Detection of specific noncovalent protein-fullerenols complexes by matrix-assisted laser desorption ionization mass spectrometry

Matrix-assisted laser desorption ionization (MALDI) mass spectrometry is difficult for the characterization of noncovalent complexes hitherto because of the limitations in acidic matrix, sample preparation, laser-induced polymerization and adduct formation with matrix. Under our experimental conditions, sinapinic acid is used as a matrix, the specific noncovalent interactions of protein with fullerenols were observed by MALDI mass spectrometry. Some mass spectrometric features, such as mass shifts, broad adduct peaks and stoichiometries, showed that the specific non-covalent complexes between protein and fullerenols have been formed at a ratio of 1 : 4 for hemoglobin-fullerenols or 1 : 1 for myoglobin-fullerenols. The results implied that fullereneols could be used to protect partly hemoglobin from decomposition in acidic media, and therefore, it is possible to realize the molecular weight determination of a quaternary protein by MALDI mass spectrometry via the addition of specific organic compound in the matrix.

Miscibility and specific interactions in poly(beta-hydroxybutyrate-co-beta-hydroxyvalerate) and poly(P-vinylphenol) blends

The blends of poly(beta-hydroxybutyrate-co-beta-hydroxyvalerate) (P(HB-co-HV)/poly(p-vinylphenol)(PVPh) were investigated by differential scanning calorimetry (DSC), Fourier transform IR (FT-IR) spectroscopy and high-resolution solid-state C-13 NMR techniques. Single glass transition temperatures existing in the whole composition range indicates that these blends are miscible. The presence of hydrogen bonding between the hydroxyl of PVPh and carbonyl of P(HB-co-HV), shown by FT-IR spectra, is the origin of the miscibility. Furthermore, results obtained by high-resolution solid-state C-13 NMR give more information about the structure of the blends. (C) 1998 Elsevier Science Ltd. All rights reserved.

Compatibility and specific interactions in poly(beta-hydroxybutyrate) and poly(p-vinylphenol) blends

The miscibility and specific interactions in poly (beta-hydroxybutyrate) (PHB)/poly(p-vinylphenol) (PVPh) blends were studied by differential scanning calorimetry(DSC) , fourier transform infrared(FTIR) spectrometer and high resolution solid state C-13 NMR, A single composition-dependent glass transition temperatures were obtained by DSC which indicate the blends of PHB/PVPh were miscible in the melt state, The experimental glass transition temperatures were fitted quite well with those obtained from Couchman-Karasz equation. The FTIR study shows that the strong intermolecular hydrogen bonding exists in blends of PHB with strong proton acceptor and PVPh with strong proton donor and is the origin of its compatibility. The CPMAS C-13 NMR spectra also show that the strong hydrogen bonding exists in PHB/PVPh blends. From the T-1 rho(H) relaxation time it follows that the blends of PHB/PVPh(40/60, 20/80) studied are completely homogeneous on the scale of about 3.2 nm.

TWO SPECIFIC CAUSES OF CELL MORTALITY IN FREEZE-THAW CYCLE OF YOUNG THALLI OF PORPHYRA YEZOENSIS (BANGIALES, RHODOPHYTA)1

Porphyra yezoensis Ueda is an important marine aquaculture crop with single-layered gametophytic thalli. In this work, the influences of thallus dehydration level, cold-preservation (freezing) time, and thawing temperature on the photosynthetic recovery of young P. yezoensis thalli were investigated employing an imaging pulse-amplitude-modulation (PAM) fluorometer. The results showed that after 40 d of frozen storage when performing thallus thawing under 10 degrees C, the water content of the thalli showed obvious effects on the photosynthetic recovery of the frozen thalli. The thalli with absolute water content (AWC) of 10%-40% manifested obvious superiority compared to the thalli with other AWCs, while the thalli thawed at 20 degrees C showed very high survival rate (93.10%) and no obvious correlation between thallus AWCs and thallus viabilities. These results indicated that inappropriate thallus water content contributed to the cell damage during the freeze-thaw cycle and that proper thawing temperature is very crucial. Therefore, AWC between 10% and 40% is the suitable thallus water content range for frozen storage, and the thawing process should be as short as possible. However, it is also shown that for short-term cold storage the Porphyra thallus water content also showed no obvious effect on the photosynthetic recovery of the thalli, and the survival rate was extremely high (100%). These results indicated that freezing time is also a paramount contributor of the cell damage during the freeze-thaw cycle. Therefore, the frozen nets should be used as soon as time permits.

