by < 90% nucleotide similarity of the N gene region: I (4 isolates, USA), II (2 isolates, South America), III (3 isolates, Africa), IV (52 strains, Europe, Middle East, Africa and South America), V (16 isolates, North America and Arctic), VI (17 isolates, Africa), VII (1 isolate, Africa), VIII (6 isolates, Thailand and Malaysia) and IX (1 isolate, Sri Lanka). A unique group of rabies viruses from Thailand and clusters of isolates corresponding to their geographic origin also were determined. The simple and rapid single-step RT-PCR proved to be useful for identifying rabies viruses, and for grouping the viruses into clades by sequence analysis.

Matched MeSH terms: Molecular Sequence Data
Fulltext Molecular characterization of a novel proto-type antimicrobial protein galectin-1 from striped murrel

Arasu A, Kumaresan V, Sathyamoorthi A, Chaurasia MK, Bhatt P, Gnanam AJ, et al.

Microbiol Res, 2014 Nov;169(11):824-34.
PMID: 24780642 DOI: 10.1016/j.micres.2014.03.005

In this study, we reported a molecular characterization of a novel proto-type galectin-1 from the striped murrel Channa striatus (named as CsGal-1). The full length CsGal-1 was identified from an established striped murrel cDNA library and further we confirmed the sequence by cloning. The complete cDNA sequence of CsGal-1 is 590 base pairs (bp) in length and its coding region encoded a poly peptide of 135 amino acids. The polypeptide contains a galactoside binding lectin domain at 4-135. The domain carries a sugar binding site at 45-74 along with its signatures (H(45)-X-Asn(47)-X-Arg(49) and Trp(69)-X-X-Glu(72)-X-Arg(74)). CsGal-1 shares a highly conserved carbohydrate recognition domain (CRD) with galectin-1 from other proto-type galectin of teleosts. The mRNA expressions of CsGal-1 in healthy and various immune stimulants including Aphanomyces invadans, Aeromonas hydrophila, Escherchia coli lipopolysaccharide and poly I:C injected tissues of C. striatus were examined using qRT-PCR. CsGal-1 mRNA is highly expressed in kidney and is up-regulated with different immune stimulants at various time points. To understand its biological activity, the coding region of CsGal-1 gene was expressed in an E. coli BL21 (DE3) cloning system and its recombinant protein was purified. The recombinant CsGal-1 protein was agglutinated with mouse erythrocytes at a concentration of 4μg/mL in a calcium independent manner. CsGal-1 activity was inhibited by d-galactose at 25mM(-1) and d-glucose and d-fructose at 100mM(-1). The results of microbial binding assay showed that the recombinant CsGal-1 protein agglutinated only with the Gram-negative bacteria. Interestingly, we observed no agglutination against Gram-positive bacteria. Overall, the study showed that CsGal-1 is an important immune gene involved in the recognition and elimination of pathogens in C. striatus.

Matched MeSH terms: Molecular Sequence Data
Isolation and characterization of the early nodule-specific protein homologue (Hev b 13), an allergenic lipolytic esterase from Hevea brasiliensis latex

Arif SA, Hamilton RG, Yusof F, Chew NP, Loke YH, Nimkar S, et al.

