Topoisomerases in Immune Cell Development and Function.

DNA topoisomerases (TOPs) are complex enzymatic machines with extraordinary capacity to maintain DNA topology during torsion-intensive steps of replication and transcription. Recently, TOPs have gained significant attention for their tissue-specific function, and the vital role of TOPs in immune homeostasis and dysfunction is beginning to emerge. TOPs have been implicated in various immunological disorders such as autoimmunity, B cell immunodeficiencies, and sepsis, underscoring their importance in immune regulation. However, much remains unknown about immunological underpinnings of TOPs, and a deeper understanding of the role of TOPs in the immune system will be critical for yielding significant insights into the etiology of immunological disorders. In this review, we first discuss the recent literature highlighting the contribution of TOPs in the development of immune cells, and we further provide an overview of their importance in immune cell responses.

Anti-biofilm properties and immunological response of an immune molecule lectin isolated from shrimp Metapenaeus monoceros.

The study is carried out to understand the antimicrobial and immunological response of a potential immune molecule lectin, MmLec isolated from haemolymph of Speckled shrimp, Metapenaeus monoceros. MmLec was purified using mannose coupled Sepharose CL-4B affinity chromatography, which was further subjected on SDS-PAGE to ascertain the distribution of their molecular weight. Sugar binding specificity assay was conducted at various pH and temperatures to investigate the binding affinity of MmLec towards the specific carbohydrate molecule. Functional analysis of immune molecule MmLec included haemagglutination assays performed using human erythrocytes and yeast agglutination activity against Saccharomyces cerevisiae which, were analyzed using light microscopy. In order to study the antimicrobial activity, two Gram-negative (Vibrio parahaemolyticus and Aeromonas hydrophila) and two Gram-positive (Staphylococcus aureus and Enterococcus faecalis) bacteria were treated with purified MmLec. Moreover, these bacterial species were also treated at different concentration of the MmLec to speculate the antibiofilm properties of MmLec which was analyzed under Light Microscopy and Confocal Laser Scanning Microscopy. In addition, other functional characterization of MmLec showed the uniqueness of MmLec in agglutination of human erythrocyte as well as the cells of yeast Saccharomyces cerevisiae. Also, the phenoloxidase activity and encapsulation assay was evaluated. MTT assay displayed that MmLec are potent in anticancer activity. The study will help to understand the immunological interference and antimicrobial nature of MmLec which would be supportive in establishing a potential therapeutic tool and to develop better and novel disease control strategies in shrimp and farmed aquaculture industries as well as in health management.

Green synthesis of anisotropic zinc oxide nanoparticles with antibacterial and cytofriendly properties.

Zinc oxide nanoparticles (ZnONPs) exhibit abundant biomedical applications. Anisotropic ZnONPs with a defined shape and size were synthesized using Bacillus megaterium (NCIM 2326) cell free extract as a bio-reductant. The study investigated the multidimensional effect of ZnONPs on Helicobacter pylori strains and assessed its biosafety in normal human mesenchymal stem cells (hMSc). The highly stable ZnONPs were produced using B. megaterium and Zinc nitrate as a precursor. The phase of ZnONPs formation and structural characterization were performed by UV- visible (UV-Vis), Fourier transform infrared (FTIR) spectroscopy, X-ray diffraction (XRD) and Field Emission Scanning electron microscopy (FESEM) analysis. Furthermore, the ZnONPs exhibited higher biocompatibility against human mesenchymal stem cells (hMSC) and proved to be potentially safe in mammalian cells. Corroborating the current investigation, we described the anti-H. Pylori dosage of ZnONPs was safe to hMSC and could efficiently use as nano-antibiotic.

Differences in structure and changes in gene regulation of murrel molecular chaperone HSP family during epizootic ulcerative syndrome (EUS) infection.

