Study of energy transfer between rare earth ions in CaS host by photoluminescence spectra

When CaS:Sm3+, Eu2+ is excited at 476.5 nm (Ar+), the emission spectra taken at room temperature and at 77 K are different, indicating that there are two competitive energy transfer processes-Sm3+ --> Eu2+ and Eu2+ --> Sm3+ with phonon participation. So, the luminescence intensity of Sm3+ increases first, and then decreases as the concentration of Eu2+ is increasing. (C) 2001 Elsevier Science Ltd. All rights reserved.

Crystal field splitting of Nd3+ ion and chemical bonds on the host crystals

In terms of the theory of chemical bonds on complex crystals(CBCC), the crystal structure and coordination, the chemical bond parameters of a group of ABO(4)-type crystals were calculated in detail, The results show that the relation between the crystal field splitting of Nd3+ ion and the covalency of the crystal is linear.

HOST-GUEST INTERACTIONS OF THIAMINE WITH ANIONS - CRYSTAL-STRUCTURE OF THIAMINE IODIDE SESQUIHYDRATE

The crystal structure of thiamine iodide sesquihydrate has been determined by X-ray diffraction methods as a host-guest model for coenzyme-substrate interactions. The asymmetric unit contains two chemical units. Both the thiamine molecules A and B, which are crystallographically independent, assume the usual F conformation and have a disordered hydroxyethyl side chain. An iodide anion (or a water molecule) bridges the pyrimidine and thiazolium rings of molecule A (or B) by forming a hydrogen bond with the amino group and an electrostatic contact with the thiazolium ring to stabilize the molecular conformation. In the crystal the thiamine molecules self-associate to form a pipe-like polymeric structure, in which four thiamine hosts surround an iodide guest and hold it through C(2)-H...I hydrogen bonds and thiazolium...I electrostatic interactions. Crystal data: C12H17N4OS+.I- . 1.5 H2O, monoclinic, P2(1)/c, a = 12.585(2), b = 25.303(5), c = 12.030(2) angstrom, beta = 115.15(1)degrees, V = 3468(1) angtrom3, Z = 8, D(c) = 1.606 g cm-3, R = 0.045 for 3328 observed reflections.

A clip domain serine protease (cSP) from the Chinese mitten crab Eriocheir sinensis: cDNA characterization and mRNA expression

Clip domain serine protease (cSP), characterized by conserved clip domains, is a new serine protease family identified mainly in arthropod, and plays important roles in development and immunity. In the present study, the full-length cDNA of a cSP (designated EscSP) was cloned from Chinese mitten crab Eriocheir sinensis by expressed sequence tags (ESTs) and PCR techniques. The 1380 bp EscSP cDNA contained a 1152 bp open reading frame (ORF) encoding a putative cSP of 383 amino acids, a 5'-untranslated region (UTR) of 54 bp, and a 3'-UTR of 174 bp. Multiple sequence alignment presented twelve conserved cysteine residues and a canonical catalytic triad (His(185), Asp(235) and Ser(332)) critical for the fundamental structure and function of EscSP. Two types of cSP domains, the clip domain and tryp_spc domain, were identified in the deduced amino acids sequence of EscSP. The conservation characteristics and similarities with previously known cSPs indicated that EscSP was a member of the large cSP family. The mRNA expression of EscSP in different tissues and the temporal expression in haemocytes challenged by Listonella anguillarum were measured by real-time RT-PCR. EscSP mRNA transcripts could be detected in all examined tissues, and were higher expressed in muscle than that in hepatopancreas. gill, gonad, haemocytes and heart. The EscSP mRNA expression in haemocytes was up-regulated after L anguillarum challenge and peaked at 2 h (4.96 fold, P < 0.05) and 12 h (9.90 fold, P < 0.05). Its expression pattern was similar to prophenoloxidase (EsproPO), one of the components of crab proPO system found in our previous report. These results implied that EscSP was involved in the processes of host-pathogen interaction probably as one of the proPO system members. (C) 2009 Elsevier Ltd. All rights reserved.

