Fluorescence spectroscopy studies on the action of rare earths with bovine serum albumin in multimolecular system

In this paper. the interaction of tripositive terbium ions (Tb3+) with bovine serum albumin (BSA) has been investigated in the presence of some alkaline earth metals and citric acid (Cit) by using fluorescence spectroscopy. The results show that Tb-31. BSA and Cit can form ternary complex BSA . Tb-2. Cit(4) in mu =0.1 mol/l NaCl. pH6.3 hexamethylenetetramine buffer. Other tare earths are able to compete for the same binding site in BSA with Tb3+-. The sequence of con,petition is Eu3+>Pr3+>Yb3+>Gd3+>La3+>Ca2+ and Mg2+ cannot replace Tb bound to BSA.

IN-SITU UV-VISIBLE SPECTROELECTROCHEMICAL AND CIRCULAR DICHROIC ELECTROCHEMICAL STUDY OF BILIRUBIN AND BILIRUBIN PLUS HUMAN SERUM-ALBUMIN COMPLEX

The electro-oxidation of bilirubin (BR) in aqueous solution was investigated by cyclic voltammetry and in-situ thin-layer spectroelectrochemical techniques, It was found that both oxidation processes of BR are two electron transfer reactions. A mechanism

Cloning, characterization and expression of ferritin subunit from clam Meretrix meretrix in different larval stages

Shell formation is one of the important events during larval development and metamorphosis in bivalves. However, the molecular mechanisms and environmental cues regulating shell initiation and growth are unclear. Here, we report that ferritin, a principal protein for biological iron storage and metabolism, might play a role in larval shell development of the bivalve mollusk Meretrix meretrix. A full-length ferritin subunit cDNA, named as MmeFer, was cloned and characterized. The MmeFer mRNA expression in different developmental stages, from trochophore to post larvae, was analyzed by real-time reverse transcription polymerase chain reaction (RT-PCR). MmeFer mRNA expression in larvae of later developmental stages increased at least 8-fold following trochophores. Moreover, the temporal and spatial expressions of MmeFer mRNA were examined by whole mount in situ hybridization. In the trochophore stage, MmeFer was detectable where it was supposed to be for shell initiation. In the later developmental stages, MmeFer was found near digestive glands and mantle that secret larval shell. MmeFer expression was also detected in larvae cultured in artificial seawater with different iron concentrations ranging from 0 to 100 mu M. These results suggest that ferritin may play a role in the shell formation of mollusks. (C) 2009 Elsevier Inc. All rights reserved.

Cloning, expression and identification of ferritin from Chinese shrimp, Fenneropenaeus chinensis

Ferritin, the iron storage protein, plays a key role in iron metabolism. A cDNA encoding ferritin (FcFer) was cloned from hepatopancreas of Chinese shrimp, Fenneropenaeus chinensis. The predicted protein contains 170 amino acid residues with a predicted molecular weight (MW) about 19, 422.89 Da and theoretical isoelectric point (PI) of 4.73. Amino acid alignment of FcFer revealed 97% homology with Litopenaeus vannamei ferritin. Results of the RT-PCR showed that the expression of FcFer mRNA was up-regulated after shrimp was challenged with either white spot syndrome virus (WSSV) or heavy metal ions (Zn2+ and Cu2+) in the laboratory. A fusion protein containing FcFer was produced and the purified recombinant protein exhibited similar function of iron uptake in vitro. The result of in-gel digestion and identification using LC-ESI-MS showed that two peptide fragments (-DDVALPGFAK- and -LLEDEYLEEQVDS1KK-) of the recombinant protein were identical to the corresponding sequence of L. vannamei ferritin. The recombinant FcFer protein will be proved useful for study on the structure and function of ferritin in F chinensis. (c) 2006 Elsevier B.V. All rights reserved.