Heat shock proteins (HSPs) are immunogenic, ubiquitous class of molecular chaperones, which are induced in response to various environmental and microbial stressful conditions. It plays a vital role in maintaining cellular protein homeostasis in eukaryotic cells. In this study, we described a comprehensive comparative data by bioinformatics approach on three different full length cDNA sequences of HSP family at molecular level. The cDNA sequences of three HSPs were identified from constructed cDNA library of Channa striatus and named as CsCPN60, CsHSP60 and CsHSP70. We have conducted various physicochemical study, which showed that CsHSP70 (666 amino acid) possessed a larger polypeptides followed by CsCPN60 (575) and CsCPN60 (542). Three dimensional structural analysis of these HSPs showed maximum residues in α-helices and least in ß-sheets; also CsHSP60 lacks ß-sheet and formed helix-turn-helix structure. Further analysis indicated that each HSP carried distinct domains and gene specific signature motif, which showed that each HSP are structurally diverse. Homology and phylogenetic study showed that the sequences taken for analysis shared maximum identity with fish HSP family. Tissue specific mRNA expression analysis revealed that all the HSPs showed maximum expression in one of the major immune organ such as CsCPN60 in kidney, CsHSP60 in spleen and CsHSP70 in head kidney. To understand the function of HSPs in murrel immune system, the elevation in mRNA expression level was analyzed against microbial oxidative stressors such as fungal (Aphanomyces invadans) and bacterial (Aeromonas hydrophila). It is interesting to note that all the HSP showed a different expression pattern and reached maximum up-regulation at 48 h post-infection (p.i) during fungal stress, whereas in bacterial stress only CsCPN60 showed maximum up-regulation at 48 h p.i, but CsHSP60 and CsHSP70 showed maximum up-regulation at 24 h p.i. The differential expression pattern showed that each HSP is diverse in function. Overall, the elevation in expression levels showed that HSPs might have potential involvement in murrel immune protection thus, protecting the organism against various external stimuli including environmental and microbial stress.

In-silico analysis and mRNA modulation of detoxification enzymes GST delta and kappa against various biotic and abiotic oxidative stressors.

This study reports the comprehensive comparative information of two different detoxification enzymes such as glutathione S-transferases (GSTs) delta and kappa from freshwater giant prawn Macrobrachium rosenbergii (designated as MrGSTD and MrGSTK) by investigating their in-silico characters and mRNA modulation against various biotic and abiotic oxidative stressors. The physico-chemical properties of these cDNA and their polypeptide structure were analyzed using various bioinformatics program. The analysis indicated the variation in size of the polypeptides, presence or absence of domains and motifs and structure. Homology and phylogenetic analysis revealed that MrGSTD shared maximum identity (83%) with crustaceans GST delta, whereas MrGSTK fell in arthropods GST kappa. It is interesting to note that MrGSTD and MrGSTK shared only 21% identity; it indicated their structural difference. Structural analysis indicated that MrGSTD to be canonical dimer like shape and MrGSTK appeared to be butterfly dimer like shape, in spite of four ß-sheets being conserved in both GSTs. Tissue specific gene expression analysis showed that both MrGSTD and MrGSTK are highly expressed in immune organs such as haemocyte and hepatopancreas, respectively. To understand the role of mRNA modulation of MrGSTD and MrGSTK, the prawns were inducted with oxidative stressors such as bacteria (Vibrio harveyi), virus [white spot syndrome virus (WSSV)] and heavy metal, cadmium (Cd). The analysis revealed an interesting fact that both MrGSTD and MrGSTK showed higher (P < 0.05) up-regulation at 48 h post-challenge, except MrGSTD stressed with bacteria, where it showed up-regulation at 24 h post-challenge. Overall, the results suggested that GSTs are diverse in their structure and possibly conferring their potential involvement in immune protection in crustaceans. However, further study is necessary to focus their functional differences at proteomic level.

Molecular importance of prawn large heat shock proteins 60, 70 and 90.

