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Human Serum (human + serum)
Kinds of Human Serum Terms modified by Human Serum Selected AbstractsElectrochemical Evaluation of Nucleoside Analogue Lamivudine in Pharmaceutical Dosage Forms and Human SerumELECTROANALYSIS, Issue 20 2005Burcu Dogan Abstract Lamivudine (LAM) is a synthetic nucleoside analogue with activity against human immunodeficiency virus-type 1 (HIV-1) and Hepatitis B virus (HBV). The aim of this study was to determine LAM levels in serum and pharmaceutical formulations, by means of electrochemical methods using hanging mercury drop electrode (HMDE). On this electrode, LAM undergoes irreversible reduction at the peak potential near Ep,1.26,V (vs. Ag/AgCl/3,M KCl). Reduction LAM signals were measured by cyclic voltammetry (CV), differential pulse voltammetry (DPV) and square-wave voltammetry (OSW). DPV and OSW techniques for the determination of LAM in acetate buffer at pH,4.5, which allows quantitation over the 4×10,6 to 1×10,4,M range in supporting electrolyte for both methods, were proposed. The linear response was obtained in acetate buffer in the ranges of 2×10,6 to 2×10,4,M for spiked serum samples at pH,4.5 for both techniques. The repeatability and reproducibility of the methods for all media were determined. The standard addition method was used in serum. Precision and accuracy were also checked in all media. No electroactive interferences from the endogenous substances were found in serum. With respect to side effects of high doses and short half-life of LAM, a fast and simple detection method is described in this study. [source] Electrophoretically mediated microanalysis for the evaluation of interspecies variation in cholinesterase metabolismELECTROPHORESIS, Issue 14 2010Joana Moura Abstract This study describes an electrophoretically mediated microanalysis method, suitable for the preclinical evaluation of the hydrolysis of ester drugs by the serum of different animals and for further characterization of human,animal correlation. Dog, cat, cow, horse, sheep, rat and human serum were diluted (25%) in the appropriate buffer and replaced the enzyme solution usually used in electrophoretically mediated microanalysis methods for the study of enzyme kinetics. They were then compared in terms of the ability to hydrolyze acetylthiocholine and butyrylthiocholine (0.25,mM) by in-capillary reaction. Human serum afforded the highest conversion rates (52% butyryltiocholine and 34% acetylthiocholine) followed by horse (31 and 35%), dog (26 and 24%), cat (22 and 14%), rat (11 and 15%) and sheep (8 and 8%). Hydrolysis by bovine serum was negligible. The method is fast (under 8,min including rinsing steps), sensitive (under 25,,M substrate could be quantified) and repeatable (RSD,2%), only requiring minute amounts of sample. [source] A Glassy Carbon Electrode Modified with Multiwalled Carbon Nanotube/Chitosan Composite as a New Sensor for Simultaneous Determination of Acetaminophen and Mefenamic Acid in Pharmaceutical Preparations and Biological SamplesELECTROANALYSIS, Issue 15 2010Ali Babaei Abstract A new chemically modified electrode is constructed based on multiwalled carbon nanotube/chitosan modified glassy carbon electrode (MWCNTs-CHT/GCE) for simultaneous determination of acetaminophen (ACT) and mefenamic acid (MEF) in aqueous buffered media. The measurements were carried out by application of differential pulse voltammetry (DPV), cyclic voltammetry (CV) and chronoamperometry (CA) methods. Application of DPV method showed that the linear relationship between oxidation peak current and concentration of ACT and MEF were 1,,M to 145,,M, and 4,,M to 200,,M, respectively. The analytical performance of this sensor has been evaluated for detection of ACT and MEF in human serum, human urine and a pharmaceutical preparation with satisfactory results. [source] Simultaneous Determination of Dopamine and Ascorbic Acid Using the Nano-Gold Self-Assembled Glassy Carbon ElectrodeELECTROANALYSIS, Issue 10 2009Guangzhi Hu Abstract Electrochemical behavior of dopamine (DA) was investigated at the gold nanoparticles self-assembled glassy carbon electrode (GNP/LC/GCE), which was fabricated by self-assembling gold nanoparticles on the surface of L -cysteine (LC) modified glassy carbon electrode (GCE) via successive cyclic voltammetry (CV). A pair of well-defined redox peaks of DA on the GNP/LC/GCE was obtained at Epa=0.197,V and Epc=0.146,V, respectively. And the peak separation between DA and AA is about 0.2,V, which is enough for simultaneous determination of DA and AA. The peak currents of DA and AA were proportional with their concentrations in the range of 6.0×10,8,8.5×10,5 mol L,1 and 1.0×10,6,2.5×10,3 mol L,1, with the detection limit of 2.0×10,8 mol L,1 and 3.0×10,7 mol L,1 (S/N=3), respectively. The modified electrode exhibits an excellent reproducibility, sensibility and stability for simultaneous determination of DA and AA in human serum with satisfactory result. [source] Electrophoretically mediated microanalysis for the evaluation of interspecies variation in cholinesterase metabolismELECTROPHORESIS, Issue 14 2010Joana Moura Abstract This study describes an electrophoretically mediated microanalysis method, suitable for the preclinical evaluation of the hydrolysis of ester drugs by the serum of different animals and for further characterization of human,animal correlation. Dog, cat, cow, horse, sheep, rat and human serum were diluted (25%) in the appropriate buffer and replaced the enzyme solution usually used in electrophoretically mediated microanalysis methods