Molecular basis of transferrin polymorphism in goldfish (Carassius auratus)

Transferrin (TF) polymorphism was investigated in a color variety of goldfish (Carassius auratus), and its molecular basis analyzed. Three TF variants (A(1), A(2) and B-1) were identified from an inbred strain of the goldfish, of which A(1) and B-1 displayed a large electrophoretic difference on both native and SDS-PAGE gels. The TF cDNAs corresponding to variants A(1) and B-1 were cloned and sequenced from A(1)A(1), A(1)B(1) and B1B1 individuals, and their deduced amino acid sequences were analyzed. Substantial amino acid variation occurred between variants A(1) and B-1, with significant differences in peptide length, theoretical molecular weight (Mw) and isoelectric point (pI). No potential glycosylation sites were observed in the two amino acid sequences, which excluded the possibility that carbohydrate difference might cause electrophoretic variation among the TF variants. Further analysis suggested that the distinct electrophoretic mobility of the two variants A(1) and B-1 by SDS-PAGE resulted from their Mw difference, while the difference by the native PAGE could be explained by their pI variation. Furthermore, genomic DNA fragments containing the transferrin alleles were amplified and subjected to RFLP analysis in A(1)A(1), A(1)B(1) and B1B1 individuals. The data revealed characteristic banding patterns for each TF genotype, and demonstrated that the TF alleles A(1) and B-1 could be used as a co-dominant marker system. The initial work relating to the goldfish TF variants will benefit the understanding of the evolutionary and functional significance of TF polymorphism in fish.

Differential screening and characterization analysis of the egg envelope glycoprotein ZP3 cDNAs between gynogenetic and gonochoristic crucian carp

Gynogenetic silver crucian carp, Carassius auratus gibelio, is an intriguing model. system. In the present work, a systemic study has been initiated by introducing suppression subtractive hybridization technique into this model system to identify the differentially expressed genes in oocytes between gynogenetic silver crucian carp and its closely related gonochoristic color crucian carp. Five differential cDNA fragments were identified from the preliminary screening, and two of them are ZP3 homologues. Moreover, the full length ZP3 cDNAs were cloned from their oocyte cDNA libraries. The length of ZP3 cDNAs were 1378 bp for gyno-carp and 1367 bp for gono-carp, and they can be translated into proteins with 435 amino acids. Obvious differences are not only in the composition of amino acids, but also in the number of potential O-linked oligosaccharide sites. In addition, gyno-carp ZP3 amino acid sequence has an unexpected higher identity value with common carp (83.5%) than that with the closely related gono-carp (74.7%). The unique homology may be originated from the ancient hybridization. Northern blot analysis confirmed that expression of the ZP3 gene occurred exclusively in the oocytes. Because O-linked oligosaccharides on ZP3 have been demonstrated to play very important roles in fertilization, it is suggested that the extra O-linked glycosylation sites may be related to the unique sperm-egg recognition mechanism in gynogenesis.

