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Sample records for labelled human serum

  1. 99M-technetium labeled macroaggregated human serum albumin pharmaceutical

    DOEpatents

    Winchell, Harry S.; Barak, Morton; Van Fleet, III, Parmer

    1977-05-17

    A reagent comprising macroaggregated human serum albumin having dispersed therein particles of stannous tin and a method for instantly making a labeled pharmaceutical therefrom, are disclosed. The labeled pharmaceutical is utilized in organ imaging.

  2. Characterizing Active Pharmaceutical Ingredient Binding to Human Serum Albumin by Spin-Labeling and EPR Spectroscopy.

    PubMed

    Hauenschild, Till; Reichenwallner, Jörg; Enkelmann, Volker; Hinderberger, Dariush

    2016-08-26

    Drug binding to human serum albumin (HSA) has been characterized by a spin-labeling and continuous-wave (CW) EPR spectroscopic approach. Specifically, the contribution of functional groups (FGs) in a compound on its albumin-binding capabilities is quantitatively described. Molecules from different drug classes are labeled with EPR-active nitroxide radicals (spin-labeled pharmaceuticals (SLPs)) and in a screening approach CW-EPR spectroscopy is used to investigate HSA binding under physiological conditions and at varying ratios of SLP to protein. Spectral simulations of the CW-EPR spectra allow extraction of association constants (KA ) and the maximum number (n) of binding sites per protein. By comparison of data from 23 SLPs, the mechanisms of drug-protein association and the impact of chemical modifications at individual positions on drug uptake can be rationalized. Furthermore, new drug modifications with predictable protein binding tendency may be envisaged. PMID:27460503

  3. Functionalized gold nanoclusters as fluorescent labels for immunoassays: Application to human serum immunoglobulin E determination.

    PubMed

    Alonso, María Cruz; Trapiella-Alfonso, Laura; Fernández, José M Costa; Pereiro, Rosario; Sanz-Medel, Alfredo

    2016-03-15

    A quantitative immunoassay for the determination of immunoglobulin E (IgE) in human serum using gold nanoclusters (AuNCs) as fluorescent label was developed. Water soluble AuNCs were synthesized using lipoic acid and then thoroughly characterized. The obtained AuNCs have a particle size of 2.7 ± 0.1 nm and maximum fluorescence emission at 710 nm. The synthesized AuNCs showed very good stability of the fluorescent signal with light exposure and at neutral and slightly basic media. A covalent bioconjugation of these AuNCs with the desired antibody was carried out by the carbodiimide reaction. After due optimization of such bioconjugation reaction, a molar ratio 1:3 (antibody:AuNCs) was selected. The bioconjugate maintained an intense luminescence emission, slightly red-shifted as compared to the free AuNCs. Two typical immunoassay configurations, competitive and sandwich, were assayed and their performance for IgE determination critically compared. After the different immunoassay steps were accomplished, the fluorescence emission of the bioconjugate was measured. While the sandwich format provided a detection limit (DL) of 10 ng/mL and a linear range between 25 and 565 ng/mL of IgE, the competitive format revealed a DL of 0.2 ng/mL with a linear range between 0.3 and 7.1 ng/mL The applicability of the more sensitive competitive fluorescent immunoassay was assessed by successful analysis of the IgE in human serum and comparison of results with those from a commercial kit. The main advantages of the proposed AuNCs-based fluorimetric method include a low DL and a simple immunoassay protocol involving few reagents. PMID:26547433

  4. TDPAC studies of181Hf-labelled transferrin: Comparison between human and rat serum transferrin

    NASA Astrophysics Data System (ADS)

    Appel, H.; Duffield, J.; Taylor, D. M.; Then, G. M.; Thies, W.-G.

    1987-04-01

    A fast BaF2 TDPAC setup was used to study the binding of181Hf to serum transferrin. Two well-defined binding configurations were observed, which are characterized by high EFGs and large asymetry parameter values. The distribution between these configurations depends essentially on the pH of the serum. Small but significant differences between human and rat serum transferrin can be deduced from the electric quadrupole interaction (QI) parameters.

  5. Analysis of liposoluble carboxylic acids metabolome in human serum by stable isotope labeling coupled with liquid chromatography-mass spectrometry.

    PubMed

    Zhu, Quan-Fei; Zhang, Zheng; Liu, Ping; Zheng, Shu-Jian; Peng, Ke; Deng, Qian-Yun; Zheng, Fang; Yuan, Bi-Feng; Feng, Yu-Qi

    2016-08-19

    Fatty acids (FAs) are groups of liposoluble carboxylic acids (LCAs) and play important roles in various physiological processes. Abnormal contents or changes of FAs are associated with a series of diseases. Here we developed a strategy with stable isotope labeling combined with liquid chromatography-tandem mass spectrometry (IL-LC-MS) analysis for comprehensive profiling and relative quantitation of LCAs in human serum. In this strategy, a pair of isotope labeling reagents (2-dimethylaminoethylamine (DMED)) and d4-2-dimethylaminoethylamine (d4-DMED) were employed to selectively label carboxyl groups of LCAs. The DMED and d4-DMED labeled products can lose four characteristic neutral fragments of 45 and 49Da or 63 and 67Da in collision-induced dissociation. Therefore, quadruple neutral loss scan (QNLS) mode was established and used for non-targeted profiling of LCAs. The peak pairs of DMED and d4-DMED labeling with the same retention time, intensity and characteristic mass differences were extracted from the two NLS spectra respectively, and assigned as potential LCA candidates. Using this strategy, 241 LCA candidates were discovered in the human serum; 156 carboxylic acid compounds could be determined by searching HMDB and METLIN databases (FAs are over 90%) and 21 of these LCAs were successfully identified by standards. Subsequently, a modified pseudo-targeted method with multiple reaction monitoring (MRM) detection mode was developed and used for relative quantification of LCAs in human serum from type 2 diabetes mellitus (T2DM) patients and healthy controls. As a result, 81 LCAs were found to have significant difference between T2DM patients and healthy controls. Taken together, the isotope labeling combined with tandem mass spectrometry analysis demonstrated to be a powerful strategy for identification and quantification of LCA compounds in serum samples. PMID:27432792