for the study of enzyme kinetics. They were then compared in terms of the ability to hydrolyze acetylthiocholine and butyrylthiocholine (0.25,mM) by in-capillary reaction. Human serum afforded the highest conversion rates (52% butyryltiocholine and 34% acetylthiocholine) followed by horse (31 and 35%), dog (26 and 24%), cat (22 and 14%), rat (11 and 15%) and sheep (8 and 8%). Hydrolysis by bovine serum was negligible. The method is fast (under 8,min including rinsing steps), sensitive (under 25,,M substrate could be quantified) and repeatable (RSD,2%), only requiring minute amounts of sample. [source] Improved disc SDS-PAGE for extraction of low molecular weight proteins from serumELECTROPHORESIS, Issue 6 2010Tiechun Li Abstract The low molecular weight proteins can provide a lot of valuable information of biomarkers. To study these proteins, the high abundance and high molecular weight proteins must be removed prior to analysis. In this work, a simple and inexpensive disc SDS-PAGE to extract low molecular weight proteins from human serum and cutoff proteins larger than 30,kDa was developed. Some experimental conditions were examined. The experimental results obtained by plate SDS-PAGE and MALDI-TOF MS showed that the molecular weight of extracted proteins was about in the range from 0.3 to 28,kDa. Some experiments, including precipitation of proteins in organic solvents, SPE and cytochrome C test, were carried out and the experimental results demonstrated successful recovery of proteins/peptides with molecular weight from several hundreds of dalton to about 30,kDa. The experimental results obtained by plate SDS-PAGE indicated the repeatability was satisfactory. [source] Analysis of aristolochic acids by CE-MS with carboxymethyl chitosan-coated capillaryELECTROPHORESIS, Issue 10 2009Xiaofang Fu Abstract A CE-MS method for rapid determination of aristolochic acid-I and aristolochic acid-II (AA-II) in traditional Chinese medicines and biological samples was described in the present paper. AA-I and AA-II can be baseline separated within 6,min by CE-MS with carboxymethyl-chitosan-coated capillary. CZE conditions including pH, concentration of buffer, applied voltage, and capillary temperature were systematically investigated, and the composition and flow rate of sheath liquid were also optimized for CE-MS. Furthermore, the CE-UV method without any additives in BGE solution was established and compared with the CE-MS method. The results showed that the two methods could achieve satisfactory separation efficiency, repeatability, and linearity, while the LOD was 0.6,,g/mL for CE-UV and 0.05,,g/mL for CE-MS. Compared with the CE-UV method, the sensitivity of CE-MS was significantly improved, in addition to the structure information provided by MS detection at the same time. As an application example, a spiked sample in human serum was analyzed by the CE-MS method, indicating that the new CE-MS method can be applied to analyze AAs in biological samples. [source] Cover Picture: Electrophoresis 22'2008ELECTROPHORESIS, Issue 22 2008Article first published online: 26 NOV 200 Regular issues provide a wide range of research and review articles covering all aspects of electrophoresis. Here you will find cutting-edge articles on methods and theory, instrumentation, nucleic acids, CE and CEC, miniaturization and microfluidics, proteomics and two-dimensional electrophoresis. Selected topics of issue 22 are: Microfluidics: Applications for analytical purposes in chemistry and biochemistry ((http://doi.wiley.com/10.1002/elps.200800121)) Simultaneous laser-induced fluorescence and retro-reflected beam interference detection for CE ((http://doi.wiley.com/10.1002/elps.200800292)) Quantitative Proteomics by Fluorescent Labeling of Cysteine Residues using a Set of Two Cyanine-based or Three Rhodamine-based Dyes ((http://doi.wiley.com/10.1002/elps.200800092)) Chemometric resolution of fully overlapped capillary electrophoresis peaks: quantitation of carbamazepine in human serum in the presence of several interferences ((http://doi.wiley.com/10.1002/elps.200800400)) Identification of inorganic ions in post-blast explosive residues using portable capillary electrophoresis instrumentation and capacitively-coupled contactless conductivity detection ((http://doi.wiley.com/10.1002/elps.200800226)) [source] Cover Picture: Electrophoresis 21'2008ELECTROPHORESIS, Issue 21 2008Article first published online: 14 NOV 200 This issue has an emphasis on "Proteomics and Related Topics". It comprises 11 research articles including the "Fast Track" article on the topic of proteomics, glycoproteomics, proteins and peptides. The "Fast Track" article describes a CE-LIF detection-based assay for the simultaneous measurements of the electrophoretic mobility, catalytic activity and the variation of activity over time of the individual enzymes molecules of Escherichia coli beta-galactosidase. The remaining 10 research articles of the Emphasis deal with the development of sensitive fluorescent staining for proteomic analysis, depletion of high abundance proteins form human serum, lectin affinity chromatography in the identification of rat urinary glycoproteome, lab-on-chip screening strategy of mouse serum samples prior to proteomics analysis, identification of proteins from membrane preparations, capillary coating for CE of proteins, characterization of rabbit liver apothioneins by CE-ESI-MS, quantitative analysis of recombinant protein charge heterogeneity by imaging CIEF, dye staining and immunodetection of proteins on a PVDF membrane, and separation of multiphosphorylated peptide