The construction of a cDNA library enriched for immune genes and the analysis of 7535 ESTs from Chinese mitten crab Eriocheir sinensis

Chinese mitten crab Eriocheir sinensis is one of the most important aquaculture crustacean species in China. A cDNA library was constructed from hemocytes of E. sinensis challenged with the mixture of Listonella anguillarum and Staphylococcus aureus, and randomly sequenced to collect genomic information and identify genes involved in immune defense response. Single-pass 5' sequencing of 10368 clones yielded 7535 high quality ESTs (Expressed Sequence Tags) and these ESTs were assembled into 2943 unigenes. BLAST analysis revealed that 1706 unigenes (58.0% of the total) or 4593 ESTs (61.0% of the total) were novel genes that had no significant matches to any protein sequences in the public databases. The rest 1237 unigenes; (42.0% of the total) were closely matched to the known genes or sequences deposited in public databases, which could be classed into 20 or 23 classifications according to "molecular function" or "biological process" respectively based on the Gene Ontology (GO). And 221 unigenes (7.5% of all 2943 unigenes, 17.9% of matched unigenes) or 969 ESTs (12.9% of all 7535 ESTs, 32.9% of matched ESTs) were identified to be immune genes. The relative higher proportion of immune-related genes in the present cDNA library than that in the normal library of E. sinensis and other crustaceans libraries, and the differences and changes in percentage and quantity of some key immune-related genes especially the immune inducible genes between two E. sinensis cDNA libraries may derive from the bacteria challenge to the Chinese mitten crab. The results provided a well-characterized EST resource for the genomics community, gene discovery especially for the identification of host-defense genes and pathways in crabs as well as other crustaceans. (C) 2009 Elsevier Ltd. All rights reserved.

A review of advances in research on marine molluscan antimicrobial peptides and their potential application in aquaculture

Antimicrobial peptides (AMPs) are important components of the host innate immune response against microbial invasion. They are usually characterized by their small-size, heat-stability and broad range of antimicrobial activity. This review covers research advances on marine mollusc AMPs, specifically those isolated from mussels, scallops, oysters, venerid clams and abalone, which mainly include MGD, mytilin, myticin, mytimycin, big defensin, and RPD-1. Their structural characteristics, antibacterial activity, and expression pattern as well as peptide distribution and their release following microbial challenge are also discussed. In addition, the prospect of the application of AMPs as food additives or their use in immunostimulation to prevent diseases of aquatic animals, as well as their potential hazards, are also discussed.

Cloning and characterization of a novel C-type lectin gene from shrimp Litopenaeus vannamei

In invertebrates, C-type lectins play crucial roles in innate immunity responses by mediating the recognition of host cells to pathogens and clearing microinvaders, which interact with carbohydrates and function as pattern recognition receptors (PRRs). A novel C-type lectin gene (LvLec) cDNA was cloned from hemocytes of Litopenaeus vannamei by expressed sequence tag (EST) and rapid amplification of cDNA ends (RACE) PCR. The full-length cDNA of LvLec was of 618 bp, consisting of a 5'-terminal untranslated region (UTR) of 60 bp and a 3'-UTR of 87 bp with a poly (A) tail. The deduced amino acid sequence of LvLec possessed all conserved features critical for the fundamental structure, such as the four cysteine residues (Cys(53), Cys(128), Cys(144), Cys(152)) involved in the formation of disulfides bridges and the potential Ca2+/carbohydrate-binding sites. The high similarity and the close phylogenetic relationship of LvLec shared with C-type lectins from vertebrates and invertebrates. The structural features of LvLec indicated that it was an invertebrate counterpart of the C-type lectin family. The cDNA fragment encoding the mature peptide of LvLec was recombined and expressed in Escherichia coli BL21(DE3)-pLysS. The recombinant protein (rLvLec) could agglutinate bacteria E. coli JM109 depending on Ca2+, and the agglutination could be inhibited by mannose and EDTA. These results indicated that LvLec was a new member of C-type lectin family and involved in the immune defence response to Gram negative bacteria in Litopenaeus vannamei. (C) 2008 Elsevier Ltd. All rights reserved.