  6. Dansyl labeling to modulate the relative affinity of bile acids for the binding sites of human serum albumin.

    PubMed

    Rohacova, Jana; Sastre, German; Marin, M Luisa; Miranda, Miguel A

    2011-09-01

    Binding of natural bile acids to human serum albumin (HSA) is an important step in enterohepatic circulation and provides a measure of liver function. In this article, we report on the use of four dansyl (Dns) derivatives of cholic acid (ChA) to demonstrate a regiodifferentiation in their relative affinity for the two binding sites of HSA. Using both steady-state and time-resolved fluorescence, formation of Dns-ChA@HSA complexes was confirmed; the corresponding binding constants were determined, and their distribution between bulk solution and HSA microenvironment was estimated. By means of energy transfer from Trp to the Dns moiety, donor-acceptor distances were estimated (21-25 Å) and found to be compatible with both site 1 and site 2 occupancies. Nevertheless, titration using warfarin and ibuprofen as specific displacement probes clearly indicated that 3α- and 3β-Dns-ChA bind to HSA at site 2, whereas their C-7 regioisomers bind to HSA at site 1. Furthermore, the C-3-labeled compounds are displaced by lithocholic acid, whereas they are insensitive to ChA, confirming the assumption that the former binds to HSA at site 2. Thus, Dns labeling provides a useful tool to modulate the relative affinity of ChA to the major binding sites of HSA and, in combination with other fluorescent ChA analogs, to mimic the binding behavior of natural bile acids. PMID:21797258

  7. Quantification of human growth hormone in serum with a labeled protein as an internal standard: essential considerations.

    PubMed

    Pritchard, Caroline; Groves, Kate J; Biesenbruch, Sabine; O'Connor, Gavin; Ashcroft, Alison E; Arsene, Cristian; Schulze, Dirk; Quaglia, Milena

    2014-07-01

    To manage and inform diagnostic or therapeutic decisions, measurement results which are accurate, specific, and comparable between laboratories are required. Two challenges associated with this are the definition of the measurand and the commutability of the reference standard used. Once the measurand is defined, the next step in improving standardization is developing traceable quantification methods for proteins in biological fluids. A novel reference method for the quantification of recombinant human growth hormone (rhGH) in serum has been developed using multistep sample cleanup at the protein level, tryptic digestion, and isotope dilution mass spectrometry (IDMS). Critical considerations for using isotopically labeled rhGH as the internal standard are described. A bulk serum sample was prepared at the clinically relevant level of 10 ng/g and quantified using the method described to give results traceable to the International System of Units (SI) with a total measurement uncertainty of <20%. Results compared favorably with an orthogonal traceable method using total tryptic digestion, peptide separation, and isotope dilution mass spectrometry. PMID:24856175

  8. Determination of reduced homocysteine in human serum by elemental labelling and liquid chromatography with ICP-MS and ESI-MS detection.

    PubMed

    Espina, Juan Gómez; Montes-Bayón, Maria; Blanco-González, Elisa; Sanz-Medel, Alfredo

    2015-10-01

    Analytical methods allowing sensitive determination of reduced homocysteine (rHcy), one of the so-called biothiols, in human serum is a topic of growing interest due to its close relation to several human disorders, mainly cardiovascular diseases. Although most widely used analytical strategies to determine total Hcy involve derivatization by means of fluorescent labels, this work proposes the use of ebselen, a Se-containing labelling agent to derivatize the reactive sulfhydryl group of the Hcy molecule in its "free" reduced form, which is more likely to play different roles in disease pathogenesis. Optimization of the derivatization and separation conditions by high-performance liquid chromatography (HPLC) to isolate the excess of derivatizing reagent is carried out here using UV/VIS detection. Further, the study of the Se labelling reaction by electrospray ionization tandem mass spectrometry (ESI-MS/MS) provides a stoichiometry of the derivative of 1:1. The main advantage of using ebselen as a labelling agent is the presence of the Se atom in the molecule that allows the use of inductively coupled plasma mass spectrometry (ICP-MS) as a sensitive and selective Se detector. The coupling of HPLC with ICP-MS provided excellent features for the determination of Se-derivatized rHcy (detection limit of 9.6 nM) in real samples. Quantification was accomplished by using post-column isotope dilution (ID) of Se in serum samples, after precipitation of the main serum proteins. Quantitative results for "free" rHcy turned out to be around 0.18-0.22 μM in serum samples from healthy individuals that could be directly analyzed without sample preconcentration. PMID:26362154

  9. TDPAC studies of the metal-binding sites in serum transferrin: comparison between 181Hf-labeled human- and rat-serum transferrin.