isomers by CZE. [source] SPE and large-volume sample stacking in MEKC for determination of doxycycline in biological fluids: Comparison of direct injection to SPE-MEKCELECTROPHORESIS, Issue 21 2008Rade Injac Abstract A novel and simple method has been developed for the determination of doxycycline (DOX) in biological fluids. The method is based on SPE, large-volume sample stacking (LVSS) and MEKC with UV-DAD detection. Six SPE cartridges have been used in investigation for sample clean up and pre-concentration (Supelco® LC-8, LC-18, LC-SCX, and LC-WCX, as well as StrataÔ-X and X-C). DOX was determined on a 56,cm (effective length 50,cm)×50,,m id fused-silica capillary. The BGE was 20,mM borate buffer, pH 9.3, containing 80,mM SDS and 7.5%,v/v of methanol (30,s×50,mbar), and the temperature and voltage were 25°C and 30,kV, respectively. The analytical wavelength was set at 210,nm. Under optimized conditions it is possible to determine DOX in human serum, urine, semen, tears and saliva with recovery of 97.5% (RSD 2.5%). The method was shown to be sensitive (LOD is 1,,g/L) and precise (intra-day RSD 0.2 and 2.4%; inter-days 0.4 and 3.5% for migration time and peak area, respectively). Results for developed SPE-LVSS-MEKC were compared with LVSS-MEKC method with direct sample injection. The new LVSS-MEKC method is presented as a useful technique for rapid determination without extraction procedure of DOX in human urine and serum, using 80,mM of SDS, 10%,v/v of methanol and 40,mM borate buffer (pH 9.3; 30,s×50,mbar; 25°C; 30,kV; 350,nm), but not for the other biological fluids, according to lower sensitivity of the method and because of the sample composition. [source] A simple and rapid CZE method for the analysis of mycophenolic acid and its phenol glucuronide metabolite in human serum,ELECTROPHORESIS, Issue 17 2008Kaname Ohyama Abstract A simple and rapid capillary electrophoretic method was developed for simultaneous determination of mycophenolic acid and its metabolite, phenol glucuronide, in human serum. The sample preparation in the proposed method required only the precipitation by acetonitrile. Separation was performed by capillary zone electrophoresis using 75,mM phosphate buffer (pH 7.5) as running buffer, an applied voltage of 10,kV, and UV detection at 217,nm. Each serum sample analysis was completed within 15,min. The optimized method demonstrated good performance concerning specificity, linearity (r>0.9955), accuracy (95.9,113%), precision (<6.4%) and enough sensitivity for therapeutic drug monitoring. This method was successfully applied to measurements of mycophenolic acid and mycophenolic acid glucuronide in renal transplant patient samples and was a useful alternative to high-performance liquid chromatography-based methods. [source] High-sensitive determination of human ,-thrombin by its 29-mer aptamer in affinity probe capillary electrophoresisELECTROPHORESIS, Issue 12 2008Yilin Li Abstract ACE technique provides an effective tool for the separation and identification of disease-related biomarkers in clinical analysis. In recent years, a couple of synthetic DNA or RNA oligonucleotides, known as aptamers, rival the specificity and affinity for targets to antibodies and are employed as one kind of powerful affinity probe in ACE. In this work, based on high affinity between antithrombin aptamer and thrombin (their dissociation constant is 0.5,nM), a carboxyfluorescein-labeled 29-nucleotide (nt) aptamer (F29-mer) was used and an aptamer-based affinity probe CE (aptamer-based APCE) method was successfully established for high-sensitive detection and quantitative analysis of thrombin. Experimental conditions including incubation temperature and time, buffer composition, and concentration of cations were investigated and optimized. Under the optimized condition, the linear range was from 0 to 400,nM and the LOD was 2,nM (74,ng/mL, S/N,=,3), i.e., 40,amol, both in running buffer and in 5% v/v human serum. This LOD is the lowest one than those achieved by the previous APCE methods but based on a 15-mer aptamer. This approach offers a promising method for the rapid, selective, and sensitive detection of thrombin in practical utility. Further binding experiments using one carboxyfluorescein-labeled aptamer and the other nonlabeled aptamer or vice versa were carried out to deduce the formation of ternary complex when these two aptamers coexisted in the free solution with thrombin. [source] Determination of ethyl glucuronide in human serum by hyphenation of capillary isotachophoresis and zone electrophoresisELECTROPHORESIS, Issue 8 2008Michaela Nováková Abstract The determination of ethyl glucuronide (EtG), a marker of recent alcohol consumption, in human serum by hyphenation of capillary ITP (CITP) and CZE is reported. For CITP step, 1×10,2,M hydrochloric acid adjusted with ,-aminocaproic acid (EACA) to pH,4.4 was used as the leading electrolyte, and 1×10,2,M nicotinic acid with EACA, pH,4.4, was used as the terminating electrolyte (TE). All electrolytes contained 0.2% hydroxypropylcellulose to suppress electroosmosis. In CITP, EtG was separated from fast serum macrocomponents chloride, phosphate, lactate, and acetate. Zones of microcomponents including EtG that migrated between acetate and nicotinate were forwarded to the second capillary filled with a BGE identical with the TE. Conductivity detection was used in the CITP step. Sensitive detection in the CZE step was performed using indirect spectrophotometric detection at 254,nm. The assay is based on a 1:5 dilution of serum with deionized water and has a concentration LOD