2-C-支链-1,6-脱水吡喃葡萄糖的开环反应及吡喃烯糖的合成研究

在糖化学合成中，1,6-脱水吡喃糖不仅是合成具有生物活性低聚糖、糖共体、抗原、抗体以及天然产物等化合物重要原料，而且还是许多具有生物活性的天然产物的结构单元。同时，它还具有[3,2,1]的双环缩醛结构，使其在糖化学合成中具有高的立体选择性和区域选择性，同时减少了C-1 和C-6 位的保护和去保护的优点。此外，环内的缩醛开环后，又可以相应地在C-1 和C-6 位进行官能团转化以及糖苷化反应。 本文报道了一种新的1,6-脱水吡喃糖的合成方法，并设计合成了2-C-支链-1,6-脱水吡喃葡萄糖1-195、1-197、1-198 以及2-C-支链-6-硫代1,6-脱水吡喃葡萄糖1-225。到目前为止，1,6-脱水糖开环并进行糖苷化反应，存在选择性较差、产率低的缺点。我们发现，在乙腈做溶剂的条件下，NiCl5 能高立体选择性高产率地催化化合物1-195、1-197、1-198 开环并与ROH、RSH 发生糖苷化反应。在NiCl5-乙腈条件下，合成了一系列2-C-支链-α-糖苷和2-C-支链-β-硫代糖苷，并对2-C-支链1,6-脱水吡喃葡萄糖的生成机理以及开环机理进行了探讨。 烯糖在糖化学合成中是重要的起始原料，从Fischer 首次合成烯糖至今，一直不断地有新的合成方法出现。但目前文献报道的方法存在所用试剂有毒、价格贵和操作繁琐等缺点。我们对Fischer-Zach 方法进行了改进， 发现Zn-NaH2PO4-H2O 和Zn-PEG600-H2O 体系都能很好地合成烯糖。该方法具有条件温和、绿色环保、操作简单的优点。在Zn-NaH2PO4 溶液或Zn-PEG600 条件下，以溴代糖为原料，高产率地合成一系列的烯糖。 The 1,6-anhydrohexopyranoses are crucial subunits of myriad bioactive nature products, as well as important syntons of carbohydrate chemistry which have been extensively used to prepare the biologically potential oligosaccharides, glycoconjugates, antibiotics, and structurally varied nature products. Their particular [3.2.1] bicyclic skeleton makes them have high regio- and stereo-control in a variety of reactions, and such structure avoids protecting hydroxyl groups at C1 and C6.Additionally, the cleavage of the internal acetal under acidic conditions could be beneficial for further transformations of functional group and glycosylation of the corresponding pyranosyl sugar at the C6 or C1 site. Herein we developed a novel approach to prepare the 1,6-anhydrohexopyranose, and synthesized the 2-C-branched-1,6-anhydrohexopyranose 1-195, 1-197, 1-198 and 2-C-branched-6-thio-1,6-anhydrohexopyranose 1-225. Until now, glycosylation of 1,6-anhydrohexopyranoses has been limited because of the low yields and low stereoselectivity. In this paper, we found that NiCl5-MeCN system could selectively cleave the ring of 1,6-anhydrohexopyranoses with alcohols and thiols at room temperature in high yields. A series of 2-C-branched-α-glycosides and 2-C-branched-β-thioglycosides have been synthesized via NiCl5-catalyzed. Furthermore, we investigated the formation and ring-opening mechanism of 2-C-acetylmethyl-1,6-anhydrohexopyranose. Glycals are significant starting material in carbohydrate chemistry. After the Fischer-Zach method for forming glucal was reported for the first time, the numerous synthetic methods for glycals have been explored. However, there are several drawbacks in the existing methods, such as the usage of very expensive and toxic reagents, intricate operation, and the influence of acid-sensitive and base-sensitive functional group. We improved the Fischer-Zach method and developed a facile, mild and environmentally benign methodology towards the synthesis of the glycals in Zn-NaH2PO4-H2O or Zn-PEG600-H2O system. Our method involves the treatment of glycosyl bromides with Zn in NaH2PO4 aqueous solution or PEG600-H2O at room temperature, affording various glycals in excellent yields.

Facile synthesis of ginsenoside Ro

Two concise synthetic routes, being different in the glycosylation sequence, toward ginsenoside Ro (1) are developed. These syntheses feature the elaboration of the glucuronide residue at a later stage via the TEMPO-mediated selective oxidation and the installation of AZMB as a benzoylic neighboring participating group capable of being selectively removed afterward.

Evaluation of flavonoids binding to DNA duplexes by electrospray ionization mass spectrometry

In this study, electrospray ionization mass spectrometry (ESI-MS) was used to investigate the binding interactions of ten flavonoid aglycones and ten flavonoid glycosides with DNA duplexes. Relative binding affinities of the flavonoids toward DNA duplexes were estimated based on the fraction of bound DNA. The results revealed that the 4'-OH group of flavonoid aglycones was essential for their DNA-binding properties. Flavonoid glycosides with sugar chain linked on ring A or ring B showed enhanced binding toward the duplexes over their aglycone counterparts, whereas glycosylation of the flavonol quercetin on ring C exhibited a less pronounced effect.

A study of the non-covalent interaction between flavonoids and DNA triplexes by electrospray ionization mass spectrometry

The binding interactions of 22 flavonoids (9 aglycones and 13 glycosides) with DNA triplexes were investigated using electrospray ionization mass spectrometry (ESI-MS). The results revealed that the hydroxyl positions of aglycones. the locations and numbers of saccharide, as well as the aglycone skeletons play roles in the triplex-binding properties of flavonoids. The presence of 3-OH, or 3'-OH, or replacement of 4'-OH with methoxy group in aglycones decreased the fraction of bound DNA sharply. Flavonoid glycosides exhibit higher binding affinities towards the DNA triplexes than their aglycone counterparts. Glycosylations of flavones at the 8-C position and isoflavones at the 7-O position show higher binding affinities than those on the other positions of ring A of aglycones. Glycosylation with a disaccharide on 0 position of flavonol results in higher binding affinity than that with monosaccharide. Flexibility of the ring B is favorable for its interaction with DNA triplex. According to sustained off-resonance irradiation collision-induced dissociation (SORI-CID) experiments, glycosylation and non-planarity of flavonoid aglycones lead to different dissociation pathways of the flavonoid/triplex complexes.