Discovery of genes involved in defense/immunity functions in a haemocytes cDNA library from Fenneropenaeus chinensis by ESTs annotation

Crustacean haemocytes play important roles in the host immune response including recognition, phagocytosis, melanization, cytotoxicity and inter-cellular signal communication. Expressed sequence tags (ESTs) analysis is proved to be an efficient approach not only for gene discovery, but also for gene expression profiles performance. In order to further understand the innate immune system and defense mechanisms of Chinese shrimp at molecular level, complementary DNA library is constructed from the haemocyte tissue of Fenneropenaeus chinensis. A total of 2371 cDNA clones are successfully sequenced and the average sequence length is 460 bp. About 50% are identified as orthologs of known genes from other organisms by BLASTx and BLASTn program. By sequences comparability and analysis, 34 important genes including 177 ESTs are identified that may be involved in defense or immune functions in shrimp based on the known knowledge. These genes are categorized into five categories according to their putative functions in shrimp immune system: 13 genes are different types of antimicrobial peptides (AMP, penaeidin, antilipopolysaccharide factor, etc.), and their proportion is about 3 8%; 11 genes belong to prophenoloxidase system (prophenoloxidase, serine proteinase, serine proteinase inhibitor, etc.), and their proportion is about 32%; five genes have high homology with clotting protein (lectin, transglutaminase, etc), and their proportion is about 15%; three genes may be involved in inter-cell signal communication (peroxinectin, integrin), and their proportion is about 9%; two genes have been identified to be chaperone proteins (Hsc70, thioredoxin peroxidase), and their proportion is about 6%. These EST sequences enrich our understanding of the immune genes of F chinensis and will help farther experimental research into immune factors and improve our knowledge of the immune mechanisms of shrimp. (c) 2007 Elsevier B.V. All rights reserved.

Preliminary study on a potential antibacterial peptide derived from histone H2A in hemocytes of scallop Chlamys farreri

Histone H2A is reported to participate in host defense response through producing novel antimicrobial peptides (AMPs) from its N-terminus in vertebrates and invertebrates, while the AMPs derived from H2A have not to our knowledge been reported in mollusca. In the present study, gene cloning, mRNA expression of H2A from scallop Chlamys farreri, and the recombinant expression of its N-terminus were conducted to investigate whether a similar mechanism exists in mollusca. The full-length DNA of H2A was identified by the techniques of homology cloning and genomic DNA walking, The full-length DNA of the scallop H2A was 696 bp long, including a 5'-terminal untranslated region (UTR) of 90 bp, a 3'-terminal UTR of 228 bp with a stem-loop structure and a canonical polyadenylation signal sequence AATAAA, and an open reading frame of 375 bp encoding a polypeptide of 125 amino acids. The mRNA expression of H2A in the hemocytes of scallop challenged by microbe was measured by semi-quantitative RT-PCR. The expression of H2A was not upregulated after stimulation, suggesting that H2A did not participate in immunity response directly. The DNA fragment of 117 bp encoding 39 amino acids corresponding to the N-terminus of scallop H2A, which was homologous to buforin I in vertebrates, was cloned into Pichia pastoris GS115. The transformants (His(+) Mut(+)) containing multi-copy gene insertion were selected with increasing concentration of antibiotic G418. The peptide of 39 amino acids was expressed by induction of 0.5% methanol. The recombinant product exerted antibacterial activity against both Gram-positive (G(+)) and Gram-negative (G(-)) bacteria. The antibacterial activity toward G(+) bacteria was 2.5 times more than that against G(-) bacteria. The results elucidated that N-terminus of H2A was a potential AMP and provided a promising candidate for a new antibiotic screening. However, whether H2A is really involved in scallop immune response mechanisms needs to be further investigated. (C) 2006 Elsevier Ltd. All rights reserved.

Cloning and characterization of a novel C-type lectin from Zhikong scallop Chlamys farreri