for EtG in diluted sample of 9.8×10,9,M. The method was used for the determination of EtG in sera of volunteers consuming alcohol. [source] An automated, sheathless capillary electrophoresis-mass spectrometry platform for discovery of biomarkers in human serumELECTROPHORESIS, Issue 7-8 2005Alexander P. Sassi Abstract A capillary electrophoresis-mass spectrometry (CE-MS) method has been developed to perform routine, automated analysis of low-molecular-weight peptides in human serum. The method incorporates transient isotachophoresis for in-line preconcentration and a sheathless electrospray interface. To evaluate the performance of the method and demonstrate the utility of the approach, an experiment was designed in which peptides were added to sera from individuals at each of two different concentrations, artificially creating two groups of samples. The CE-MS data from the serum samples were divided into separate training and test sets. A pattern-recognition/feature-selection algorithm based on support vector machines was used to select the mass-to-charge (m/z) values from the training set data that distinguished the two groups of samples from each other. The added peptides were identified correctly as the distinguishing features, and pattern recognition based on these peptides was used to assign each sample in the independent test set to its respective group. A twofold difference in peptide concentration could be detected with statistical significance (p -value < 0.0001). The accuracy of the assignment was 95%, demonstrating the utility of this technique for the discovery of patterns of biomarkers in serum. [source] Determination of ribavirin in human serum and plasma by capillary electrophoresisELECTROPHORESIS, Issue 10-11 2004Michael C. Breadmore Abstract The electrophoretic separation of ribavirin and 5-methylcytidine (internal standard) by capillary electrophoresis was examined. Separation was achieved using reverse polarity in a 100 mM borate electrolyte, pH 9.1, with 5 mM spermine added to reduce the electroosmotic flow. Sample preparation based on acetonitrile protein precipitation was found to be unsuitable for ribavirin analysis in patient samples due to insufficient sensitivity and interferences. Solid-phase extraction employing phenyl boronic acid cartridges provided cleaner separations. Using this approach with 500 ,L sample and reconstitution of the dried extract into 100 ,L of 33% v/v 100 mM phosphate buffer, pH 6.4 / 67% v/v acetonitrile, the detection and quantitation limits were determined to be 0.05 and 0.10 ,g/mL, respectively, a sensitivity that is suitable for therapeutic drug monitoring of ribavirin in human plasma and serum samples. The method was validated and compared to a high-performance liquid chromatography (HPLC) method, showing excellent agreement between the two for a set of samples that stemmed from patients being treated with ribavirin and interferon-,-2b for a hepatitis C virus infection. [source] Capillary zone electrophoresis with a dynamic double coating for analysis of carbohydrate-deficient transferrin in human serum: Impact of resolution between disialo- and trisialotransferrin on reference limitsELECTROPHORESIS, Issue 24 2003Christian Lanz Abstract Capillary electrophoresis with a dynamic double coating formed by charged polymeric reagents represents a very effective tool for the separation of iron-saturated transferrin (Tf) isoforms and thus the determination of carbohydrate-deficient transferrin (CDT) in human serum. The resolution between di- and trisialo-Tf is dependent on the applied voltage and capillary temperature. With a 50 ,m inside diameter (ID) capillary of about 60 cm total length mounted into the P/ACE MDQ, 28 kV and 40°C, the resolution of the two Tf isoforms is shown to be between 1.0 and 1.4, whereas with reduced voltage and/or temperature, increased resolution at the expense of elongated run times is observed. Best data with complete resolution (Rs , 1.4) are obtained at 20 kV and 30°C. For the determination of CDT in serum, incomplete separation of di- and trisialo-Tf is demonstrated to have an impact on the reference limits. Analysis of the sera of 54 healthy individuals with no or moderate alcohol consumption and using valley-to-valley peak integration, the upper (lower) reference limits for CDT in relation to total Tf at the two power levels are 1.33 (0.52) and 1.57 (0.81)%, respectively, representing intervals that are significantly different (P < 0.001). Furthermore, the reference intervals are shown to be strongly dependent on the peak integration approach used. Valley-to-valley peak integration should only be employed for conditions with complete resolution between disialo- and trisialo-Tf. [source] The alpha-amino group of l -arginine mediates its antioxidant effectEUROPEAN JOURNAL OF CLINICAL INVESTIGATION, Issue 2 2001S. Wallner Antioxidant effects may constitute part of the possible antiatherogenic effects of the amino acid l -arginine. These antioxidant properties were further characterized in a model of lipoprotein oxidation. Oxidation of lipoproteins in unfractionated human serum was continuously monitored by a fluorescent probe. The antioxidant effects of l -arginine, N -,-acetyl-arginine and vitamin E in combination with l -arginine were measured after initiation of free radical generation with either copper or 2,2,-azobis(2-amidinopropane) hydrochloride (AAPH). The half-time of the fast propagation rate for copper-induced lipoprotein oxidation increased after incubation with l -arginine in a dose-dependent manner (P < 0·01). N -,-acetyl-arginine did