    PubMed

    Appel, H; Duffield, J; Taylor, D M; Then, G M; Thies, W G

    1987-12-01

    The binding of hafnium to human serum transferrin was studied using the time differential perturbed angular correlation (TDPAC-) technique. The samples were prepared in vitro by adding 181Hf-NTA solution to human serum. Two specific electric quadrupole interactions were observed, which correspond to two well-defined binding configurations. Their relative intensities depend on the pH, salt- and hafnium-concentrations, and on the incubation time. The present data may be compared with the results of a previous rat serum study, where the hafnium binding to transferrin behaved rather similarly. Small but significant differences, however, can be deduced from the TDPAC-parameters for these human and rat transferrin species. For either binding configuration, the electric field gradient (EFG) is slightly higher in the case of rat transferrin. The most characteristic difference, however, concerns the asymmetry parameter eta 2 of the second binding configuration, which is about 10% smaller for rat serum transferrin. The TDPAC-technique might be used as a sensitive and reliable analytical method to study the metal-binding sites of different transferrin species. PMID:3437277

  10. TDPAC studies of the metal-binding sites in serum transferrin: comparison between /sup 181/Hf-labeled human- and rat-serum transferrin

    SciTech Connect

    Appel, H.; Duffield, J.; Taylor, D.M.; Then, G.M.; Thies, W.G.

    1987-12-01

    The binding of hafnium to human serum transferrin was studied using the time differential perturbed angular correlation (TDPAC-) technique. The samples were prepared in vitro by adding /sup 181/Hf-NTA solution to human serum. Two specific electric quadrupole interactions were observed, which correspond to two well-defined binding configurations. Their relative intensities depend on the pH, salt- and hafnium-concentrations, and on the incubation time. The present data may be compared with the results of a previous rat serum study, where the hafnium binding to transferrin behaved rather similarly. Small but significant differences, however, can be deduced from the TDPAC-parameters for these human and rat transferrin species. For either binding configuration, the electric field gradient (EFG) is slightly higher in the case of rat transferrin. The most characteristic difference, however, concerns the asymmetry parameter eta 2 of the second binding configuration, which is about 10% smaller for rat serum transferrin. The TDPAC-technique might be used as a sensitive and reliable analytical method to study the metal-binding sites of different transferrin species.

  11. Label-free electrochemical impedance biosensor to detect human interleukin-8 in serum with sub-pg/ml sensitivity.

    PubMed

    Sharma, R; Deacon, S E; Nowak, D; George, S E; Szymonik, M P; Tang, A A S; Tomlinson, D C; Davies, A G; McPherson, M J; Wälti, C

    2016-06-15

    Biosensors with high sensitivity and short time-to-result that are capable of detecting biomarkers in body fluids such as serum are an important prerequisite for early diagnostics in modern healthcare provision. Here, we report the development of an electrochemical impedance-based sensor for the detection in serum of human interleukin-8 (IL-8), a pro-angiogenic chemokine implicated in a wide range of inflammatory diseases. The sensor employs a small and robust synthetic non-antibody capture protein based on a cystatin scaffold that displays high affinity for human IL-8 with a KD of 35 ± 10 nM and excellent ligand specificity. The change in the phase of the electrochemical impedance from the serum baseline, ∆θ(ƒ), measured at 0.1 Hz, was used as the measure for quantifying IL-8 concentration in the fluid. Optimal sensor signal was observed after 15 min incubation, and the sensor exhibited a linear response versus logarithm of IL-8 concentration from 900 fg/ml to 900 ng/ml. A detection limit of around 90 fg/ml, which is significantly lower than the basal clinical levels of 5-10 pg/ml, was observed. Our results are significant for the development of point-of-care and early diagnostics where high sensitivity and short time-to-results are essential. PMID:26897263

  12. Label-free electrochemical impedance biosensor to detect human interleukin-8 in serum with sub-pg/ml sensitivity

    PubMed Central

    Sharma, R.; Deacon, S.E.; Nowak, D.; George, S.E.; Szymonik, M.P.; Tang, A.A.S.; Tomlinson, D.C.; Davies, A.G.; McPherson, M.J.; Wälti, C.

    2016-01-01

    Biosensors with high sensitivity and short time-to-result that are capable of detecting biomarkers in body fluids such as serum are an important prerequisite for early diagnostics in modern healthcare provision. Here, we report the development of an electrochemical impedance-based sensor for the detection in serum of human interleukin-8 (IL-8), a pro-angiogenic chemokine implicated in a wide range of inflammatory diseases. The sensor employs a small and robust synthetic non-antibody capture protein based on a cystatin scaffold that displays high affinity for human IL-8 with a KD of 35±10 nM and excellent ligand specificity. The change in the phase of the electrochemical impedance from the serum baseline, ∆θ(ƒ), measured at 0.1 Hz, was used as the measure for quantifying IL-8 concentration in the fluid. Optimal sensor signal was observed after 15 min incubation, and the sensor exhibited a linear response versus logarithm of IL-8 concentration from 900 fg/ml to 900 ng/ml. A detection limit of around 90 fg/ml, which is significantly lower than the basal clinical levels of 5–10 pg/ml, was observed. Our results are significant for the development of point-of-care and early diagnostics where high sensitivity and short time-to-results are essential. PMID:26897263

  13. Preparation and biodistribution of 188Re-labeled folate conjugated human serum albumin magnetic cisplatin nanoparticles (188Re-folate-CDDP/HSA MNPs) in vivo

    PubMed Central

    Tang, Qiu-Sha; Chen, Dao-Zhen; Xue, Wen-Qun; Xiang, Jing-Ying; Gong, Yong-Chi; Zhang, Li; Guo, Cai-Qin