Lung alveolar proteomics of bronchoalveolar lavage from a pulmonary alveolar proteinosis patient using high-resolution FTICR mass spectrometry

High-resolution Fourier transform ion cyclotron resonance (FTICR) mass spectrometry was developed and applied to the proteome analysis of bronchoalveolar lavage fluid (BALF) from a patient with pulmonary alveolar proteinosis. With use of 1-D and 2-D gel electrophoresis, surfactant protein A (SP-A) and other surfactant-related lung alveolar proteins were efficiently separated and identified by matrix-assisted laser desorption/ionization FTICR mass spectrometry . Low molecular mass BALF proteins were separated using a gradient 2-D gel. An efficient extraction/precipitation system was developed and used for the enrichment of surfactant proteins. The result of the BALF proteome analysis show the presence of several isoforms of SP-A, in which an N-non-glycosylierte form and several proline hydroxylations were identified. Furthermore, a number of protein spots were found to contain a mixture of proteins unresolved by 2-D gel electrophoresis, illustrating the feasibility of high-resolution mass spectrometry to provide identifications of proteins that remain unseparated in 2-D gels even upon extended pH gradients.

cDNA cloning and mRNA expression of the translationally controlled tumor protein (TCTP) gene from Japanese sea perch (Lateolabrax japonicus)

A homologue of the lower vertebrates translationally controlled tumor protein (TCTP) was cloned from the marine fish Japanese sea perch (Lateolabrax japonicus) by the technology of homology cloning. The full-length cDNA sequence of the sea perch TCTP gene contained a 5' untranslated region (UTR) of 47 bp, a 3' UTR of 433 bp, and a putative open reading frame (ORF) of 510 bp encoding a polypeptide of 170 amino acids. The deduced amino acid sequence of the sea perch TCTP gene showed a high similarity to that of zebrafish, rohu, rabbit, chicken and human. Sequence analysis revealed there were a signature sequence of TCTP family, an N-glycosylation site, and five Casein kinase phosphorylation sites in the sea perch TCTP. The temporal expression of TCTP genes in healthy and lipopolysaccharide (LPS) challenged fishes was measured by semi-quantitative reverse transcription-PCR (RT-PCR). The results indicated that LPS could up-regulate the expression of sea perch TCTP in the examined tissues, including head-kidney, spleen and liver.

Molecular cloning and expression of interleukin 1beta (IL-1 beta) from red seabream (Pagrus major)

The interleukin 1beta (IL-1beta) cDNA was cloned from the red seabream (Pagrus major) by homology cloning strategy. A cDNA fragment was amplified by PCR using two degenerated primers, which were designed according to the conserved regions of other known IL-1beta sequences, and elongated by 3' ends and 5' ends RACE PCR to get the full length coding sequence of red seabream IL-1beta (RS IL-1beta). The sequence contained 1252 nucleotides that included a 5' untranslated region (UTR) of 84 bp, a 3' UTR of 410 bp and an open reading frame (ORF) of 759 nucleotides which could be translated into a putative peptide of 253 amino acids with molecular weight of 28.6 kD and putative isoelectric point pI of 5.29. The deduced peptide contained two potential N-glycosylation sites and an identifiable IL1 family signature, but lacked the signal peptide and the clear ICE cut site, which were common in other nonmammalian IL-1beta genes. The RS IL-1beta had the highest homology with piscine IL-1beta according to phylogenetic tree analysis. The transcript expression was detected in blood, brain, gill, heart, head kidney, kidney, liver, muscle and spleen in the pathogen challenged and healthy red seabream by RTPCR. Results showed that the RS IL-1beta mRNA was constitutively expressed in most of the tissues both in stimulated and un-stimulated fish, and the expression could be enhanced by pathogen challenging.