J Biol Chem, 2004 Jun 04;279(23):23933-41.
PMID: 15024009

Recurring reports of a highly allergenic 42-46-kDa protein in Hevea brasiliensis latex appeared to have been resolved with the discovery of a 43-kDa allergenic latex protein that was a homologue to patatin. However, the low to moderate prevalence of sensitization to the protein, designated Hev b 7, among latex-allergic patients could not adequately explain the frequent observations of the 42-46-kDa allergen. This led to the hypothesis that another, more allergenic protein of a similar molecular mass existed in Hevea latex. We report the isolation and purification of a 42.98-kDa latex glycoprotein showing homology to the early nodule-specific protein (ENSP) of the legumes Medicago sativa, Medicago truncatula, and Glycine max. The protein is allergenic, being recognized by immunoglobulin E (IgE) in sera from latex-allergic patients. The IgE epitope resides on the carbohydrate moiety of the protein, and the presence of a similar carbohydrate component on potato tuber patatin enables the latter to inhibit IgE binding to the ENSP homologue. The cDNA encoding the ENSP homologue was isolated by reverse transcription-PCR and cloned. The protein predicted from the cDNA sequence has 391 amino acids, the first 26 of which constitute a putative signal peptide. The deduced molecular mass of the mature protein is 40.40 kDa, while its isoelectric point is estimated at 5.0. The discrepancy between the predicted and observed molecular mass might be due to glycosylation, for which three N-sites on the protein are predicted. The purified protein showed lipase and esterase activities and may be involved in plant defense.

Matched MeSH terms: Molecular Sequence Data
Cloning and characterization of cDNAs encoding three isoforms of phospholipase A2 in Malayan spitting cobra (Naja naja sputatrix) venom

Armugam A, Earnest L, Chung MC, Gopalakrishnakone P, Tan CH, Tan NH, et al.

Toxicon, 1997 Jan;35(1):27-37.
PMID: 9028006

cDNAs encoding three phospholipase A2 (PLA2) isoforms in Naja naja sputatrix were cloned and characterized. One of them encoded an acidic PLA2 (APLA) while the others encoded neutral PLA2 (NPLA-1 and NPLA-2). The specific characteristics of APLA and NPLA were attributed to mutations at nt139 and nt328 from G to C and G to A, respectively, resulting in amino acid substitutions from Asp20 and 83 in APLA to His20 and Asn83 in NPLA. Amino acid sequencing of purified protein also showed the presence of this Asp20 and His20 in APLA and NPLA, respectively. The cDNA encoding one of the PLA2 (NAJPLA-2A), when expressed in Escherichia coli, yielded a protein that exhibited PLA2 activity.

Matched MeSH terms: Molecular Sequence Data
Macrobrachium rosenbergii mannose binding lectin: synthesis of MrMBL-N20 and MrMBL-C16 peptides and their antimicrobial characterization, bioinformatics and relative gene expression analysis

Arockiaraj J, Chaurasia MK, Kumaresan V, Palanisamy R, Harikrishnan R, Pasupuleti M, et al.

Fish Shellfish Immunol, 2015 Apr;43(2):364-74.
PMID: 25575476 DOI: 10.1016/j.fsi.2014.12.036

Mannose-binding lectin (MBL), an antimicrobial protein, is an important component of innate immune system which recognizes repetitive sugar groups on the surface of bacteria and viruses leading to activation of the complement system. In this study, we reported a complete molecular characterization of cDNA encoded for MBL from freshwater prawn Macrobrachium rosenbergii (Mr). Two short peptides (MrMBL-N20: (20)AWNTYDYMKREHSLVKPYQG(39) and MrMBL-C16: (307)GGLFYVKHKEQQRKRF(322)) were synthesized from the MrMBL polypeptide. The purity of the MrMBL-N20 (89%) and MrMBL-C16 (93%) peptides were confirmed by MS analysis (MALDI-ToF). The purified peptides were used for further antimicrobial characterization including minimum inhibitory concentration (MIC) assay, kinetics of bactericidal efficiency and analysis of hemolytic capacity. The peptides exhibited antimicrobial activity towards all the Gram-negative bacteria taken for analysis, whereas they showed the activity towards only a few selected Gram-positive bacteria. MrMBL-C16 peptides produced the highest inhibition towards both the Gram-negative and Gram-positive bacteria compared to the MrMBL-N20. Both peptides do not produce any inhibition against Bacillus sps. The kinetics of bactericidal efficiency showed that the peptides drastically reduced the number of surviving bacterial colonies after 24 h incubation. The results of hemolytic activity showed that both peptides produced strong activity at higher concentration. However, MrMBL-C16 peptide produced the highest activity compared to the MrMBL-N20 peptide. Overall, the results indicated that the peptides can be used as bactericidal agents. The MrMBL protein sequence was characterized using various bioinformatics tools including phylogenetic analysis and structure prediction. We also reported the MrMBL gene expression pattern upon viral and bacterial infection in M. rosenbergii gills. It could be concluded that the prawn MBL may be one of the important molecule which is involved in antimicrobial mechanism. Moreover, MrMBL derived MrMBL-N20 and MrMBL-C16 peptides are important antimicrobial peptides for the recognition and eradication of viral and bacterial pathogens.