    2011-01-01

    Background The purpose of this study was to develop intraperitoneal hyperthermic therapy based on magnetic fluid hyperthermia, nanoparticle-wrapped cisplatin chemotherapy, and magnetic particles of albumin. In addition, to combine the multiple-killing effects of hyperthermal targeting therapy, chemotherapy, and radiotherapy, the albumin-nanoparticle surfaces were linked with radionuclide 188Re-labeled folic acid ligand (188Re-folate-CDDP/HSA). Methods Human serum albumin was labeled with 188Re using the pre-tin method. Reaction time and optimal conditions of labeling were investigated. The particles were intravenously injected into mice, which were sacrificed at different time points. Radioactivity per gram of tissue of percent injected dose (% ID/g) was measured in vital organs. The biodistribution of 188Re-folate-CDDP/HAS magnetic nanoparticles was assessed. Results Optimal conditions for 188Re-labeled folate-conjugated albumin combined with cisplatin magnetic nanoparticles were: 0.1 mL of sodium gluconate solution (0.3 mol/L), 0.1 mL of concentrated hydrochloric acid with dissolved stannous chloride (10 mg/mL), 0.04 mL of acetic acid buffer solution (pH 5, 0.2 mol/L), 30 mg of folate-conjugated albumin combined with cisplatin magnetic nanoparticles, and 188ReO4 eluent (0.1 mL). The rate of 188Re-folate-CDDP-HSA magnetic nanoparticle formation exceeded 90%, and radiochemical purity exceeded 95%. The overall labeling rate was 83% in calf serum at 37°C. The major uptake tissues were the liver, kidney, intestine, and tumor after the 188Re-folate-CDDP/HSA magnetic nanoparticles were injected into nude mice. Uptake of 188Re-folate-CDDP/HSA magnetic nanoparticles increased gradually after injection, peaked at 8 hours with a value of 8.83 ± 1.71, and slowly decreased over 24 hours in vivo. Conclusion These results indicate that 188Re-folate-CDDP/HSA magnetic nanoparticles can be used in radionuclide-targeted cancer therapy. Surface-modified albumin nanoparticles with

  14. Catabolism of (64)Cu and Cy5.5-labeled human serum albumin in a tumor xenograft model.

    PubMed

    Kang, Choong Mo; Kim, Hyunjung; Koo, Hyun-Jung; Park, Jin Won; An, Gwang Il; Choi, Joon Young; Lee, Kyung-Han; Kim, Byung-Tae; Choe, Yearn Seong

    2016-07-01

    Human serum albumin (HSA), the most abundant protein in blood plasma, has been used as a drug carrier for the last few decades. Residualizingly radiolabeled serum albumin has been reported to be avidly taken up by tumors of sarcoma-bearing mice and to most likely undergo lysosomal degradation. In this study, we prepared (64)Cu-1,4,7,10-tetraazacyclododecane-N,N',N″,N'″-tetraacetic acid (DOTA) and Cy5.5-conjugated HSA (dual probe), and evaluated its tumor uptake and catabolism. Two dual probes were prepared using different DOTA conjugation sites of HSA (one via Lys residues and the other via the Cys residue). (64)Cu-DOTA-Lys-HSA-Cy5.5 (dual probe-Lys) exhibited higher uptake by RR1022 sarcoma cells in vitro than (64)Cu-DOTA-Cys-HSA-Cy5.5 (dual probe-Cys). In RR1022 tumor-bearing mice, the two dual probes showed a similar level of tumor uptake, but uptake of dual probe-Lys was reduced in the liver and spleen compared to dual probe-Cys, probably because of the presence of a higher number of DOTA molecules in the former. At 24 and 48 h after injection, dual probe-Lys was intact or partially degraded in blood, liver, kidney, and tumor samples, but (64)Cu-DOTA-Lys was observed in the urine using radioactivity detection. Similarly, Cy5.5-Lys was observed in the urine using fluorescence detection. These results indicate that dual probe-Lys may be useful for predicting the catabolic fate of drug-HSA conjugates. PMID:27098932

  15. Carbon dots as a fluorescent probe for label-free detection of physiological potassium level in human serum and red blood cells.

    PubMed

    Zhang, Lingyang; Chen, Shenna; Zhao, Qian; Huang, Haowen

    2015-06-23

    A unique photoluminescence carbon dots (CDs) with larger size were prepared by microwave-assisted method. Complex functional groups on the surface of the CDs facilitate the nanoparticles to form affinity with some metal ions. Taking advantage of the effective fluorescence quenching effect of K(+), a highly sensitive CD-based fluorescence analytical system for label-free detection of K(+) with limit of detection (LOD) 1.0×10(-12) M was established. The concentrations of potassium ion in biological samples such as human serum are usually found at millimolar levels or even higher. The proposed method begins with a substantial dilution of the sample to place the K(+) concentration in the dynamic range for quantification, which covers 3 orders of magnitude. This offers some advantages: the detection of K(+) only needs very small quantities of biological samples, and the dilution of samples such as serum may effectively eliminate the potential interferences that often originate from the background matrix. The determined potassium levels were satisfactory and closely comparable with the results given by the hospital, indicating that this fluorescent probe is applicable to detection of physiological potassium level with high accuracy. Compared with other relative biosensors requiring modified design, bio-molecular modification or/and sophisticated instruments, this CD-based sensor is very simple, cost-effective and easy detection, suggesting great potential applications for successively monitoring physiological potassium level and the change in biological system. PMID:26092345

  16. The Human Serum Metabolome

    PubMed Central

    Psychogios, Nikolaos; Hau, David D.; Peng, Jun; Guo, An Chi; Mandal, Rupasri; Bouatra, Souhaila; Sinelnikov, Igor; Krishnamurthy, Ramanarayan; Eisner, Roman; Gautam, Bijaya; Young, Nelson; Xia, Jianguo; Knox, Craig; Dong, Edison; Huang, Paul; Hollander, Zsuzsanna; Pedersen, Theresa L.; Smith, Steven R.; Bamforth, Fiona; Greiner, Russ; McManus, Bruce; Newman, John W.; Goodfriend, Theodore; Wishart, David S.