Characterization of a new lectin involved in the protoplast regeneration of Bryopsis hypnoides

A group of coenocytic marine algae differs from higher plants, whose totipotency depends on an intact cell (or protoplast). Instead, this alga is able to aggregate its extruded protoplasm in sea water and generate new mature individuals. It is thought that lectins play a key role in the aggregation process. We purified a lectin associated with the aggregation of cell organelles in Bryopsis hypnoides. The lectin was ca. 27 kDa with a pI between pH 5 and pH 6. The absence of carbohydrate suggested that the lectin was not a glycoprotein. The hemagglutinating activity (HA) of the lectin was not dependent on the. presence of divalent cations and was inhibited by N-Acetylgalactosamine, N-Acetylglucosamine, and the glycoprotein bovine submaxillary mucin. The lectin preferentially agglutinated Gram-negative bacterium. The HA of this lectin was stable between pH 4 to pH 10. Cell organelles outside the cytoplasm were agglutinated by the addition of lectin solution (0.5 mg ml(-1)). Our results suggest that the regeneration of B. hypnoides is mediated by this lectin. We also demonstrated that the formation of cell organelle aggregates was inhibited by nigericin in natural seawater (pH 8.0). Given that nigericin dissipates proton gradients across the membrane, we hypothesize that the aggregation of cell organelles was proton-gradient dependent.

A novel C-type lectin (Cflec-3) from Chlamys farreri with three carbohydrate-recognition domains

C-type lectins are a superfamily of carbohydrate-recognition proteins which play crucial roles in the innate immunity. In this study, the gene of a C-type lectin with multiple carbohydrate-recognition domains (CRDs) from scallop Chlamys farreri (designated as Cflec-3) was cloned by rapid amplification of cDNA ends (RACE) approach based on expression sequence tag (EST) analysis. The full-length cDNA of Cflec-3 was of 2256 bp. The open reading frame encoded a polypeptide of 516 amino acids, including a signal sequence and three CRDs. The deduced amino acid sequence of Cflec-3 showed high similarity to members of C-type lectin superfamily. By fluorescent quantitative real-time PCR, the Cflec-3 mRNA was mainly detected in hepatopancreas, adductor, mantle, and marginally in gill, gonad and hemocytes of healthy scallops. After scallops were challenged by Listonella anguillarum, the mRNA level of Cflec-3 in hemocytes was up-regulated and was significantly higher than that of blank at 8 h and 12 h post-challenge. The function of Cflec-3 was investigated by recombination and expression of the cDNA fragment encoding its mature peptide in Escherichia coli BL21 (DE3)-pLysS. The recombined Cflec-3 (rCflec-3) agglutinated Gram-negative bacteria Pseudomonas stutzeri. The agglutinating activity was calcium-dependent and could be inhibited by D-mannose. These results collectively suggested that Cflec-3 was involved in the immune response against microbe infection and contributed to nonself-recognition and clearance of bacterial pathogens in scallop. (C) 2009 Elsevier Ltd. All rights reserved.

Cflec-4, a multidomain C-type lectin involved in immune defense of Zhikong scallop Chlamys farreri

C-type lectins are a superfamily of carbohydrate-recognition proteins which play crucial roles in the innate immunity. In this study, a novel multidomain C-type lectin gene from scallop Chlamys farreri (designated as Cflec-4) was cloned by RACE approach based on EST analysis. The full-length cDNA of Cflec-4 was of 2086 bp. The open reading frame was of 1830 bp and encoded a polypeptide of 609 amino acids, including a signal sequence and four dissimilar carbohydrate-recognition domains (CRDs). The deduced amino acid sequence of CflecA shared high similarities to other C-type lectin family members. The phylogenetic analysis revealed the divergence between the three N-terminal CRDs and the C-terminal one, suggesting that the four CRDs in Cflec-4 originated by repeated duplication of different primordial CRD. The potential tertiary structure of each CRD in Cflec-4 was typical double-loop structure with Ca2+-binding site 2 in the long loop region and two conserved disulfide bridges at the bases of the loops. The tissue distribution of Cflec-4 mRNA was examined by fluorescent quantitative real-time PCR. In the healthy scallops, the Cflec-4 transcripts could be only detected in gonad and hepatopancreas, whereas in the Listonella anguillarum challenged scallops, it could be also detected in hemocytes. These results collectively suggested that CflecA was involved in the immune defense of scallop against pathogen infection and provided new insight into the evolution of C-type lectin superfamily. (C) 2009 Elsevier Ltd. All rights reserved.