Matched MeSH terms: Molecular Sequence Data
A novel single-domain peptide, anti-LPS factor from prawn: synthesis of peptide, antimicrobial properties and complete molecular characterization

Arockiaraj J, Kumaresan V, Bhatt P, Palanisamy R, Gnanam AJ, Pasupuleti M, et al.

Peptides, 2014 Mar;53:79-88.
PMID: 24269604 DOI: 10.1016/j.peptides.2013.11.008

In this study, we reported a complete molecular characterization including bioinformatics features, gene expression, peptide synthesis and its antimicrobial activities of an anti-lipopolysaccharide (LPS) factor (ALF) cDNA identified from the established cDNA library of freshwater prawn Macrobrachium rosenbergii (named as MrALF). The mature protein has an estimated molecular weight of 11.240 kDa with an isoelectric point of 9.46. The bioinformatics analysis showed that the MrALF contains an antimicrobial peptide (AMP) region between T54 and P77 with two conserved cysteine residues (Cys55 and Cys76) which have an anti-parallel β-sheet confirmation. The β-sheet is predicted as cationic with hydrophobic nature containing a net charge of +5. The depicted AMP region is determined to be amphipathic with a predicted hydrophobic face 'FPVFI'. A highest MrALF gene expression was observed in hemocytes and is up-regulated with virus [white spot syndrome baculovirus (WSBV)], bacteria (Aeromonas hydrophila) and Escherichia coli LPS at various time points. The LPS binding region of MrALF peptide was synthesized to study the antimicrobial property, bactericidal efficiency and hemolytic capacity. The peptide showed antimicrobial activity against both the Gram-negative and Gram-positive bacteria. The bactericidal assay showed that the peptide recognized the LPS of bacterial cell walls and binding on its substrate and thereby efficiently distinguishing the pathogens. The hemolytic activity of MrALF peptide is functioning in a concentration dependant manner. In summary, the comprehensive analysis of MrALF showed it to be an effective antimicrobial peptide and thus it plays a crucial role in the defense mechanism of M. rosenbergii.

Matched MeSH terms: Molecular Sequence Data
First report on interferon related developmental regulator-1 from Macrobrachium rosenbergii: bioinformatic analysis and gene expression

Arockiaraj J, Easwvaran S, Vanaraja P, Singh A, Othman RY, Bhassu S

Fish Shellfish Immunol, 2012 May;32(5):929-33.
PMID: 22361112 DOI: 10.1016/j.fsi.2012.02.011

This study reports the first full length gene of interferon related developmental regulator-1 (designated as MrIRDR-1), identified from the transcriptome of Macrobrachium rosenbergii. The complete gene sequence of the MrIRDR-1 is 2459 base pair long with an open reading frame of 1308 base pairs and encoding a predicted protein of 436 amino acids with a calculated molecular mass of 48 kDa. The MrIRDR-1 protein contains a long interferon related developmental regulator super family domain between 30 and 330. The mRNA expressions of MrIRDR-1 in healthy and the infectious hypodermal and hematopoietic necrosis virus (IHHNV) infected M. rosenbergii were examined using qRT-PCR. The MrIRDR-1 is highly expressed in hepatopancreas along with all other tissues (walking leg, gills, muscle, haemocyte, pleopods, brain, stomach, intestine and eye stalk). After IHHNV infection, the expression is highly upregulated in hepatopancreas. This result indicates an important role of MrIRDR-1 in prawn defense system.