    2011-01-01

    Continuing improvements in analytical technology along with an increased interest in performing comprehensive, quantitative metabolic profiling, is leading to increased interest pressures within the metabolomics community to develop centralized metabolite reference resources for certain clinically important biofluids, such as cerebrospinal fluid, urine and blood. As part of an ongoing effort to systematically characterize the human metabolome through the Human Metabolome Project, we have undertaken the task of characterizing the human serum metabolome. In doing so, we have combined targeted and non-targeted NMR, GC-MS and LC-MS methods with computer-aided literature mining to identify and quantify a comprehensive, if not absolutely complete, set of metabolites commonly detected and quantified (with today's technology) in the human serum metabolome. Our use of multiple metabolomics platforms and technologies allowed us to substantially enhance the level of metabolome coverage while critically assessing the relative strengths and weaknesses of these platforms or technologies. Tables containing the complete set of 4229 confirmed and highly probable human serum compounds, their concentrations, related literature references and links to their known disease associations are freely available at http://www.serummetabolome.ca. PMID:21359215

  17. Photoaffinity labeling of human serum vitamin D binding protein and chemical cleavage of the labeled protein: Identification of an 11. 5-kDa peptide containing the putative 25-hydroxyvitamin D sub 3 binding site

    SciTech Connect

    Ray, R.; Holick, M.F. ); Bouillon, R.; Baelen, H.V. )

    1991-07-30

    In this paper, the authors describe photoaffinity labeling and related studies of human serum vitamin D binding protein (hDBP) with 25-hydroxyvitamin D{sub 3} 3{beta}-3{prime}-(N-(4-azido-2-nitrophenyl)amino)propyl ether (25-ANE) and its radiolabeled counterpart, i.e., 25-hydroxyvitamin D{sub 3} 3{beta}-3{prime}-(N-(4-azido-2-nitro-(3,5-{sup 3}H)phenyl)amino)propyl ether ({sup 3}H-25-ANE). They have carried out studies to demonstrate that (1) 25-ANE competes with 25-OH-D{sub 3} for the binding site of the latter in hDBP and (2) {sup 3}H-25-ANE is capable of covalently labeling the hDBP molecule when exposed ot UV light. Treatment of a sample of purified hDBP, labeled with {sup 3}H-25-ANE, with BNPS-skatole produced two Coomassie Blue stained peptide fragments, and the majority of the radioactivity was assoicated with the smaller of the two peptide fragments (16.5 kDa). On the other hand, cleavage of the labeled protein with cyanogen bromide produced a peptide (11.5 kDa) containing most of the covalently attached radioactivity. Considering the primary amino acid structure of hDBP, this peptide fragment (11.5 kDa) represents the N-terminus through residue 108 of the intact protein. Thus, the results tentatively identify this segment of the protein containing the binding pocket for 25-OH-D{sub 3}.

  18. Silver vanadate nanoribbons: A label-free bioindicator in the conversion between human serum transferrin and apotransferrin via surface-enhanced Raman scattering

    NASA Astrophysics Data System (ADS)

    Zhou, Qing; Shao, Mingwang; Que, Ronghui; Cheng, Liang; Zhuo, Shujuan; Tong, Yanhua; Lee, Shuit-Tong

    2011-05-01

    Silver vanadate nanoribbons were synthesized via a hydrothermal process, which exhibited surface-enhanced Raman scattering effect. This surface-enhanced substrate was stable and reproducible for identifying human serum transferrin and human serum apotransferrin in the concentration of 1×10-5 M, which further exhibited significant sensitivity in monitoring the conversion of these two proteins in turn. This result showed that the silver vanadate nanoribbon might be employed as biomonitor in such systems.

  19. Comparative serum proteome analysis of human lymph node negative/positive invasive ductal carcinoma of the breast and benign breast disease controls via label-free semiquantitative shotgun technology.

    PubMed

    Hu, Xiaofang; Zhang, Yan; Zhang, Aili; Li, Yingzi; Zhu, Zhenmin; Shao, Zhimin; Zeng, Rong; Xu, Lisa X

    2009-08-01

    Serum proteomics provides a useful tool to identify potential biomarkers associated with cancer progression. In the present study, a two-dimensional liquid chromatography-tandem mass spectrometry (2D-LC-MS/MS) on a linear ion trap was utilized to identify and compare serum proteins from breast cancer patients. Three groups of 21 human sera, 7 from patients with lymph node-negative invasive ductal carcinoma (IDCB), 7 from patients with lymph node-positive IDCB, and 7 controls from patients with benign breast diseases, were analyzed. Through proteomic analysis, a total of 2,078 proteins were identified with at least two unique peptide hits. By quantification with label-free spectral counting, a fruitful list of serum proteins with significant differences in abundance accompanying the progression of breast cancer was found. Through hierarchical cluster analysis based on the differently expressed proteins in selection, we found that different groups of sera could be distinguished. Among the selected proteins, tenascin-XB (TNXB) was further validated by the ELISA method in 131 serum samples as a promising biomarker for early metastasis of breast cancer. These experiments revealed the valuable potential of label-free quantitative 2D-LC-MS/MS for identification of novel biomarkers for disease progression. PMID:19624269

  20. Imaging of experimental amyloidosis with /sup 131/I-labeled serum amyloid P component

    SciTech Connect

    Caspi, D.; Zalzman, S.; Baratz, M.; Teitelbaum, Z.; Yaron, M.; Pras, M.; Baltz, M.L.; Pepys, M.B.