not show such effects. Vitamin E and l -arginine show different effects on copper-induced oxidation, the former increasing only lag-time, the latter increasing only propagation rate, and do not have reciprocal effects. In contrast to copper-induced oxidation, l -arginine increased the lag-time of AAPH-induced lipoprotein oxidation (P < 0·01), with no effect on the propagation rate at physiological concentrations. Again, N -,-acetyl-arginine did not show any antioxidant effects. Our experiments provide further evidence that mechanisms other than serving as a substrate for the NO-synthase could be involved in the antiatherosclerotic effect of l -arginine. In addition, our experiments clearly show, that the antioxidant effect of l -arginine is due to a chemical moiety different from that serving as the substrate for NO biosynthesis. [source] In Vitro Formation of Nanocrystalline Carbonate Apatite , A Structural and Morphological Analogue of Atherosclerotic PlaquesEUROPEAN JOURNAL OF INORGANIC CHEMISTRY, Issue 26 2007Lars-Fride Olsson Abstract The in vitro formation of carbonate apatite in solutions with ion concentrations comparable to those in human serum was studied. The composition and morphology of the resulting apatite precipitate displayed a hierarchical assembly of elongated plate-shaped nanocrystals of carbonate apatite analogous to previously characterized bioapatites formed in vivo. The main conclusion is that so-called bioapatites may form in vitro and that precipitation inhibitors most likely are essential for the prevention of spontaneous calcification at normal human serum ion concentrations. (© Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2007) [source] Tyrosine sulfation and N-glycosylation of human heparin cofactor II from plasma and recombinant Chinese hamster ovary cells and their effects on heparin bindingFEBS JOURNAL, Issue 3 2002Christoph Böhme The structure of post-translational modifications of human heparin cofactor II isolated from human serum and from recombinant Chinese hamster ovary cells and their effects on heparin binding have been characterized. Oligosaccharide chains were found attached to all three potential N-glycosylation sites in both protein preparations. The carbohydrate structures of heparin cofactor II circulating in blood are complex-type diantennary and triantennary chains in a ratio of 6 : 1 with the galactose being > 90% sialylated with ,2,6 linked N-acetylneuraminic acid. About 50% of the triantennary structures contain one sLex motif. Proximal ,1,6 fucosylation of oligosacharides from Chinese hamster ovary cell-derived HCII was detected in >,90% of the diantennary and triantennary glycans, the latter being slightly less sialylated with exclusively ,2,3-linked N -acetylneuraminic acid units. Applying the ESI-MS/ MS-MS technique, we demonstrate that the tryptic peptides comprising tyrosine residues in positions 60 and 73 were almost completely sulfated irrespective of the protein's origin. Treatment of transfected Chinese hamster ovary cells with chlorate or tunicamycin resulted in the production of heparin cofactor II molecules that eluted with higher ionic strength from heparin,Sepharose, indicating that tyrosine sulfation and N-linked glycans may affect the inhibitor's interaction with glycosaminoglycans. [source] Toxicity to Candida albicans mediated by human serum and peripheral blood mononuclear cellsFEMS IMMUNOLOGY & MEDICAL MICROBIOLOGY, Issue 3 2006Joseph M. Bliss Abstract This study evaluates the conditions in which peripheral blood mononuclear cells mediate toxicity to Candida albicans opsonized with heat-inactivated human serum. Serum concentrations as low as 1% resulted in 50% inhibition of C. albicans metabolic activity after incubation with peripheral blood mononuclear cells at an effector to target ratio of 8. Measurable inhibition was also achieved at lower effector to target ratios and lower serum concentrations, and at least a portion of the metabolic inhibition reflected fungal cell death. Depletion of C. albicans -specific antibody decreased the toxic effect while opsonization with purified human IgG restored toxicity, and cell,cell contact between peripheral blood mononuclear cells and fungus was required. Depletion of or enrichment for monocytes from the peripheral blood mononuclear cells preparation diminished the toxic effect and the monocytic cell line, THP-1, was likewise incapable of toxicity. These studies provide evidence that antibody augments antifungal host defense and underscore the complex interrelationship between humoral and cellular immunity in these infections. [source] Serum lipids regulate dendritic cell CD1 expression and functionIMMUNOLOGY, Issue 3 2008David S. Leslie Summary Dendritic cells (DCs) are highly potent antigen-presenting cells (APCs) and play a vital role in stimulating naïve T cells. Treatment of human blood monocytes with the cytokines granulocyte,macrophage colony-stimulating factor (GM-CSF) and interleukin (IL)-4 stimulates them to develop into immature dendritic cells (iDCs) in vitro. DCs generated by this pathway have a high capacity to prime and activate resting T cells and prominently express CD1 antigen-presenting molecules on the cell surface. The presence of human serum during the differentiation of iDCs from monocytes inhibits the expression of CD1a, CD1b and CD1c, but not CD1d. Correspondingly, T cells that are restricted by CD1c showed poor responses to DCs that were generated in the presence of human serum, while the responses of CD1d-restricted T cells were enhanced. We chemically fractionated human serum to