Considering the importance of heat shock proteins (HSPs) in the innate immune system of prawn, a comparative molecular approach was proposed to study the crustacean large HSPs 60, 70 and 90. Three different large HSPs were identified from freshwater prawn Macrobrachium rosenbergii (Mr) cDNA library during screening. The structural and functional characteristic features of HSPs were studied using various bioinformatics tools. Also, their gene expression and mRNA regulation upon various pathogenic infections was studied by relative quantification using 2(-ΔΔCT) method. MrHSP60 contains a long chaperonin 60 domain at 46-547 which carries a chaperonin 60 signature motif between 427 and 438, whereas MrHSP70 contains a long HSP70 domain at 21-624 and MrHSP90 carries a HSP90 domain at 188-719. The two dimensional analysis showed that MrHSP60 contains more amino acids (52%) in helices, whereas MrHSP70 (40.6%) and MrHSP90 (51.8%) carried more residues in coils. Gene expression results showed significant (P < 0.05) expression of MrHSP60, 70 and 90 in haemocyte, gill and hepatopancreas, respectively. Further, the expression level was up-regulated upon bacterial (Aeromonas hydrophilla and Vibrio harveyi) and viral [white spot syndrome virus (WSSV) and M. rosenbergii nodo virus (MrNV)] infections during various time periods. The gene expression results exhibited the potential involvement of these three HSPs in the immune system of prawn. The study indicated the potentiality of these molecules, thereby protecting cells against pathogens as well as severe cellular and environmental stresses in crustaceans.

Molecular profiles and pathogen-induced transcriptional responses of prawn B cell lymphoma-2 related ovarian killer protein (BOK).

In this study, we have reported a molecular characterization of the first B cell lymphoma-2 (BCL-2) related ovarian killer protein (BOK) from freshwater prawn Macrobrachium rosenbergii (Mr). BOK is a novel pro-apoptotic protein of the BCL-2 family that entails in mediating apoptosis to remove cancer cells. A cDNA sequence of MrBOK was identified from the prawn cDNA library and its full length was obtained by internal sequencing. The coding region of MrBOK yields a polypeptide of 291 amino acids. The analysis revealed that MrBOK contains a transmembrane helix at V(261)-L(283) and a putative BCL-2 family domain at V(144)-W(245). MrBOK also possessed four putative BCL-2 homology domains including BH1, BH2, BH3 and weak BH4. The BH3 contains 21 binding sites and among them five residues are highly conserved with the aligned BOK proteins. The homology analysis showed that MrBOK shared maximum similarity with the Caligus rogercresseyi BOK A. The topology of the phylogenetic tree was classified into nine sister groups which includes BOK, BAK, BAX, BAD, BCL-2, BCL-XL, NR13 and MCL members. The BOK protein group further sub-grouped into vertebrate and invertebrate BOK, wherein MrBOK located within insect monophyletic clad of invertebrate BOK. The secondary structural analysis showed that MrBOK contains 11 α-helices (52.2%) which are connected over random coils (47.7%). The 3D structure of MrBOK showed three central helices (α6, α7 and α8) which formed the core of the protein and are flanked on one side by α1, α2 and α3, and on the other side by α4, α5 and α11. MrBOK mRNA is expressed most abundantly (P < 0.05) in ovary compared to other tissues taken for analysis. Hence ovary was selected to study the possible roles of MrBOK mRNA regulation upon bacterial (Aeromonas hydrophila and Vibrio harveyi) and viral [white spot syndrome virus (WSSV) and M. rosenbergii nodovirus] infection. During bacterial and viral infection, the highest MrBOK mRNA transcription was varied at different time points. In bacterial infected ovary tissue, the highest mRNA expression was at 24 h post-infection, whereas in viral infection, the expression was highest at 48 h post-infection. Thus we can conclude that MrBOK functions as an apoptotic protein in intracellular programmed cell-death pathway to counteract the anti-apoptotic proteins released by bacterial and viral pathogens at the time of infection. This is the first study that emphasizes the importance of BOK during bacterial and viral infection in crustacean.

Macrobrachium rosenbergii mannose binding lectin: synthesis of MrMBL-N20 and MrMBL-C16 peptides and their antimicrobial characterization, bioinformatics and relative gene expression analysis.

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.

A redox active site containing murrel cytosolic thioredoxin: analysis of immunological properties.