C-type lectin is a family of Ca2+ dependent carbohydrate-recognition proteins which play crucial roles in the innate immunity of invertebrates by mediating the recognition of host cells to pathogens and clearing microinvaders as a pattern recognition protein (PRP). The cDNA of Zhikong scallop Chlamys farreri C-type lectin (designated CFLec-1) was cloned by expressed sequence tag (EST) and RACE techniques. The full-length cDNA of CFLec-1 was 1785 bp, consisting of a 5'-terminal untranslated region (UTR) of 66 bp and an unusually long 3' UTR of 1040 bp with seven polyadenylation signal sequences AATAAA and a poly(A) tail. The CFLec-1 cDNA encoded a polypeptide of 221 amino acids with a putative signal peptide of 15 amino acid residues and a mature protein of 206 amino acids. Analysis of the protein domain features indicated a typical long-form carbohydrate-recognition domain (CRD) of 130 residues in the CFLec-1 deduced amino acid sequence. The expression pattern of CFLec-1 transcripts in healthy and bacterial challenged scallops was studied by semi-quantitative RT-PCR. mRNA transcripts of CFLec-1 could be mainly detected in the tissues of haemocytes, gill, gonad and mantle of unchallenged scallops, whereas the expression of CFLec-1 transcripts was increased in all the tested tissues after heat-killed Vibrio anguillarum challenge. The temporal expression of CFLec-1 mRNA in haemolymph challenged by Micrococcus luteus and V anguillarum was both up-regulated and reached the maximum level at 8 and 16 It post stimulation, respectively, and then dropped back to the original level. In order to investigate its immune functions, CFLec- I was recombined and expressed in Escherichia coli BL21(DE3)-pLysS as a fusion protein with thioredoxin. The recombinant CFLec-1 agglutinated bacteria E. coli JM109 in vitro, and the agglutination was Ca2+ dependent which could be inhibited by EDTA. But it did not agglutinate M. luteus, Candida lipolytica and animal erythrocytes including rabbit, rat, mouse, chicken, human group A, human group B, human group O. Meanwhile, the recombinant CFLec-1 could inhibit the growth of both E. coli JM 109 and M. luteus, but no inhibition activity against V anguillarum. These result indicated that CFLec-1 was a constitutive and inducible PRP which was involved in the reorganization and clearance of invaders in scallop. (c) 2006 Elsevier Ltd. All rights reserved.

Molecular cloning and expression of a novel Kazal-type serine proteinase inhibitor gene from Zhikong scallop Chlamys farreri, and the inhibitory activity of its recombinant domain

Serine proteinase inhibitors (SPIs) play important roles in host physiological and immunological processes in all multicellular organisms. A novel Kazal-type SPI gene was cloned from the Zhikong scallop Chlamys farreri (designated as CfKZSPI) by expressed sequence tag (EST) and rapid amplification of cDNA ends (RACE) approaches. The full-length cDNA of CfKZSPI was of 1788 nucleotides with a canonical polyadenylation signal sequence AATAAA and a polyA tail, and an open reading frame (ORF) encoding a polypeptide of 509 amino acids with a putative signal peptide of 22 amino acids. The deduced amino acid sequence of CfKZSPI contained 12 tandem Kazal domains with high similarity to other Kazal-type SPIs. The temporal expression of CfKZSPI in hemocytes after Vibrio anguillorum challenge was recorded by quantitative real-time RT-PCR. The relative mRNA expression level of CfKZSPI was up-regulated and reached 43.6-fold at 3 h post-challenge. After a decrease at 6 h, the expression Level increased again and reached 207.8-fold at 12 h post-challenge. The 12th Kazal domain of CfKZSPI was recombined into pET-32a(+) and expressed in Escherichia coli Rosetta-gami (DE3) to investigate its inhibitory activity. The purified recombinant protein (rCf KZSPI-1 2) showed significant inhibitory activity against trypsin but no activity against thrombin. When the molar ratio of inhibitor to trypsin reached 1:1, almost 90% of the enzyme activity could be inhibited, which suggested that one molecule of rCfKZSPI-12 was able to inhibit one molecule of trypsin. Kinetics analysis with Dixon plot showed that the inhibition constant (K-i) of rCfKZSPI-12 to trypsin was 173 nmol L-1. These results indicated that CfKZSPI was a novel Kazal-type SPI with significant inhibitory activity against trypsin, and was suspected to be involved in scallop immune response. (c) 2008 Elsevier Ltd. All rights reserved.

A toll receptor from Chinese shrimp Fenneropenaeus chinensis is responsive to Vibrio anguillarum infection

Toll-like receptors (TLRs) are an evolutionarily ancient family of pattern recognition receptors (PRRs), playing a crucial role in innate immune responses. Here we present a Toll homolog from Chinese shrimp Fenneropenaeus chinensis, designated FcToll. The full-length cDNA of FcToll is 4115 bp including a poly A-tail of 16 bp, encoding a putative protein of 931 amino acids. The predicted protein consists of an extracellular domain with a potential signal peptide, 16 leucine-rich repeats (LRR), two LRR-C-terminal (LRR-CT) motifs, and two LRR-N-terminal (LRR-NT) motifs, followed by a transmembrane segment of 23 amino acids, and a cytoplasmic Toll/Interteukin-IR (TIR) domain of 139 residues. Genomic structure of FcToll gene contains five exons and four introns. Phylogenetic analysis revealed that it belongs to insect-type invertebrate Toll family. Transcripts of FcToll gene were constitutively expressed in various tissues, with predominant level in lymphoid organ. Real-time PCR assays demonstrated that expression patterns of FcToll were distinctly modulated after bacterial or viral stimulation, with significant enhancement after 5 h post-Vibrio anguillorum challenge but markedly reduced levels immediately after white spot syndrome virus (WSSV) exposure. These results suggest that FcToll might be involved in innate host defense, especially against the pathogen V. anguillarum. (c) 2008 Elsevier Ltd. All rights reserved.