Identification and analysis of a Scophthalmus maximus ferritin that is regulated at transcription level by oxidative stress and bacterial infection

Ferritins are conserved Iron storage proteins that exist in most living organisms and play an essential role in Iron homeostasis. In this study, we reported the identification and analysis a ferritin M subunit, SmFerM, from turbot Scophthalmus maximus. The full length cDNA of SmFerM contains a 5'-untranslated region (UTR) of 232 bp, an open reading frame (ORF) of 531 bp, and a 3'-UTR of 196 bp The ORF encodes a putative protein of 176 amino acids, which shares extensive sequence identities with the M terrains of several fish species. In silico analysis identified in SmFerM both the ferroxidase center of mammalian H ferritins and the iron nucleation site of mammalian L ferritins. Quantitative real time reverse transcriptase-PCR analysis indicated that SmFerM expression was highest in muscle and lowest in heart and responded positively to experimental challenges with bacterial pathogens and poly(I center dot C) Exposure of cultured turbot hepatocytes to treatment of stress inducers (iron, copper, and H2O2) significantly upregulated the expression of SmFerM in a dose dependent manner. Iron chelating analysis showed that recombinant SmFerM purified from Escherichia coli exhibited apparent iron binding activity. These results suggest that SmFerM is a functional M ferritin and is likely to play a role in iron sequestration and protection against oxidative stress and microbial infection (C) 2010 Elsevier Inc All rights reserved

Purification and characterisation of a natural lectin from the serum of the shrimp Litopenaeus vannamei

A natural lectin from the serum of the shrimp Litopenaeus vannamei was purified to homogeneity by a single-step affinity chromatography using fetuin-coupled agarose. The purified serum lectin (named LVL) showed a strong affinity for human A/B/O erythrocytes (RBC), mouse RBC, chicken RBC and its haemagglutinating (HA) activity was specifically dependent on Ca2+ and reversibly sensitive to EDTA. LVL inactive form had a molecular mass estimate of 172 kDa and was composed of two non-identical subunits (32 and 38 kDa) cross-linked by interchain disulphide bonds. Significant LVL activity was observed between pH 7 and 11. In HA-inhibition assays performed with several carbohydrates and glycoproteins, LVL showed a distinct and unique specificity for GalNAc/GluNAc/NeuAc which had an acetyl group, while glycoproteins fetuin and bovine submaxillary mucin (BSM) had sialic acid. Moreover, this agglutinin appeared to recognise the terminal N- and O-acetyl groups in the oligosaccharide chain of glycoconjugates. The HA activity of L. vannamei lectin was also susceptible to inhibition by lipopolysaccharides from diverse Gram-negative bacteria, which might indicate a significant in vivo role of this humoral agglutinin in the host immune response against bacterial infections. (C) 2006 Elsevier Ltd. All rights reserved.

Enhancement of atmospheric pressure chemical ionization for the determination of free and glycine-conjugated bile acids in human serum

A highly sensitive and accurate method based on the precolumn derivatization of bile acids (BA) with a high ionization efficiency labeling reagent 1,2-benzo-3,4-dihydrocarbazole-9-ethyl-benzenesulfonate (BDEBS) coupled with LC/MS has been developed. After derivatization, BA molecules introduced a weak basic nitrogen atom into the molecular core structure that was readily ionized in commonly used acidic HPLC mobile phases. Derivatives were sufficiently stable to be efficiently analyzed by atmospheric pressure chemical ionization (APCI)-MS/MS in positive-ion mode. The MS/MS spectra of BA derivatives showed an intense protonated molecular ion at m/z [M + H](+). The collision-induced dissociation of the molecular ion produced fragment ions at [MH - H2O](+), [MH - 2H(2)O](+), [MH - 3H(2)O](+). The characteristic fragment ions were at m/z 320.8, 262.8, and 243.7 corresponding to a cleavage of N - CO, O - CO, and C - OCC, respectively, and bonds of derivatized molecules. The selected reaction monitoring, based on the m/z [M + H]+ -> [MH - H2O](+), [MH - H2O](+), [MH - 2H(2)O](+), [MH-3H(2)O](+), 320.8, 262.8, and 243.7 transitions, was highly specific for the BA derivatives. The LODs for APCI in a positive-ion mode, at an S/N of 5, were 44.36-153.6 fmol. The validation results showed high accuracy in the range of 93-107% and the mean interday precision for all standards was < 15% at broad linear dynamic ranges (0.0244-25nmol/mL). Good linear responses were observed with coefficients of > 0.9935 in APCI/MS detection. Therefore, the facile BDEBS derivatization coupled with mass spectrometric analysis allowed the development of a highly sensitive and specific method for the quantitation of trace levels of the free and glycine-conjugated BA from human serum samples.