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.

Identification of a C-type lectin from the bay scallop Argopecten irradians

C-type lectins are Ca2+ dependent carbohydrate-recognition proteins that play crucial roles in the invertebrate innate immunity, such as nonself recognition, activation of proPO system, antibacterial activity, promotion of phagocytosis and nodule formation. In this study, a novel C-type lectin of bay scallops Argopecten irradians (Ai Lec) was identified using expressed sequence tag (EST) and RACE techniques. The Ai Lec cDNA encoded a polypeptide of 171 amino acids with a putative signal peptide of 21 amino acid residues and a mature protein of 150 amino acids. The deduced amino acid sequence of Ai Lec was highly similar to those of the C-type lectins from other animals and contained a typical carbohydrate-recognition domain (CRD) of 131 residues, which has four conserved disulfide-bonded cysteine residues that define the CRD and two additional cysteine residues at the amino terminus. The expression of Ai Lec transcript was dominantly detected in the hepatopancreas and slightly detected in the haemocytes of normal scallops. 6 h after Vibrio anguillarum-challenge and 8 h after Micrococcus luteus-challenge, the temporal expression of Ai Lec mRNA in hemocytes was increased by 4.4- and 3.6-folds, respectively. The results suggested that Ai Lec was a constitutive and inducible acute-phase protein and might be involved in immune response to Gram-negative and Gram-positive microbial infection in bay scallop A. irradians.

An electrochemical DNA sensor based on electrodepositing aluminum ion films on stearic acid-modified carbon paste electrode and its application for the detection of specific sequences related to bar gene and CP4 Epsps gene

An electrochemical DNA biosensor was fabricated by immobilizing DNA probe on aluminum ion films that were electrodeposited on the surface of the stearic acid-modified carbon paste electrode (CPE). DNA immobilization and hybridization were characterized with cyclic voltammetry (CV) by using methylene blue (MB) as indicator. MB has a couple of well-defined voltammetric redox peaks at the CPE. The currents of redox peaks of MB decreased after depositing aluminum ion films on the CPE (Al(III)/CPE) and increased dramatically after immobilizing DNA probe (ssDNA/Al(III)/CPE). Hybridization of DNA probe led to a marked decrease of the peak currents of MB, which can be used to detect the target single-stranded DNA. The conditions for the preparation of Al(III)/CPE, and DNA immobilization and hybridization were optimized. The specific sequences related to bar transgene in the transgenic corn and the PCR amplification of CP4 epsps gene from the sample of transgenic roundup ready soybean were detected by differential pulse voltammetry (DPV) with this new electrochemical DNA biosensor. The difference between the peak currents of MB at ssDNA/Al(III)/CPE and that at hybridization DNA modified electrode (dsDNA/Al(III)/CPE) was applied to determine the Specific sequence related to the target bar gene with the dynamic range comprised between 1.0 X 10(-7) mol/L to 1.0 x 10(-4) mol/L. A detection limit of 2.25 x.10(-8) mol/L. of oligonucleotides can be estimated.

Haematopoietic lineage cell-specific protein 1 (HS1) promotes actin-related protein (Arp) 2/3 complex-mediated actin polymerization

HS1 (haematopoietic lineage cell-specific gene protein 1), a prominent substrate of intracellular protein tyrosine kinases in haematopoietic cells, is implicated in the immune response to extracellular stimuli and in cell differentiation induced by cytokines. Although HS1 contains a 37-amino acid tandem repeat motif and a C-terminal Src homology 3 domain and is closely related to the cortical-actin-associated protein cortactin, it lacks the fourth repeat that has been shown to be essential for cortactin binding to filamentous actin (F-actin). In this study, we examined the possible role of HS1 in the regulation of the actin cytoskeleton. Immunofluorescent staining demonstrated that HS1 co-localizes in the cytoplasm of cells with actin-related protein (Arp) 2/3 complex, the primary component of the cellular machinery responsible for de novo actin assembly. Furthermore, recombinant HS1 binds directly to Arp2/3 complex with an equilibrium dissociation constant (K-d) of 880 nM. Although HS1 is a modest F-actin-binding protein with a Kd of 400 nM, it increases the rate of the actin assembly mediated by Arp2/3 complex, and promotes the formation of branched actin filaments induced by Arp2/3 complex and a constitutively activated peptide of N-WASP (neural Wiskott-Aldrich syndrome protein). Our data suggest that HS1, like cortactin, plays an important role in the modulation of actin assembly.