    1987-11-01

    /sup 131/I-labeled human serum amyloid P component, which was injected into mice with experimentally induced systemic AA amyloidosis and into controls, became specifically localized and was retained in amyloidotic organs. In comparison, it was rapidly and completely eliminated from unaffected tissues and from control animals. Distinctive images of this amyloid-specific deposition of labeled serum amyloid P component were derived from whole body scanning, in vivo, of amyloidotic mice. These findings suggest that such imaging may have applications for the diagnosis and quantitation of amyloid deposits in humans.

  1. A novel quantification method for analysis of twenty natural amino acids in human serum based on N-phosphorylation labeling using reversed-phase liquid chromatography-tandem mass spectrometry.

    PubMed

    Chen, Xiaowu; Gao, Dan; Liu, Feng; Gao, Xiang; Wang, Shujuan; Zhao, Yufen; Liu, Hongxia; Jiang, Yuyang

    2014-07-11

    A novel method based on the strategy of N-phosphorylation labeling is described for quantification of twenty natural amino acids in human serum by reversed-phase liquid chromatography-electrospray tandem mass spectrometry (RP-LC/ESI-MS). The derivatization reaction was easily performed in one-pot reaction under mild conditions within 30min. The reaction mixture was then evaporated to dryness, redissolved, desalted by C18 SPE. The twenty N-phosphoryl amino acids were separated on an RP-C18 column within 20min by isocratic elution (0.1% formic acid-acetonitrile, v/v 7:3). At the same time, multiple reaction monitoring (MRM) MS enabled quantitation of twenty natural amino with the LOD of 0.0005-0.15μM and LOQ of 0.0020-0.5μM in human serum. The linear range was from 0.025 to 25μM (except Cys and Trp) with R>0.99. The recovery range was determined to be 85.5-117.4% with the relative standard deviation (RSD) in the range of 1.3-13.9%. All twenty amino acids were successfully detected in human serum samples with the concentration from 5.7 to 577.9μM, which indicates potential of the developed method for determination of amino acids in complex biological samples, hence for screening of amino acid metabolite related diseases. PMID:24974871

  2. Polymerized soluble venom--human serum albumin

    SciTech Connect

    Patterson, R.; Suszko, I.M.; Grammer, L.C.

    1985-03-01

    Extensive previous studies have demonstrated that attempts to produce polymers of Hymenoptera venoms for human immunotherapy resulted in insoluble precipitates that could be injected with safety but with very limited immunogenicity in allergic patients. We now report soluble polymers prepared by conjugating bee venom with human serum albumin with glutaraldehyde. The bee venom-albumin polymer (BVAP) preparation was fractionated on Sephacryl S-300 to have a molecular weight range higher than catalase. /sup 125/I-labeled bee venom phospholipase A was almost completely incorporated into BVAP. Rabbit antibody responses to bee venom and bee venom phospholipase A were induced by BVAP. Human antisera against bee venom were absorbed by BVAP. No new antigenic determinants on BVAP were present as evidenced by absorption of antisera against BVAP by bee venom and albumin. BVAP has potential immunotherapeutic value in patients with anaphylactic sensitivity to bee venom.

  3. The label free picomolar detection of insulin in blood serum.

    PubMed

    Xu, Mengyun; Luo, Xiliang; Davis, Jason J

    2013-01-15

    Insulin, a polypeptide hormone secreted by pancreatic cells, is a key regulator in glucose homeostasis. Its deficiency leads to insulin-dependent (type I) diabetes whereas resistance to insulin is common in type II diabetes, obesity and a range of endocrine disorders. Its determination is of considerable value, particularly in the clinical diagnosis of diabetes mellitus and the doping control of athletes. It has, additionally, been noted as a potential breast cancer marker (serum insulin levels being found to be raised in comparison to control patients). Electrochemical assays are potentially very cheap, highly sensitive, and very readily transposed to a point of care. Though there exist numerous examples of label free impedimetric or capacitative assaying of biomolecules, these are rarely demonstrated to be effective in complex biological mixtures or to be applicable to low molecular weight targets (since they operate through the interfacial displacement of water/ions and/or the steric blocking of a redox probe). We report herein an ultrasensitive electrochemical and label-free biosensor for insulin in blood serum with a clinically relevant linear range and detection limit of 1.2pM. The transducing surfaces, based on readily prepared, antibody modified, polyethylene glycol monolayer modified polycrystalline gold surfaces, respond in a highly specific and re-useable manner to the target in up to 50% blood serum. PMID:22840329

  4. In Vivo Labeling of Serum Albumin for PET

    PubMed Central

    Niu, Gang; Lang, Lixin; Kiesewetter, Dale O.; Ma, Ying; Sun, Zhongchan; Guo, Ning; Guo, Jinxia; Wu, Chenxi; Chen, Xiaoyuan

