Capillary liquid chromatographic-high-resolution mass spectrometric analysis of ribonucleotides

A method of capillary HPLC-high-resolution MS was developed for the trace analysis of ATP, GTP, dATP and dGTP Dimethylhexylamine (DMHA) was used as ion-pairing agent for the HPLC retention and separation of the nucleotides and positive ion electrospray time-of-flight MS was used for the detection. The application of capillary HPLC allowed minimal usage of DMHA while providing excellent peak retention and resolution, which significantly reduced the ion suppression in electrospray ionization-MS analysis and thus increased the sensitivity. Adduct ions of nucleotides and DMHA were used as quantitative ions in order to achieve the best sensitivity. DMHA concentration at 5 mM in the aqueous mobile phase at pH 7 was found to be the optimal conditions for the C Is capillary column. The method was applied to determine ATP level in cultured C6 glioma cells that were treated with toxic concentrations of Zn. The results showed that the cellular ATP level decreased from 2.7 pmol/cell (<10% cell death) in average control cell samples to 0.36 pmol/cell as the concentration of Zn increased to 120 mg/l (>35% cell death) in culture medium.

Quantitative structure-property relationship research to the analysis of photoionization sensitivity of organic compounds in gas chromatography

The A(m) index and molecular connectivity index were used for studying the photoionization sensitivity of some organic compounds in gas chromatography. The analysis of structure-property relationship between the photoionization sensitivity of the compounds and the A(m) indices or molecular connectivity indices has been carried out. The genetic algorighm was used to build the correlation model in this field. The results demonstrate that the property of compounds can be described by both A(m) indices and molecular connectivity indices, and the mathematical model obtained by the genetic algorithm was better than that by multivariate regression analysis.

Selection for variables in quantitative structure-activity/property relationship studies - Comparison between methods of orthogonal descriptors and leaps-and-bounds regression analysis

In this paper, the comparison of orthogonal descriptors and Leaps-and-Bounds regression analysis is performed. The results obtained by using orthogonal descriptors are better than that obtained by using Leaps-and-Bounds regression for the data set of nitrobenzenes used in this study. Leaps-and-Bounds regression can be used effectively for selection of variables in quantitative structure-activity/property relationship(QSAR/QSPR) studies. Consequently, orthogonalisation of descriptors is also a good method for variable selection for studies on QSAR/QSPR.

Quantitative analysis of pethidine using liquid secondary ion and tandem mass spectrometry

A method for the quantatitive determination of pethidine in human urine by liquid secondary ion and tandem mass spectrometry is presented. Quantification was carried out by using ketamine as internal standard. It was found that the collision-induced dissociation (CID) spectrum of the [M + H](+) ion of pethidine exhibited a prominent daughter ion at mit 220 and ketamine also yielded the same daughter ion at nit 220, For ((quadrupole)) quantitative analysis, the first quadrupole mass filter was set to transmit mit 220 and a narrow-range magnet scan yielded a spectrum of parents, including mit 238 and 248, corresponding to ketamine and pethidine, respectively. Copyright (C) 1999 John Wiley & Sons, Ltd.

Quantitative structure-property relationships for colour reagents and their colour reactions with ytterbium using regression analysis and computational neural networks

In this paper, the new topological indices A(x1)-A(x3) suggested in our laboratory and molecular connectivity indices have been applied to multivariate analysis in structure-property studies. The topological indices of twenty asymmetrical phosphono bisazo derivatives of chromotropic acid have been calculated. The structure-property relationships between colour reagents and their colour reactions with ytterbium have been studied by A(x1)-A(x3) indices and molecular connectivity indices with satisfactory results. Multiple regression analysis and neural networks were employed simultaneously in this study.

QUANTITATIVE STRUCTURE-ACTIVITY-RELATIONSHIPS FOR TOXICITY OF PHENOLS USING REGRESSION-ANALYSIS AND COMPUTATIONAL NEURAL NETWORKS

Quantitative structure-toxicity models were developed that directly link the molecular structures of a et of 50 alkYlated and/or halogenated phenols with their polar narcosis toxicity, expressed as the negative logarithm of the IGC50 (50% growth inhibitor

ANALYSIS OF A NEW SUPERCONDUCTING PHASE IN THE BISRCACUO SYSTEM

By doping Sb, Ba and In to the Bi-system and with the variation of nominal atomic ratio of Cu, Sr and Ca, a new possible phase is found with T(c) almost-equal-to 105 K, but with a structure of 2212 phase. Effects of post-sintering treatment from air-quenched to O2-annealed were investigated for the approximately 105 K new phase.