In this study, we have reported the immunological properties of cDNA encoding thioredoxin which is obtained from the database of Channa striatus (named as CsTRx) cDNA library. The analysis showed that the CsTRx polypeptide contains a thioredoxin domain between Val(2) and Asn(106). The domain possessed a thioredoxin active family at 24­42 along with a redox active site (also known as catalytic center) at (31)WCGPC(35). The analysis showed that the catalytic center is responsible for the control of protein function. Phylogenetic study showed that CsTRx clustered together with vertebrate TRx-1. Based on the phylogenetic analysis and other bioinformatics analysis, it is confirmed that the characterized CsTRx belongs to TRx-1 family. In addition, the sub-cellular localization prediction analysis showed that CsTRx is a cytosol thioredoxin. The highest gene expression was observed in gill (P < 0.05). Further, its transcriptional modulation was evaluated under fungal (Aphanomyces invadans), bacterial (Aeromonas hydrophila) and H2O2 challenges. The recombinant CsTRx protein was over-expressed and purified using an Escherichia coli expression vector system. We conducted a H2O2 peroxidase assay using recombinant CsTRx protein under various pH and temperature. Further, we studied the influence of recombinant CsTRx protein on C. striatus spleen leukocyte activation. The recombinant CsTRx protein enhanced the cell proliferation in a concentration dependant manner. The results of antioxidant analysis showed that the antioxidant capacity of recombinant CsTRx protein was determined to be 4.2 U/mg protein. We conducted an insulin disulfides assay to study the enzymatic oxidoreductase activity of CsTRx and we observed no activity in the control group. But the recombinant CsTRx protein addition rapidly increased the enzymatic oxidoreductase activity. Over all, the results showed that the CsTRx may contain potential antioxidant properties, which could regulate the oxidative stress created by various biological pathogens as well as chemical stress in the immune system of C. striatus, thus protecting it.

Molecular cloning, characterization and gene expression of murrel CXC chemokine receptor 3a against sodium nitrite acute toxicity and microbial pathogens.

CXCR3 is a CXC chemokine receptor 3 which binds to CXC ligand 4 (CXCL4), 9, 10 and 11. CXC chemokine receptor 3a (CXCR3a) is one of the splice variants of CXCR3. It plays crucial role in defense and other physiological processes. In this study, we report the molecular cloning, characterization and gene expression of CXCR3a from striped murrel Channa striatus (Cs). The full length CsCXCR3a cDNA sequence was obtained from the constructed cDNA library of striped murrel by cloning and sequencing using an internal sequencing primer. The full length sequence is 1425 nucleotides in length including an open reading frame of 1086 nucleotides which is encoded with a polypeptide of 361 amino acids (mol. wt. 40 kDa). CsCXCR3a domain analysis showed that the protein contains a G protein coupled receptor between 55 and 305 along with its family signature at 129-145. The transmembrane prediction analysis showed that CsCXCR3a protein contains 7 transmembrane helical regions at 34-65, 80-106, 113-146, 154-181, 208-242, 249-278 and 284-308. The 'DRY' motif from CsCXCR3a protein sequence at (140)Asp-(141)Arg-(142)Tyr which is responsible for G-protein binding is also highly conserved with CXCR3 from other species. Phylogenetic tree showed that the CXC chemokine receptors 3, 4, 5 and 6, each formed a separate clade, but 1 and 2 were clustered together, which may be due to the high similarity between these receptors. The predicted 3D structure revealed cysteine residues, which are responsible for 'CXC' motif at 116 and 198. The CsCXR3a transcript was found to be high in kidney, further its expression was up-regulated by sodium nitrite acute toxicity exposure, fungal, bacterial and poly I:C challenges. Overall, these results supported the active involvement of CsCXCR3a in inflammatory process of striped murrel during infection. However, further study is necessary to explore the striped murrel chemokine signaling pathways and their roles in defense system.

A murrel interferon regulatory factor-1: molecular characterization, gene expression and cell protection activity.

In this study, we have reported a first murrel interferon regulatory factor-1 (designated as Murrel IRF-1) which is identified from a constructed cDNA library of striped murrel Channa striatus. The identified sequence was obtained by internal sequencing method from the library. The Murrel IRF-1 varies in size of the polypeptide from the earlier reported fish IRF-1. It contains a DNA binding domain along with a tryptophan pentad repeats, a nuclear localization signal and a transactivation domain. The homologous analysis showed that the Murrel IRF-1 had a significant sequence similarity with other known fish IRF-1 groups. The phylogenetic analysis exhibited that the Murrel IRF-1 clustered together with IRF-1 members, but the other members including IRF-2, 3, 4, 5, 6, 7, 8, 9 and 10 were clustered individually. The secondary structure of Murrel IRF-1 contains 27% α-helices (85 aa residues), 5.7% ß-sheets (19 aa residues) and 67.19% random coils (210 aa residues). Furthermore, we predicted a tertiary structure of Murrel IRF-1 using I-Tasser program and analyzed the structure on PyMol surface view. The RNA structure of the Murrel IRF-1 along with its minimum free energy (-284.43 kcal/mol) was also predicted. The highest gene expression was observed in spleen and its expression was inducted with pathogenic microbes which cause epizootic ulcerative syndrome in murrels such as fungus, Aphanomyces invadans and bacteria, Aeromonas hydrophila, and poly I:C, a viral RNA analog. The results of cell protection assay suggested that the Murrel IRF-1 regulates the early defense response in C. striatus. Moreover, it showed Murrel IRF-1 as a potential candidate which can be developed as a therapeutic agent to control microbial infections in striped murrel. Overall, these results indicate the immune importance of IRF-1, however, the interferon signaling mechanism in murrels upon infection is yet to be studied at proteomic level.