Matched MeSH terms: Molecular Sequence Data
Immunological role of thiol-dependent peroxiredoxin gene in Macrobrachium rosenbergii

Arockiaraj J, Easwvaran S, Vanaraja P, Singh A, Othman RY, Bhassu S

Fish Shellfish Immunol, 2012 Jul;33(1):121-9.
PMID: 22565019 DOI: 10.1016/j.fsi.2012.04.010

In this study, we have reported a full length of peroxiredoxin (designated MrPrdx) gene, identified from the transcriptome of freshwater prawn Macrobrachium rosenbergii. The complete gene sequence of the MrPrdx is 940 base pairs in length, and encodes 186 amino acids. MrPrdx contains a long thioredoxin domain in the amino acid sequence between 34 and 186. The gene expressions of MrPrdx in healthy and the infectious hypodermal and hematopoietic necrosis virus (IHHNV) challenged M. rosenbergii were examined using quantitative real time polymerase chain reaction. MrPrdx is highly expressed in all the other tissues of M. rosenbergii considered for analysis and the highest in gills. The expression is strongly up-regulated in gills after IHHNV infection. To understand MrPrdx functional properties, the recombinant MrPrdx protein was expressed in Escherichia coli BL21 (DE3) and purified. A peroxidise activity assay was conducted using recombinant MrPrdx protein at different concentrations. This peroxidises activity showed that the recombinant MrPrdx is a thiol-dependant protein. Additionally, this result showed that recombinant MrPrdx protein, as a secretory protein can remove H₂O₂ and protect DNA damage. This finding leads a possible way to propose the recombinant MrPrdx protein as an effective medicine for reactive oxygen species (ROS) related diseases.

Matched MeSH terms: Molecular Sequence Data
Bioinformatic characterization and gene expression pattern of apoptosis inhibitor from Macrobrachium rosenbergii challenged with infectious hypodermal and hematopoietic necrosis virus

Arockiaraj J, Vanaraja P, Easwvaran S, Singh A, Othman RY, Bhassu S

Fish Shellfish Immunol, 2011 Dec;31(6):1259-67.
PMID: 21945707 DOI: 10.1016/j.fsi.2011.09.008

Apoptosis is genetically programmed cellular killing processes that execute unnecessary or infected cells. It plays an important role in embryogenesis, homeostasis, insect metamorphosis and immunity. Apoptosis inhibitor (MrIAP) was sequenced from the freshwater giant prawn Macrobrachium rosenbergii using Illumina Solexa Genome Analyzer Technique. MrIAP consisted of 1753 base pair nucleotides encoded 535 polypeptide with an estimated molecular mass of 60 kDa. MrIAP amino acid sequence contains IAP superfamily domain between 5 and 490. The deduced amino acid sequences of the MrIAP were aligned with the other IAP family members. The highest sequence similarity was observed in IAP-5 from ant Camponotus floridanus (67%) followed by IAP from body louse Pediculus humanus corporis (66%) and the lowest (62%) in IAP-5 isoform-5 from common chimpanzee Pan troglodytes and IAP-5 from Aedes aegypti. The IAP phylogenetic tree showed that MrIAP closely related to other arthropod blacklegged tick Ixodes scapularis, formed a sister group with IAP from a hemichordate acorn worm Saccoglossus kowalevskii and finally clustered together with IAPs from fish groups. The quantitative real time PCR analysis revealed that significantly (P < 0.05) highest expression was noticed in hepatopancreas and significantly (P < 0.05) lowest expression in pleopods. Based on the results of gene expression analysis, MrIAP mRNA transcription in M. rosenbergii challenged to infectious hypodermal and hematopoietic necrosis virus (IHHNV) was highly induced in hepatopancreas. The collective results of this study indicate that the MrIAP is an essential immune gene and influences the immune response against IHHNV infection in M. rosenbergii.

Matched MeSH terms: Molecular Sequence Data

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