isolate the bioactive factors that modulate surface expression of CD1 proteins during monocyte to DC differentiation. The human serum components that affected CD1 expression partitioned with polar organic soluble fractions. Lysophosphatidic acid and cardiolipin were identified as lipids present in normal human serum that potently modulate CD1 expression. Control of CD1 expression was mediated at the level of gene transcription and correlated with activation of the peroxisome proliferator-activated receptor (PPAR) nuclear hormone receptors. These findings indicate that the ability of human DCs to present lipid antigens to T cells through expression of CD1 molecules is sensitively regulated by lysophosphatidic acid and cardiolipin in serum, which are ligands that can activate PPAR transcription factors. [source] The Rubino test for leprosy is a ,2 -glycoprotein 1-dependent antiphospholipid reactionIMMUNOLOGY, Issue 1 2000A. Panunto-Castelo Summary We describe the isolation and identification of three components required for the Rubino reaction (RR), which is the rapid sedimentation of formalinized sheep red-blood cells (SRBC) initiated by serum from leprosy patients with defective Mycobacterium leprae -specific cell immunity. The Rubino reaction factor (RRF) required for this phenomenon, previously identified as an immunoglobulin M (IgM), was purified from leprosy patient serum by adsorption to formalinized SRBC. Purified RRF IgM, when added to formalinized SRBC, did not produce a positive RR. However, when the contact was carried out in the presence of normal human serum (NHS), cells rapidly sedimented. The purified cofactor from NHS contained two components of 70 000 and 50 000 molecular weight (MW), as determined by sodium dodecyl sulphate,polyacrylamide gel electrophoresis (SDS,PAGE). The latter was recognized by the RRF IgM on immunoblot and its N-terminal sequence indicated that it was ,2 -glycoprotein 1 (,2 -GP1), an anionic phospholipid-binding protein. Methanol-treated formalinized SRBC did not support the RR. Thin-layer chromatography of an extract of membranes indicated that the SRBC ligand was a cell-surface phospholipid. Cardiolipin inhibited the RR. These data demonstrate that the RR involves a trimolecular interaction in which IgM, ,2 -GP1 and an SRBC phospholipid participate. By analogy with the antiphospholipid antibodies (anti-PL) that occur in autoimmune processes, serum samples from 29 systemic lupus erythematosus patients with high levels of anticardiolipin antibodies were submitted to the RR. A positive RR was obtained for 45% (13 of 29 patients). These results modify the paradigm of the absolute specificity of the RR for leprosy and demonstrate that RRF IgM is a ,2 -GP1-dependent anti-PL. [source] Receptor for the globular heads of C1q (gC1q-R, p33, hyaluronan-binding protein) is preferentially expressed by adenocarcinoma cellsINTERNATIONAL JOURNAL OF CANCER, Issue 5 2004Daniel B. Rubinstein Abstract Combinatorial Ig libraries with phage display allow in vitro generation of human Ig fragments without the need to maintain hybridomas in ongoing cell culture or to select circulating Ig from human serum. Identifying tumor-associated antigens on the surface of intact tumor cells, as opposed to purified proteins, presents a challenge due to the difficulty of preserving complex 3-D epitopic sites on the cell surface, the variable expression of antigens on different malignant cell types and the stereotactic interference of closely associated proteins on the intact membrane surface limiting accessibility to antigenic sites. A combinatorial Ig library of 1010 clones was generated from the cDNA of PBMCs derived from patients with breast adenocarcinoma. Following subtractive panning, the library was enriched for Ig (Fab fragment) binding to intact adenocarcinoma cells and the resultant Fabs were screened against a cDNA expression library, itself generated from breast cancer cells. Using this approach, we isolated clones from the cDNA library expressing gC1q-R, a glycoprotein comprising the major structure of C1, the first component of the complement system. gC1q-R is a 33 kDa glycoprotein expressed not only on the cell surface but also intracellularly, with motifs that target it to mitochondria and complete homology with HABP and human HeLa cell protein p32, which is copurified with pre-mRNA SF2. Sequencing of the gene encoding tumor-associated gC1q-R did not reveal any consistent tumor-specific mutations. However, histochemical staining with anti-gC1q-R MAb demonstrated marked differential expression of gC1q-R in thyroid, colon, pancreatic, gastric, esophageal and lung adenocarcinomas compared to their nonmalignant histologic counterparts. In contrast, differential expression was not seen in endometrial, renal and prostate carcinomas. Despite high expression in breast carcinoma, gC1q-R was also expressed in nonmalignant breast tissue. Although the precise relation of gC1q-R to carcinogenesis remains unclear, our finding of tumor overexpression and the known multivalent binding of gC1q-R to not only C1q itself but also a variety of circulating plasma proteins as well as its involvement in cell-to-cell interactions suggest that gC1q-R may have a role in tumor metastases and potentially serve in molecule-specific targeting of malignant cells. © 2004 Wiley-Liss, Inc. [source] The extraordinary ligand binding properties of human serum albuminIUBMB LIFE, Issue 12 2005Mauro Fasano Abstract Human serum albumin (HSA), the most prominent protein in plasma, binds different classes of ligands at multiple sites. HSA provides a