CDNA cloning and mRNA expression of the lipopolysaccharide- and beta-1,3-glucan-binding protein gene from scallop Chlamys farreri

Lipopolysaccharide and beta-1,3-glucan-binding protein (LGBP) play a crucial role in the innate immune response of invertebrates as a pattern recognition protein (PRP). The scallop LGBP gene was obtained from Chlamys farreri challenged by Vibrio anguillarum by randomly sequencing cDNA clones from a whole body cDNA library, and by fully sequencing a clone with homology to known LGBP genes. The scallop LGBP consisted of 1876 nucleotides with a canonical polyadenylation signal sequence AATAAA and a poly(A) tail, encoding a polypeptide of 440 amino acids with the estimated molecular mass of 47.16 kDa and a predicted isoelectric point of 5.095. The deduced amino acid sequence showed a high similarity to that of invertebrate recognition proteins from blue shrimp, black tiger shrimp, mosquito, freshwater crayfish, earthworms, and sea urchins, with conserved features including a potential polysaccharide-binding motif, a glucanase motif, and N-glycosylation sites. The temporal expression of LGBP genes in healthy and V. anguillarum-challenged C farreri scallop, measured by real-time semiquantitative reverse transcription polymerase chain reaction (PCR), showed that expression was up-regulated initially, followed by recovery as the stimulation cleared. Results indicated that scallop LGBP was a constitutive and inducible acute-phase protein that could play a critical role in scallop-pathogen interaction. (C) 2004 Elsevier B.V. All rights reserved.

Molecular cloning and characterization of a catalase gene from Zhikong scallop Chlamys farreri

Catalase is one of the central enzymes involved in scavenging the high level of reactive oxygen species (ROS) by degradation of hydrogen peroxide to oxygen and water. The full-length catalase cDNA of Zhikong scallop Chlamys farreri (denoted as CfCAT) was identified from hemocytes by expressed sequence tag (EST) and rapid amplification of cDNA ends (RACE) approaches. The nucleotide sequence of CfCAT cDNA consisted of 3146 bp with a 5' UTR of 103 bp, an unusually long 31 UTR of 1519 bp with a canonical polyadenylation signal sequence AATAAA and a potyA tail, and an open reading frame (ORF) of 1521 bp encoding a polypeptide of 507 amino acids with predicted molecular weight of 57.5 kDa. The deduced amino acid sequence of CfCAT has significant homology to catalases from animals, plants and bacteria. Several highly conserved motifs including the proximal heme-ligand signature sequence RLFSYNDTH, the proximal active site signature FNRERIPERVVHAKGGGA, and the three catalytic amino acid residues of His(72), Asn(145) and Tyr(355) were identified in the deduced amino acid sequence of CfCAT. The CfCAT was demonstrated to be a peroxisomal glycoprotein with two potential glycosylation sites and a peroxisome targeting signal of ANL that was consistent with human, mouse and rat catalases. The time-course expression of CfCAT in hemocytes was measured by quantitative real-time PCR. The expression of CfCAT increased gradually and reached the highest point at 12 h post-Vibrio infection, then recovered to the original level at 24 h. All these results indicate that CfCAT, a constitutive and inducible protein, is a member of the catalase family and is involved in the process against ROS in scallop. (c) 2007 Published by Elsevier Ltd.

Molecular cloning and characterization of a thioester-containing protein from Zhikong scallop Chlamys farreri

Thioester-containing proteins are a family of proteins characterized by the unique intrachain beta-cysteinyl-gamma-glutamyl thioester, which play important roles in innate immune responses. The cDNA of Zhikong scallop Chlamys farreri thioester-containing protein (designated as CfTEP) was cloned by expressed sequence tag (EST) and rapid amplification of cDNA ends (RACE) approaches. The full-length cDNA of CfTEP was of 4616 bp, consisting of a 5 '-terminal untranslated region (UTR) of 30 bp and a 3 ' UTR of 140 bp with a polyadenylation signal sequence AATAAA and a poly(A) tail. The CfTEP cDNA encoded a polypeptide of 1481 amino acids with the theoretical isoelectric point of 5.98 and the predicted molecular weight of 161.4 kDa. The deduced amino acid sequence of CfTEP contained the canonical thioester motif GCGEQ, nine potential N-glycosylation sites and a C-terminal distinctive cysteine signature. It also contained a presumed catalytic histidine and proteolytic cleavage sites that were similar to C3 molecules. The high similarity of CfTEP with the thioester-containing proteins in other organisms, such as the TEPs from insects, the complement component C3, C4, C5 and the protease inhibitor alpha(2)-macroglobulin indicated that CfTEP should be a member of TEP family. The phylogenetic analysis revealed that CfTEP was closely related to TEPs from mollusc, nematodes and insects, and they formed a separate branch apart from the branches of complements factors and alpha(2)-macroglobulins. The spatial expression of CfTEP transcripts in healthy and bacterial challenged scallops was examined by semi-quantitative RT-PCR. The CfTEP transcripts were mainly detected in the tissues of hepatopancreas and gonad, and remarkably up-regulated by Microbial challenge, which suggested that CfTEP was a constitutive and inducible acute-phase protein involved in immune defense. These results provided new insights into the role of CfTEP in scallop immune responses, as well as the evolutionary origin of this important, widespread and functionally diversified family of proteins. (c) 2007 Published by Elsevier Ltd.