    2015-01-01

    The purpose of this study was to develop a novel in vivo albumin-labeling method to allow PET of cardiac function after myocardial infarction and vascular leakage and increased permeability in inflammatory diseases and malignant tumors. Methods To label albumin in vivo, we synthesized a NOTA (1,4,7-triazacyclononane-N,N′, N″-triacetic acid)-conjugated truncated form of Evans blue (NEB). 18F labeling was achieved by the formation of an 18F-aluminum fluoride (18F-AlF) complex, and 64Cu labeling was obtained by a standard chelation method. Sixty-minute dynamic PET imaging was performed on normal mice to evaluate the distribution of 18F-AlF-NEB, which was compared with in vitro–labeled mouse serum albumin (18F-fluorobenzyl-MSA). Electrocardiography-gated PET imaging was performed in a mouse model of myocardial infarction. Both dynamic and static PET scans were obtained in a mouse inflammation model induced by local injection of turpentine to evaluate vascular leakage. Tumor permeability was studied by dynamic and late-point static PET using 64Cu-NEB in a UM-22B xenograft model. Results NEB was successfully synthesized, and 18F labeling including work-up took about 20–30 min, with a radiochemical purity greater than 95% without the need for high-performance liquid chromatography purification. Most of the radioactivity was retained in the circulation system at 60 min after injection (26.35 ± 1.52 percentage injected dose per gram [%ID/g]). With electrocardiography-gated PET, ventricles of the heart and major arteries were clearly visualized. The myocardial infarction mice showed much lower left ventricular ejection fraction than the control mice. Inflammatory muscles showed significantly higher tracer accumulation than the contralateral healthy ones. UM-22B tumor uptake of 64Cu-NEB gradually increased with time (5.73 ± 1.11 %ID/g at 1 h and 8.03 ± 0.77 %ID/g at 2 h after injection). Conclusion The distribution and local accumulation of serum albumin can be

  5. 76 FR 2268 - Viruses, Serums, Toxins, and Analogous Products; Packaging and Labeling

    Federal Register 2010, 2011, 2012, 2013, 2014

    2011-01-13

    ...We are proposing to amend the Virus-Serum-Toxin Act regulations regarding the packaging and labeling of veterinary biological products to provide for the use of an abbreviated true name on small final container labeling for veterinary biologics; require labeling to bear a consumer contact telephone number; change the format used to show the establishment or permit number on labeling and......

  6. Chemical labelling of active serum thioester proteins for quantification.

    PubMed

    Holm, Lotta; Ackland, Gareth L; Edwards, Mark R; Breckenridge, Ross A; Sim, Robert B; Offer, John

    2012-02-01

    The complement serum proteins C3 and C4 and the protease inhibitor α-2 macroglobulin are all members of the C3/α-2M thioester protein family, an evolutionarily ancient and conserved family that contains an intrachain thioester bond. The chemistry of the thioester bond is a key to the function of the thioester proteins. All these proteins function by covalently linking to their target by acyl transfer of the protein via the thioester moiety. We show that the signature thioester bond can be targeted with nucleophiles linked to a bioreporter molecule, site-specifically modifying the whole, intact thioester protein. Conditions were optimised to label selectively and efficiently pull-down unprocessed thioester-containing proteins from serum. We demonstrated pull-down of full-length C3, α-2M and C4 from sera in high salt, using a biotinylated nucleophile and streptavidin-coated resin, confirmed by MALDI-TOF MS identification of the gel bands. The potential for the development of a quantitative method for measuring active C3 in serum was investigated in patient sera pre and post operation. Quantifying active C3 in clinical assays using current methods is difficult. Methods based on antibody detection (e.g. nephelometry) do not distinguish between active C3 and inactive breakdown products. C3-specific haemolytic assays can be used, but these require use of relatively unstable reagents. The current work represents a promising robust, enzyme- and antibody-free chemical method for detecting active thioester proteins in blood, plasma or serum. PMID:21852021

  7. LabeledIn: cataloging labeled indications for human drugs.

    PubMed

    Khare, Ritu; Li, Jiao; Lu, Zhiyong

    2014-12-01

    Drug-disease treatment relationships, i.e., which drug(s) are indicated to treat which disease(s), are among the most frequently sought information in PubMed®. Such information is useful for feeding the Google Knowledge Graph, designing computational methods to predict novel drug indications, and validating clinical information in EMRs. Given the importance and utility of this information, there have been several efforts to create repositories of drugs and their indications. However, existing resources are incomplete. Furthermore, they neither label indications in a structured way nor differentiate them by drug-specific properties such as dosage form, and thus do not support computer processing or semantic interoperability. More recently, several studies have proposed automatic methods to extract structured indications from drug descriptions; however, their performance is limited by natural language challenges in disease named entity recognition and indication selection. In response, we report LabeledIn: a human-reviewed, machine-readable and source-linked catalog of labeled indications for human drugs. More specifically, we describe our semi-automatic approach to derive LabeledIn from drug descriptions through human annotations with aids from automatic methods. As the data source, we use the drug labels (or package inserts) submitted to the FDA by drug manufacturers and made available in DailyMed. Our machine-assisted human annotation workflow comprises: (i) a grouping method to remove redundancy and identify representative drug labels to be used for human annotation, (ii) an automatic method to recognize and normalize mentions of diseases in drug labels as candidate indications, and (iii) a two-round annotation workflow for human experts to judge the pre-computed candidates and deliver the final gold standard. In this study, we focused on 250 highly accessed drugs in PubMed Health, a newly developed public web resource for consumers and clinicians on prevention

  8. 181Hf-Labelled rat serum transferrin: Influence of temperature on the metal-binding configurations

    NASA Astrophysics Data System (ADS)

    Appel, H.; Duffield, J.; Taylor, D. M.; Then, G. M.; Thies, W.-G.

    1987-04-01

    A181Hf-labelled rat serum transferrin sample was studied at different temperatures between 100 K and 346 K using a fast BaF2 TDPAC setup. Relaxation effects were observed in the liquid serum as well as in the frozen serum sample. The temperature dependence of the correlation time has been determined. Different relaxation modes are discussed.