A prawn core histone 4: derivation of N- and C-terminal peptides and their antimicrobial properties, molecular characterization and mRNA transcription.

This study investigates the complete molecular characterization including bioinformatics characterization, gene expression, synthesis of N and C terminal peptides and their antimicrobial activity of the core histone 4 (H4) from freshwater giant prawn Macrobrachium rosenbergii (Mr). A cDNA encoding MrH4 was identified from the constructed cDNA library of M. rosenbergii during screening and the sequence was obtained using internal sequencing primers. The MrH4 coding region possesses a polypeptide of 103 amino acids with a calculated molecular weight of 11kDa and an isoelectric point of 11.5. The bioinformatics analysis showed that the MrH4 polypeptide contains a H4 signature at (15)GAKRH(19). Multiple sequence alignment of MrH4 showed that the N-terminal (21-42) and C-terminal (87-101) antimicrobial peptide regions and the pentapeptide or H4 signature (15-19) are highly conserved including in humans. The phylogenetic tree formed two separate clades of vertebrate and invertebrate H4, wherein MrH4 was located within the arthropod monophyletic clade of invertebrate H4 groups. Three-dimensional model of MrH4 was established using I-TASSER program and the model was validated using Ramachandran plot analysis. Schiffer-Edmundson helical wheel modeling was used to predict the helix propensity of N (21-42) and C (87-101) terminal derived Mr peptides. The highest gene expression was observed in gills and is induced by viral [white spot syndrome baculovirus (WSBV) and M. rosenbergii nodovirus (MrNV)] and bacterial (Aeromonas hydrophila and Vibrio harveyi) infections. The N and C terminal peptides were synthesized and their antimicrobial and hemolytic properties were examined. Both peptides showed activity against the tested Gram negative and Gram positive bacteria; however, the highest activity was noticed against Gram negative bacteria. Among the two peptides used in this study, C-terminal peptide yielded better results than the N-terminal peptide. Therefore, C terminal peptide can be recommended for the development of an antimicrobial agent.

An anti-apoptotic B-cell lymphoma-2 (BCL-2) from Channa striatus: Sequence analysis and delayed and advanced gene expression in response to fungal, bacterial and poly I:C induction.

B-cell lymphoma-2 (BCL-2) is a suppressor of apoptosis and inhibits the caspase dependent apoptosis pathway. In this study, we report molecular characterization of a cDNA sequence encoded of BCL-2 from striped murrel, Channa striatus. A partial cDNA sequence of CsBCL-2 was identified from the striped murrel cDNA library during annotation. Subsequently, the full length CsBCL-2 cDNA sequence was obtained by an internal sequencing method using a forward primer. The sequence contains 699 nucleotide base pairs which encode 232 amino acid residues. The domain and motif analysis revealed that the CsBCL-2 polypeptide consists of BCL-2 homologous domain BH4 at the N-terminal region between 4 and 21 and the BCL-2 homologous domains BH1, BH2 and BH3 between 87 and 187. The CsBCL-2 polypeptide sequence does not have a signal peptide region, but it consists of two novel transmembrane regions at 134-152 and 209-226. The sequence analysis showed that the CsBCL-2 has highest sequence identity (70%) with BCL-2 like protein 1 (BCL-2 L1) from pufferfish Takifugu rubripes. The phylogenetic analysis showed that the CsBCL-2 was situated in the BCL-2 L1 fish clade. The secondary analysis showed that the CsBCL-2 protein consists of 132 amino acid residues in the α-helical region and 100 amino acid residues in the random coil region. The validated 3D structure of CsBCL-2 showed the active residues Gly(135) and Arg(136) in the 7th α-helical position, whereas Trp(178) is in the 9th α-helical region. CsBCL-2 mRNA transcription is predominately present in spleen and is upregulated upon being induced with fungus Aphanomyces invadans, bacteria Aeromonas hydrophila, Escherichia coli LPS, Laminaria digitata beta-1,3-glucan and poly I:C. Overall, the CsBCL-2 mRNA transcription results indicate the potential involvement of CsBCL-2 in immune system of C. striatus. However, further research at proteomic level is necessary to examine these predictions.