Expression of Scophthalmus maximus CD83 correlates with bacterial infection and antigen stimulation

CD83 is a transmembrane glycoprotein of the immunoglobulin (Ig) superfamily and a surface marker for fully matured dendritic cells (DCs) in humans and mice. In teleosts, DC-like cells and their molecular markers are largely unknown. In this report, we described the identification and expressional analysis of a CD83 homologue, SmCD83, from turbot Scophthalmus maximus. The open reading frame of SmCD83 is 639 bp, which is preceded by a S'-untranslated region (UTR) of 87 bp and followed by a 3'-UTR of 1111 bp. The SmCD83 gene is 4716 bp in length, which contains five exons and four introns. The deduced amino acid sequence of SmCD83 shares 40-50% overall identities with the CD83 of several fish species. Like typical CD83, SmCD83 possesses an Ig-like extracellular domain, a transmembrane domain, and a cytoplasmic domain. The conserved disulfide bond-forming cysteine residues and the N-linked glycosylation sites that are preserved in CD83 are also found in SmCD83. Expressional analysis showed that constitutive expression of SmCD83 was high in gill, blood, spleen, muscle, and kidney and low in heart and liver. Bacterial infection and poly(I:C) treatment enhanced SmCD83 expression in kidney in time-dependent manners. Likewise, bacterial challenge caused significant induction of SmCD83 expression in cultured macrophages. Vaccination of turbot with a bacterin and a purified recombinant subunit vaccine-induced significant SmCD83 expression during the first week following vaccination. These results demonstrate that SmCD83 expression correlates with microbial challenge and antigen stimulation, which suggests the possibility that there may exist in turbot DC-like antigen-presenting cells that express SmCD83 upon activation by antigen uptake. In addition, these results also suggest that SmCD83 may serve as a marker for activated macrophages in turbot. (C) 2010 Elsevier Ltd. All rights reserved.

Synthesis of a group of diosgenyl saponins with combined use of glycosyl trichloroacetimidate and thioglycoside donors

With the combined use of glycosyl trichloroacetimidates and thioglycosides, a group of natural diosgenyl saponins (1-6) are efficiently synthesized, in either a stepwise or a 'one-pot' manner. The trichloroacetimidate is employed as an efficient temporary hydroxy protecting group in glycosylation with the glycosyl trichloroacetimidate. The intermolecular alkylthio-group transfer is demonstrated to be a common side-reaction during glycosylation with thioglycosides.

Crystallisation and crystal forms of carbohydrate derivatives

This thesis is focused on the synthesis and solid state analysis of carbohydrate derivatives, including many novel compounds. Although the synthetic chemistry surrounding carbohydrates is well established in the literature, the crystal chemistry of carbohydrates is less well studied. Therefore this research aims to improve understanding of the solid state properties of carbohydrate derivatives through gaining more information on their supramolecular bonding. Chapter One focuses on an introduction to the solid state of organic compounds, with a background to crystallisation, including issues that can arise during crystal growth. Chapter Two is based on glucopyranuronate derivatives which are understudied in terms of their solid state forms. This chapter reports on the formation of novel glucuronamides and utilising the functionality of the amide bond for crystallisation. TEMPO oxidation was completed to form glucopyranuronates by oxidation of the primary alcohol groups of glucosides to the carboxylic acid derivatives, to increase functionality for enhanced crystal growth. Chapter Three reports on the synthesis of glucopyranoside derivatives by O-glycosylation reactions and displays crystal structures, including a number of previously unsolved acetate protected and deprotected crystal structures. More complex glycoside derivatives were also researched in an aim to study the resultant supramolecular motifs. Chapter Four contains the synthesis of aryl cellobioside derivatives including the novel crystal structures that were solved for the acetate protected and deprotected compounds. Research was carried out to determine if 1-deoxycellodextrins could act as putative isostructures for cellulose. Our research displays the presence of isostructural references with 1-deoxycellotriose shown to be similar to cellulose III11, 1-deoxycellotetraose correlates with cellulose IV11 and 1-deoxycellopentose shows isostructurality similar to that of cellulose II. Chapter Five contains the full experimental details and spectral characterisation of all novel compounds synthesised in this project and relevant crystallographic information.