Seasonal regulations of energetics, serum concentrations of leptin, and uncoupling protein 1 content of brown adipose tissue in root voles (Microtus oeconomus) from the Qinghai-Tibetan plateau

Survival of small mammals in winter requires proper adjustments in physiology, behavior and morphology. The present study was designed to examine the changes in serum leptin concentration and the molecular basis of thermogenesis in seasonally acclimatized root voles (Microtus oeconomus) from the Qinghai-Tibetan plateau. In January root voles had lower body mass and body fat mass coupled with higher nonshivering thermogenesis (NST) capacity. Consistently, cytochrome c oxidase activity and mitochondrial uncoupling protein-1 (UCP1) protein contents in brown adipose tissues were higher in January as compared to that in July. Circulating level of serum leptin was significantly lower in winter and higher in July. Correlation analysis showed that serum leptin levels were positively related with body mass and body fat mass while negatively correlated with UCP1 protein contents. Together, these data provided further evidence for our previous findings that root voles from the Qinghai-Tibetan plateau mainly depend on higher NST coupled with lower body mass to enhance winter survival. Further, fat deposition was significantly mobilized in cold winter and leptin was potentially involved in the regulation of body mass and thermogenesis in root voles. Serum leptin might act as a starvation signal in winter and satiety signal in summer.

Determination of sialic acids in the serum of cancer patients by capillary electrophoresis

A novel method for the determination of N-acetylneuraminic acid (NANA) and N-glycolylneuraminic acid (NGNA) was developed by using high-performance capillary electrophoresis (HPCE) with UV detection at 195 nm. NANA and NGNA were separated directly and analyzed without pre- or postcolumn derivation. The detection limit of NANA is 9.6 x 10(-6) mol L-1 and for mass 3.879 x 10(-14) mol (39 fmol). This method was applied for the determination of NANA in 30 normal human and 72 cancer patients. The results demonstrated that NANA in the sera of cancer patients increased significantly as compared with the normal human (P < 0.001). The new method is simple and sensitive, and is suitable for basic research and clinical application to malignant tumors.

Removal of endotoxin from human serum albumin solutions by hydrophobic and cationic charged membrane

A novel matrix of macropore cellulose membrane was prepared by chemical graft, and immobilized the cationic charged groups as affinity ligands. The prepared membrane Fan be used for the removal of endotoxin from human serum albumin (HSA) solutions. With a cartridge of 20 sheets affinity membrane of 47 mm diameter, the endotoxin level in HSA solution can be reduced ro 0.027 eu/mL. Recovery of HSA was over 95%.

Synthesis of a silica-bonded bovine serum albumin s-triazine chiral stationary phase for high-performance liquid chromatographic resolution of enantiomers

A novel method of synthesizing protein chiral stationary phase (protein-CSP) is proposed with 2,4,6-trichloro-1,3,5-triazine as the activator. The bovine serum albumin (BSA) based chiral columns (150x4.6 mm I.D.) were prepared successfully within 8 h. With tryptophan as the probe solute, it was observed that the BSA immobilized by this method had a better ability to distinguish enantiomers than that activated by glutaric dialdehyde. This may be due to the well-maintained BSA conformation and the larger amount of BSA immobilized on the silica gel. The BSA-CSP prepared by this method was relatively stable under experimental conditions, and the resolution of 13 chiral compounds was achieved. The coupling reaction in this method is mild, reliable and reproducible; it is also suitable for the immobilization of various biopolymers in the preparation of bioreactor, biosensor and affinity chromatography columns. (C) 2000 Elsevier Science B.V. All rights reserved.