White spot syndrome virus (WSSV) infects specific hemocytes of the shrimp Penaeus merguiensis

White spot syndrome virus (WSSV) was specifically detected by PCR in Penaeus merguiensis hemocytes, hemolymph and plasma. This suggested a close association between the shrimp hemolymph and the virus. Three types of hemocyte from shrimp were isolated using flow cytometry. Dynamic changes of the hemocyte subpopulations in P. merguiensis at different times after infection were observed, indicating that the WSSV infection selectively affected specific subpopulations. Immunofluorescence assay (IFA) and a Wright-Giemsa double staining study of hemocyte types further confirmed the cellular localization of the virus in the infected hemocytes. Electron microscopy revealed virus particles in both vacuoles and the nucleus of the semigranular cells (SGC), as well as in the vacuoles of the granular cells (GC). However, no virus could be detected in the hyaline cells (HC). Our results suggest that the virus infects 2 types of shrimp hemocytes-GCs and SGCs. The SGC type contains higher virus loads and exhibits faster infection rates, and is apparently more susceptible to WSSV infection.

beta-catenin in early development of the lancelet embryo indicates specific determination of embryonic polarity

The lancelet (amphioxus) embryo develops from a miolecithal egg and starts gastrulation when it is approximately 400 cells in size, in a fashion similar to that of some non-chordate deuterostomes. Throughout this type of gastrulation, the embryo develops characteristics such as the notochord and hollow nerve cord that commonly appear in chordates. beta-Catenin is an important factor in initiating body patterning. The behavior and developmental pattern of this protein in early lancelet development was examined in this study. Cytoplasmic beta-catenin was localized to the animal pole after fertilization and then was incorporated asymmetrically into the blastomeres during the first cleavage. Asymmetric distribution was observed at least until the 32-cell stage. The first nuclear localization was at the 64-cell stage, and involved all of the cells. At the initial gastrula stage, however, concentrated beta-catenin was found on the dorsal side. LiCl treatment affected the asymmetric pattern of beta-catenin during the first cleavage. LiCl also changed distribution of nuclear beta-catenin at the initial gastrula stage: distribution extended to cells on the animal side. Apparently associated with this change, expression domains of goosecoid, lhx3 and otx also changed to a radially symmetric pattern centered at the animal pole. However, LiCl-treated embryos were able to establish embryonic polarity. The present study suggests that in the lancelet embryo, polarity determination is independent of dorsal morphogenesis.

Molecular cloning, characterization and expression analysis of a putative C-type lectin (Fclectin) gene in Chinese shrimp Fenneropenaeus chinensis

Lectin is regarded as a potential molecule involved in immune recognition and phagocytosis through opsonization in crustacean. Knowledge on lectin at molecular level would help us to understand its regulation mechanism in crustacean immune system. A novel C-type lectin gene (Fclectin) was cloned from hemocytes of Chinese shrimp Fenneropenaeus chinensis by 3' and 5' rapid amplification of cDNA ends (RACE) PCR. The full-length cDNA consists of 1482 bp with an 861 bp open reading frame, encoding 287 amino acids. The deduced amino acid sequence contains a putative signal peptide of 19 amino acids. It also contains two carbohydrate recognition domains/C-type lectin-like domains (CRD1 and CRD2), which share 78% identity with each other. CRD1 and CRD2 showed 34% and 30% identity with that of mannose-binding lectin from Japanese lamprey (Lethenteron japonicum), respectively. Both CRD1 and CRD2 of Fclectin have I I amino acids residues, which are relatively invariant in animals' C-type lectin CRDs. Five residues at Ca2+ binding site I are conserved in Fclectin. The potential Ca2+/carbohydrate-binding (site 2) motif QPD, E, NP (Gln-Pro-Asp, Glu, Asn-Pro) presented in the two CRDs of Fclectin may support its ability to bind galactose-type sugars. It could be deduced that Fclectin is a member of C-type lectin superfamily. Transcripts of Fclectin were found only in hemocytes by Northern blotting and RNA in situ hybridization. The variation of mRNA transcription level in hemocytes during artificial infection with bacteria and white spot syndrome virus (WSSV) was quantitated by capillary electrophoresis after RT-PCR. An exploration of mRNA expression variation after LPS stimulation was carried out in primarily cultured hemocytes in vitro. Expression profiles of Fclectin gene were greatly modified after bacteria, LPS or WSSV challenge. The above-stated data can provide us clues to understand the probable role of C-type lectin in innate immunity of shrimp and would be helpful to shrimp disease control. (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.