CLONING AND QUANTITATIVE ANALYSIS OF THE CARBONIC ANHYDRASE GENE FROM PORPHYRA YEZOENSIS

Carbonic anhydrase (CA), an enzyme that catalyzes the interconversion of CO2 and HCO3-, has a critical role in inorganic carbon acquisition in many kingdoms, including animals, plants, and bacteria. In this study, the full-length cDNA of the CA gene from Porphyra yezoensis Ueda (denoted as PyCA) was cloned by using an expressed sequence tag (EST) and rapid amplification of cDNA ends (RACE). The nucleotide sequence of PyCA consists of 1,153 bp, including a 5' untranslated region (UTR) of 177 bp, a 3' UTR of 151 bp, and an open reading frame (ORF) of 825 bp that can be translated into a 274-amino-acid putative peptide with a molecular mass (M) of 29.8 kDa and putative isoelectric point (pI) of 8.51. The predicted polypeptide has significant homology to the beta-CA from bacteria and unicellular algae, such as Porphyridium purpureum. The mRNA in filamentous thalli, leafy thalli, and conchospores was examined, respectively, by real-time fluorescent quantitative PCR (qPCR), and the levels of PyCA are different at different stages of the life cycle. The lowest level of mRNA was observed in leafy thalli, and the level in filamentous thalli and in the conchospores was 4-fold higher and 10-fold higher, respectively.

First molluscan TNFR homologue in Zhikong scallop: Molecular characterization and expression analysis

Tumor necrosis factor receptors (TNFRs) are a superfamily of proteins characterized by the unique cysteine-rich domain (CRD) and their important roles in diverse physiological and pathological events such as inflammation, apoptosis, autoimmunity and organogenesis. The first member of the molluscan TNFR family, designated as CfTNFR, was identified from Zhikong scallop Chlamys farreri by expressed sequence tag (EST) and rapid amplification of cDNA ends (RACE) approaches. The full-length cDNA of CfTNFR was of 1334 bp, consisting of a 5' UTR of 17 bp, a 3'UTR of 69 by with a poly (A) tail, and an open reading frame (ORE) of 1248 by encoding a polypeptide of 415 amino acids with a theoretical isoelectric point of 8.33 and predicted molecular weight of 47.07 kDa. There were a signal peptide, a CRD, a transmembrane region and a death domain in the deduced amino acid sequence of CfTNFR, suggesting that it was a typical type 1 membrane protein. The high identities (22-40%) of CfTNFR with other TNFR superfamily members indicated that CfTNFR should be a member of TNFR superfamily, and moreover, it should be the first death domain-containing TNFR found in invertebrates. Phylogenetic analysis revealed that CfTNFR was closely related to TNFR-like proteins from Strongylocentrotus purpuratus, Drosophila melanogaster and Ciona intestinalis, and they formed a separate branch apart from vertebrate TNFRs. The spatial expression of CfTNFR transcripts in healthy and bacteria challenged scallops was examined by quantitative real-time PCR. CfTNFR transcripts could be detected in all tested tissues, including haemocytes, gonad, gill, mantle and hepatopancreas, and significantly up-regulated in the tissues of gonad, gill, mantle and hepatopancreas after Listonella anguillarum challenge, indicating that CfTNFR was constitutive and inducible acute-phase protein involved in immune defence. The present results suggested the existence of the TNFR-like molecules and TNF-TNFR system in low invertebrates, and provided new insights into the role of CfTNFR in scallop innate immune responses to invading microorganisms. (C) 2009 Elsevier Ltd. All rights reserved.

Quantitative traits correlative analysis and growth comparison among different populations of bay scallop, Argopecten irradians

The shell traits and weight traits are measured in cultured populations of bay scallop, Argopecten irradians. The results of regression analysis show that the regression relationships for all the traits are significant (P < 0.01). The correlative coefficients between body weight, as well as tissue weight with shell length, shell height and shell width are significant (P < 0.05). But the correlative coefficients between the anterior and posterior auricle length with body weight as well as tissue weight are not significant (P > 0.05). The multiple regression equation is obtained to estimate live body weight and tissue weight. The above traits except anterior and posterior auricle length are used for the growth and production comparison among three cultured populations, Duncan's new multiple range procedure analysis shows that all the traits in the Lingshuiqiao (LSQ) population are much more significant than those of the other two populations (P < 0.01), and there is no significant difference between the Qipanmo (QPM) and Dalijia (DLJ) populations in all traits (P > 0.05). The results indicate that the LSQ population has a higher growth rate and is expected to be more productive than the other two populations.