Immunological role of C4 CC chemokine-1 from snakehead murrel Channa striatus.

In this study, we have reported a cDNA sequence of C4 CC chemokine identified from snakehead murrel (also known as striped murrel) Channa striatus (named as CsCC-Chem-1) normalized cDNA library constructed by Genome Sequencing FLX™ Technology (GS-FLX™). CsCC-Chem-1 is 641 base pairs (bp) long that contain 438 bp open reading frame (ORF). The ORF encodes a polypeptide of 146 amino acids with a molecular mass of 15 kDa. The polypeptide contains a small cytokine domain at 30-88. The domain carries the CC motif at Cys(33)-Cys(34). In addition, CsCC-Chem-1 consists of another two cysteine residues at C(59) and C(73), which, together with C(33) and C(34), make CsCC-Chem-1 as a C4-CC chemokine. CsCC-Chem-1 also contains a 'TCCT' motif at 32-35 as CC signature motif; this new motif may represent new characteristic features, which may lead to some unknown function that needs to be further focused on. Phylogenitically, CsCC-Chem-1 clustered together with CC-Chem-1 from rock bream Oplegnathus fasciatus and European sea bass Dicentrarchus labrax. Significantly (P<0.05) highest gene expression was noticed in spleen and is up-regulated upon fungus (Aphanomyces invadans), bacteria (Aeromonas hydrophila) and virus (poly I:C) infection at various time points. The gene expression results indicate the influence of CsCC-Chem-1 in the immune system of murrel. Overall, the gene expression study showed that the CsCC-Chem-1 is a capable gene to increase the cellular response against various microbial infections. Further, we cloned the coding sequence of CsCC-Chem-1 in pMAL vector and purified the recombinant protein to study the functional properties. The cell proliferation activity of recombinant CsCC-Chem-1 protein showed a significant metabolic activity in a concentration dependent manner. Moreover, the chemotaxis assay showed the capability of recombinant CsCC-Chem-1 protein which can induce the migration of spleen leukocytes in C. striatus. However, this remains to be verified further at molecular and proteomic level.

A novel single-domain peptide, anti-LPS factor from prawn: synthesis of peptide, antimicrobial properties and complete molecular characterization.

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 ß-sheet confirmation. The ß-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.

A cytosolic glutathione s-transferase, GST-theta from freshwater prawn Macrobrachium rosenbergii: molecular and biochemical properties.

Glutathione S-transferases play an important role in cellular detoxification and may have evolved to protect cells against reactive oxygen metabolites. In this study, we report the molecular characterization of glutathione s-transferase-theta (GST-θ) from freshwater prawn Macrobrachium rosenbergii. A full length cDNA of GSTT (1417 base pairs) was isolated and characterized bioinformatically. Exposure to virus (white spot syndrome baculovirus or M. rosenbergii nodovirus), bacteria (Aeromonas hydrophila or Vibrio harveyi) or heavy metals (cadmium or lead) significantly increased the expression of GSTT (P<0.05) in hepatopancreas. Recombinant GST-θ with monochlorobimane substrate had an optimum activity at pH7.5 and 35 °C. Furthermore recombinant GST-θ activity was abolished by the denaturants triton X-100, Gua-HCl, Gua-thiocyanate, SDS and urea in a dose-dependent manner. Overall, the results suggest a potential role for M. rosenbergii GST-θ in detoxification and possibly conferring immune protection.

Molecular characterization of a novel proto-type antimicrobial protein galectin-1 from striped murrel.

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.