depot for many compounds, affects pharmacokinetics of many drugs, holds some ligands in a strained orientation providing their metabolic modification, renders potential toxins harmless transporting them to disposal sites, accounts for most of the antioxidant capacity of human serum, and acts as a NO-carrier. The globular domain structural organization of monomeric HSA is at the root of its allosteric properties which are reminiscent of those of multimeric proteins. Here, structural, functional, biotechnological, and biomedical aspects of ligand binding to HSA are summarized. [source] Induction of complement sensitivity in Escherichia coli by citric acid and low pHJOURNAL OF APPLIED MICROBIOLOGY, Issue 5 2001C. Ocaña-Morgner Aims:,The lytic functions of the complement system play an important role in the control of Gram-negative infections. Complement-resistant Escherichia coli LP1395 (O18) grown under normal conditions can survive the bactericidal action of complement present in human serum. Towards elucidating the mechanisms of complement resistance, the resistance of E. coli LP1395 grown under conditions of low pH and in the presence of citric acid was tested. Methods and Results:,E. coli LP1395 becomes sensitive to complement after growth in the presence of citric acid at pH 5. Complement resistance could be restored when the cells were transferred to pH 7 media. However, this recovery was greatly impaired when the cells were transferred to pH 7 media with chloramphenicol. This implies that protein synthesis may be involved in complement resistance. The cells exposed to citric acid at pH 5 showed no indication of a generalized outer membrane (OM) permeability when compared with those grown under normal conditions in terms of sensitivity to lysozyme, uptake of lipophilic dye, or sensitivity to a number of antibiotics. Conclusions:,Complement-resistant LP1395 may acquire a sensitivity to complement due not to a generalized disruption of the OM barrier, but possibly to the alteration of the activity of one or more normal complement resistance factors. Significance and Impact of the Study:,The elucidation of the echanisms of complement resistance of Gram-negative pathogens would bring important information about bacterial infections. Complement resistance factors could also be potential targets in antimicrobial therapies. [source] Dichlorvos, chlorpyrifos oxon and Aldicarb adducts of butyrylcholinesterase, detected by mass spectrometry in human plasma following deliberate overdoseJOURNAL OF APPLIED TOXICOLOGY, Issue 6 2010Bin Li Abstract The goal of this study was to develop a method to detect pesticide adducts in tryptic digests of butyrylcholinesterase in human plasma from patients poisoned by pesticides. Adducts to butyrylcholinesterase in human serum may serve as biomarkers of pesticide exposure because organophosphorus and carbamate pesticides make a covalent bond with the active site serine of butyrylcholinesterase. Serum samples from five attempted suicides (with dichlorvos, Aldicarb, Baygon and an unknown pesticide) and from one patient who accidentally inhaled dichlorvos were analyzed. Butyrylcholinesterase was purified from 2 ml serum by ion exchange chromatography at pH 4, followed by procainamide affinity chromatography at pH 7. The purified butyrylcholinesterase was denatured, digested with trypsin and the modified peptide isolated by HPLC. The purified peptide was analyzed by multiple reaction monitoring in a QTRAP 4000 mass spectrometer. This method successfully identified the pesticide-adducted butyrylcholinesterase peptide in four patients whose butyrylcholinesterase was inhibited 60,84%, but not in two patients whose inhibition levels were 8 and 22%. It is expected that low inhibition levels will require analysis of larger serum plasma volumes. In conclusion, a mass spectrometry method for identification of exposure to live toxic pesticides has been developed, based on identification of pesticide adducts on the active site serine of human butyrylcholinesterase. Copyright © 2010 John Wiley & Sons, Ltd. [source] Concentration of aluminium in breast cyst fluids collected from women affected by gross cystic breast diseaseJOURNAL OF APPLIED TOXICOLOGY, Issue 1 2009Ferdinando Mannello Abstract Gross cystic breast disease (GCBD) is the most common benign breast disorder, but the molecular basis of cyst formation remains to be identified. If the use of aluminium-based antiperspirant salts is involved in the etiology of gross breast cyst formation, it might be expected that aluminium would be at elevated levels in human breast cyst fluid (BCF). Aluminium was measured by ICP-MS in 48 samples of BCF, 30 samples of human blood serum and 45 samples of human breast milk at different stages of lactation (colostrum, intermediate, mature). The median level of aluminium in apocrine type I BCF (n = 27, 150 µg l,1) was significantly higher than in transudative type II BCF (n = 21, 32 µg l,1; P <0.0001). By comparison, aluminium measurements gave a median concentration of 6 µg l,1 in human serum and 25 µg l,1 in human breast milk, with no difference between colostrum, intermediate and mature milk. Levels of aluminium were significantly higher in both types of BCF than in human serum (P <0.0001). However when compared with human breast milk, aluminium levels were only significantly higher in apocrine type I BCF (P <0.0001) and not in transudative type II BCF (P = 0.152). It remains to be identified why such high levels of aluminium were found in the apocrine type I BCF and from where the aluminium originated. However, if aluminium-based