Molecular cloning, genomic organization and functional analysis of an anti-lipopolysaccharide factor from Chinese mitten crab Eriocheir sinensis

Anti-lipopolysaccharide factor (ALF) represents one kind of basic proteins, which binds and neutralizes LPS and exhibits strong antibacterial activity against Gram-negative R-type bacteria. The ALF gene of Chinese mitten crab Eriocheir sinensis (Milne Edwards, 1853) (denoted as EsALF) was identified from haemocytes by expressed sequence tag (EST) and PCR approaches. The full-length cDNA of EsALF consisted of 700 nucleotides with a canonical polyadenylation signal-sequence AATAAA, a polyA tail, and an open-reading frame of 363 bp encoding 120 amino acids. The high similarity of EsALF-deduced amino acid sequence shared with the ALFs from other species indicated that EsALF should be a member of ALF family. The mRNA expression of EsALF in the tissues of heart, gonad, gill, haemocytes, eyestalk and muscle was examined by Northern blot analysis and mRNA transcripts of EsALF were mainly detected in haemocytes, heart and gonad. The temporal expression of EsALF in haemocytes after Vibrio anguillarum challenge was recorded by quantitative real-time RT-PCR. The relative expression level of EsALF was up-regulated rapidly at 2 h post-injection and reached 3-fold to that in blank group. After a drastic decrease to the original level from 4 to 8h, the expression level increased again and reached 4-fold to that in the blank group at 12 h post-injection. The genomic DNA sequence of EsALF gene consists of 1174bp containing three exons and two introns. The coding sequence of the EsALF mature peptide was cloned and expressed in Escherichia coli BL21(DE3)-pLysS to further elucidate its biological functions. The purified recombinant product showed bactericidal activity against both Gram-positive (G(+)) and Gram-negative (G(-)) bacteria, which demonstrated that the rEsALF was a broad-spectrum antibacterial peptide. All these results indicated that EsALF was an acute-phase protein involved in the immune responses of Chinese mitten crab, and provided a potential therapeutic agent for disease control in aquaculture. (c) 2007 Elsevier Ltd. All rights reserved.

The first molluscan TCTP in Venerupis philippinarum: Molecular cloning and expression analysis

Translationally controlled tumor protein (TCTP) is one of the abundant and ubiquitously expressed proteins in metazoans In the present study, the first molluscan TCTP (denoted as VpTCTP) was identified from Venerupis philippinarum haemocytes by EST and RACE approaches The full-length cDNA of VpTCTP consisted of 1148 nucleotides with an open-reading frame of 555 bp encoding 184 amino acids The deduced amino acid sequence of VpTCTP shared high similarity with TCTPs from other species, indicating that VpTCTP should be a new member of TCTP family Several highly conserved motifs, including 5'terminal ologopyrimidine (5'TOP) starting sequence and rich AU and AUUT elements in 3'UTR, were also identified in VpTCTP The tissue and temporal expression of VpTCTP after Vi boo anguillarum challenge was recorded by quantitative real-time RT-PCR. VpTCTP transcript could be detected in all examined tissues with the highest expression level in haemocytes and the lowest in hepatopancreas Concerning the time-course expression in haemocytes, the relative expression of VpTCTP mRNA was down-regulated sharply from 6 h to 12 h post-infection. Then, the expression level was obviously up-regulated and reached 3.4-fold to that in the control group at 48 h post challenge As time progressed, the expression of VpTCTP recovered to the original level at 96 h. All these results indicated that VpTCTP was an acute-phase protein involved in the Immune response of V philippinarum (C) 2010 Elsevier Ltd. All rights reserved.

Analysis of urinary 8-hydroxydeoxyguanosine by capillary electrophoresis and solid-phase extraction

Urinary 8-hydroxydeoxyguanosine (80HdG) has been considered as an excellent marker of individuals at high risk of developing cancer. Until now, urinary 80HdG has largely been measured by high-performance liquid chromatography with electrochemical detection. A new method for the analysis of urinary 80HdG by high-performance capillary electrophoresis has been developed and optimized in our laboratory. A single step solid-phase extraction procedure was optimized and used for extracting 80HdG from human urine. Separations were performed in an uncoated silica capillary (50 cm x 50 tm i.d.) using a P/ACE MDQ system with UV detection. The separation of 80HdG from interfering urinary matrix components is optimized with regard to pH, applied voltage, pressure injection time and concentration of SDS in running buffer. The detection limit of this method is 0.4 mug/ml, the linear range is 0.8-500 mug/ml, the correlation coefficients levels is better than 0.999. The developed method is simple, fast and good reproducibility, furthermore, it requires a very small injection volumes and low costs of analysis, which makes it possible to provide a new noninvasive assay for an indirect measurement of oxidative DNA damage.