Molecular cloning of an invertebrate goose-type lysozyme gene from Chlamys farreri, and lytic activity of the recombinant protein

Lysozyme is a widely distributed hydrolase possessing lytic activity against bacterial peptidoglycan, which enables it to protect the host against pathogenic infection. In the present study, the cDNA of an invertebrate goose-type lysozyme (designated CFLysG) was cloned from Zhikong scallop Chlamys farreri by expressed sequence tag (EST) and rapid amplification of cDNA ends (RACE) techniques. The full-length cDNA of CFLysG consisted of 829 nucleotides with a canonical polyadenylation signal sequence AATAAA and a poly(A) tail, and an open reading frame (ORF) of 603 bp encoding a polypeptide of 200 amino acid residues with a predicted molecular weight of 21.92 kDa and theoretical isoelectric point of 7.76. The high similarity of CFLysG with goose-type (g-type) lysozymes in vertebrate indicated that CFLysG should be an invertebrate counterpart of g-type lysozyme family, which suggested that the origin of g-type lysozyme preceded the emergence of urochordates and even preceded the emergence of deuterostomes. Similar to most g-type lysozymes, CFLysG possessed all conserved features critical for the fundamental structure and function of g-type lysozymes, such as three catalytic residues (Glu 82, Asp 97, Asp 108). By Northern blot analysis, mRNA transcript of CFLysG was found to be most abundantly expressed in the tissues of gills, hepatopancreas and gonad, weakly expressed in the tissues of haemocytes and mantle, while undetectable in the adductor muscle. These results suggested that CFLysG could possess combined features of both the immune and digestive adaptive lysozymes. To gain insight into the in vitro lytic activities of CFLysG, the mature peptide coding region was cloned into Pichia pastoris for heterogeneous expression. Recombinant CFLysG showed inhibitive effect on the growth of both Gram-positive and Gram-negative bacteria with more potent activities against Gram-positive bacteria, which indicated the involvement of CFLysG in the innate immunity of C. farreri. (c) 2006 Elsevier Ltd. All rights reserved.

Molecular cloning, expression of a big defensin gene from bay scallop Argopecten irradians and the antimicrobial activity of its recombinant protein

Antimicrobial peptides are important components of the host innate immune responses by exerting broad-spectrum microbicidal activity against pathogenic microbes. The first mollusk big defensin (designated AiBD) cDNA was cloned from bay scallop Argopecten irradians by expressed sequence tag (EST) and rapid amplification of cDNA ends (RACE) techniques. The scallop AiBD consisted of 531 nucleotides with a canonical polyadenylation signal sequence AATAAA and a poly(A) tail, encoding a polypeptide of 122 amino acids. The high similarity of AiBD deduced amino acid sequence with big defensin from Tachypleus tridentatus and Branchiostoma belcheri tsingtaunese indicated that AiBD should be a member of big defensin family. The expression of AiBD in various tissues was measured by using Northern blotting analysis. mRNA transcripts of AiBD could be detected in haemocytes of unchallenged scallops. The temporal expression of AiBD in haemolymph after Vibrio anguilarum challenge was recorded by quantitative real time PCR. The relative expression level of AiBD in haemolymph was up-regulated evenly in the first 8 h, followed by a drastic increase, and increased 131.1-fold at 32 h post-injection. These results indicated that AiBD could be induced by bacterial challenge, and it should participate in the immune responses of A. irradians. Biological activity assay revealed that recombinant AiBD could inhibit the growth of both Gram-positive and Gram-negative bacteria, and also showed strong fungicidal activity towards the expression host. Recombinant expression of AiBD made it possible to further characterize its functions involved in immune responses, and also provided a potential therapeutic agent for disease control in aquaculture. (c) 2006 Elsevier Ltd. All rights reserved.