antiperspirants are found to be the source and to play any causal role in development of breast cysts, then it might become possible to prevent this common breast disorder. Copyright © 2008 John Wiley & Sons, Ltd. [source] Human islet-derived precursor cells can cycle between epithelial clusters and mesenchymal phenotypesJOURNAL OF CELLULAR AND MOLECULAR MEDICINE, Issue 8b 2009Behrous Davani Abstract We showed previously that undifferentiated, proliferating human islet-derived precursor cells (hIPCs) are a type of mesenchymal stem/stromal cell (MSC) that can be induced by serum deprivation to form clusters and ultimately differentiate in vitro to endocrine cells. We also demonstrated that partially differentiated hIPC clusters, when implanted under the kidney capsules of mice, continued to differentiate in vivo into hormone-producing cells. However, we noted that not all hIPC preparations yielded insulin-secreting cells in vivo and that in some animals no hormone-expressing cells were found. This suggested that the implanted cells were not always irreversibly committed to further differentiation and may even de-differentiate to a mesenchymal phenotype. In this study, we show that human cells with a mesenchymal phenotype are indeed found in the grafts of mice implanted with hIPCs in epithelial cell clusters (ECCs), which are obtained after 4-day in vitro culture of hIPCs in serum-free medium (SFM); mesenchymal cells were predominant in some grafts. We could mimic the transition of ECCs to de-differentiated mesenchymal cells in vitro by exposure to foetal bovine serum (FBS) or mouse serums, and to a significantly lesser extent to human serum. In a complementary series of experiments, we show that mouse serum and FBS are more effective stimulants of mesenchymal hIPC migration than is human serum. We found that proliferation was not needed for the transition from ECCs to de-differentiated cells because mitomycin-treated hIPCs that could not proliferate underwent a similar transition. Lastly, we show that cells exhibiting a mesenchymal phenotype can be found in grafts of adult human islets in mice. We conclude that epithelial-to-mesenchymal transition (EMT) of cells in hIPC ECCs can occur following implantation in mice. This potential for EMT of human islets or differentiated precursor cells must be considered in strategies for cell replacement therapy for diabetes. [source] Chylomicron accelerates C3 tick-over by regulating the role of Factor H, leading to overproduction of acylation stimulating proteinJOURNAL OF CLINICAL LABORATORY ANALYSIS, Issue 1 2007Takayuki Fujita Abstract Acylation stimulating protein (ASP) is a fragment of the third component of complement (C3) that is generated in the presence of chylomicron, and plays a role in the synthesis of triacylglycerol by transporting free fatty acids into adipocytes. However, the precise mechanism of ASP generation, especially the role of chylomicron in ASP generation, is unknown. We examined the mechanism through which chylomicron induces ASP generation. Ultracentrifugationally separated chylomicron was incubated with normal human serum (NHS) under various conditions, and the amounts of complement activation products and ASP in the incubation mixture were determined by enzyme-linked immunosorbent assay (ELISA). Upon incubation of NHS with various amounts of chylomicron for 120,min, ASP was generated in a dose-dependent manner. The time course of the production of ASP was similar to the time course of the C3 tick-over phenomenon that occurred by depletion of factor H from the serum. The complement activation induced by chylomicron was different from the usual complement activation that occurs under the regulation of factor H and factor I with respect to the time course and the amount of ASP produced. Our results indicate that chylomicron accelerates C3 tick-over by regulating the role of factor H, leading to the overproduction of ASP. J. Clin. Lab. Anal. 21:14,23, 2007. © 2007 Wiley-Liss, Inc. [source] Simple and simultaneous determination of sulphapyridine and acetylsulphapyridine in human serum by column-switching high-performance liquid chromatographyJOURNAL OF CLINICAL PHARMACY & THERAPEUTICS, Issue 6 2002D. Teshima PhD Summary Objective:, A high-performance liquid chromatography (HPLC) with an automated on-line column-switching system was used for the simultaneous determination of sulphapyridine and acetylsulphapyridine, two major active metabolites related to the adverse effects of sulphasalazine, in human serum. Methods:, Serum samples were directly injected into the HPLC, with the valve automatically switched on to remove serum proteins and other hydrophilic components remaining in the pre-column after elution of sulphapyridine and acetylsulphapyridine to the analytical column. Results:, Serum proteins did not interfere with the analysis of either compound. The recoveries of SLP and Ac-SLP from drug-free human serum were 93·03,99·18% and CV were 2·88,4·34%. The within-run reproducibility of assays was excellent with relative standard deviations (RSD) of 1·01,3·90% (SLP) and 0·77,5·56% (Ac-SLP). The limit of quantification of sulphapyridine and acetylsulphapyridine was 3·13 ,g/mL and 0·50 ,g/mL, respectively. The serum concentrations in a patient with ulcerative colitis, who took 1·0 g sulphasalazine twice daily, were 31·20 ,g/mL for sulphapyridine and 14·64 ,g/mL for acetylsulphapyridine at 7 h after ingestion. Conclusion:, The present simple and reproducible assay was useful for the monitoring of serum sulphapyridine and acetylsulphapyridine. [source] | ||||||||||||||||