  9. Quantitative analysis of acrylamide labeled serum proteins by LC-MS/MS.

    PubMed

    Faca, Vitor; Coram, Marc; Phanstiel, Doug; Glukhova, Veronika; Zhang, Qing; Fitzgibbon, Matthew; McIntosh, Martin; Hanash, Samir

    2006-08-01

    Isotopic labeling of cysteine residues with acrylamide was previously utilized for relative quantitation of proteins by MALDI-TOF. Here, we explored and compared the application of deuterated and (13)C isotopes of acrylamide for quantitative proteomic analysis using LC-MS/MS and high-resolution FTICR mass spectrometry. The method was applied to human serum samples that were immunodepleted of abundant proteins. Our results show reliable quantitation of proteins across an abundance range that spans 5 orders of magnitude based on ion intensities and known protein concentration in plasma. The use of (13)C isotope of acrylamide had a slightly greater advantage relative to deuterated acrylamide, because of shifts in elution of deuterated acrylamide relative to its corresponding nondeuterated compound by reversed-phase chromatography. Overall, the use of acrylamide for differentially labeling intact proteins in complex mixtures, in combination with LC-MS/MS provides a robust method for quantitative analysis of complex proteomes. PMID:16889424

  10. Isolation of human serum spreading factor.

    PubMed

    Barnes, D W; Silnutzer, J

    1983-10-25

    Serum spreading factor (SF) was isolated from human serum by a four-step procedure employing affinity chromatography on glass beads, concanavalin A-Sepharose, DEAE-agarose, and heparin-agarose. The final product was purified approximately 260-fold from the starting material and was maximally active in assays of cell spreading-promoting activity at 300 ng/ml. The isolated human SF preparation consisted of two proteins of apparent molecular weights approximately 65,000 (SF65) and 75,000 (SF75). Both SF65 and SF75 have been shown previously to exhibit cell spreading-promoting activity and to bind monoclonal antibody to human serum SF. PMID:6630199

  11. Synthesis of Fluorine-18 Radio-labeled Serum Albumins for PET Blood Pool Imaging

    PubMed Central

    Basuli, Falguni; Li, Changhui; Xu, Biying; Williams, Mark; Wong, Karen; Coble, Vincent L; Vasalatiy, Olga; Seidel, Jurgen; Green, Michael V.; Griffiths, Gary L.; Choyke, Peter L.; Jagoda, Elaine M.

    2015-01-01

    We sought to develop a practical, reproducible and clinically translatable method of radiolabeling serum albumins with fluorine-18 for use as a PET blood pool imaging agent in animals and man. Fluorine-18 radiolabeled fluoronicotinic acid-2,3,5,6-tetrafluorophenyl ester, [18F]F-Py-TFP was prepared first by the reaction of its quaternary ammonium triflate precursor with [18F]tetrabutylammonium fluoride ([18F]TBAF) according to a previously published method for peptides, with minor modifications. The incubation of [18F]F-Py-TFP with rat serum albumin (RSA) in phosphate buffer (pH 9) for 15 min at 37–40 °C produced fluorine-18-radiolabeled RSA and the product was purified using a mini-PD MiniTrap G-25 column. The overall radiochemical yield of the reaction was 18–35% (n = 30, uncorrected) in a 90-min synthesis. This procedure, repeated with human serum albumin (HSA), yielded similar results. Fluorine-18-radiolabeled RSA demonstrated prolonged blood retention (biological half-life of 4.8 hours) in healthy awake rats. The distribution of major organ radioactivity remained relatively unchanged during the 4 hour observation periods either by direct tissue counting or by dynamic PET whole-body imaging except for a gradual accumulation of labeled metabolic products in the bladder. This manual method for synthesizing radiolabeled serum albumins uses fluorine-18, a widely available PET radionuclide, and natural protein available in both pure and recombinant forms which could be scaled up for widespread clinical applications. These preclinical biodistribution and PET imaging results indicate that [18F]RSA is an effective blood pool imaging agent in rats and might, as [18F]HSA, prove similarly useful as a clinical imaging agent. PMID:25533724

  12. Flow cytometry measurements of human chromosome kinetochore labeling

    SciTech Connect

    Fantes, J.A.; Green, D.K.; Malloy, P.; Sumner, A.T.

    1989-03-01

    A method for the preparation and measurement of immunofluorescent human chromosome centromeres in suspension is described using CREST antibodies, which bind to the centromeric region of chromosomes. Fluorescein isothiocyanate (FITC)-conjugated antihuman antibodies provide the fluorescent label. Labeled chromosomes are examined on microscope slides and by flow cytometry. In both cases a dye which binds to DNA is added to provide identification of the chromosome groups. Sera from different CREST patients vary in their ability to bind to chromosome arms in addition to the centromeric region. Flow cytometry and microfluorimetry measurements have shown that with a given CREST serum the differences in kinetochore fluorescence between chromosomes are only minor. Flow cytometry experiments to relate the number of dicentric chromosomes, induced by in vitro radiation of peripheral blood cells to the slightly increased number of chromosomes with above-average kinetochore fluorescence did not produce decisive radiation dosimetry results.

  13. Thermal diffusivity of human serum and plasma

    NASA Astrophysics Data System (ADS)

    Mayén-Mondragón, R.; Yánez-Limón, J. M.; Palomares, P.; Sosa, M.; Bernal-Alvarado, J.

    2005-06-01

    Using a thermal lens experimental set up, the thermal diffusivity of human serum and plasma were measured. Several samples were studied and the results are reported as the average, including the standard deviation. The samples of serum and plasma were obtained in healthy adult donors from the Guanajuato State Blood Transfusion Center, Mexico; the donors were clinically tested and they were free of hepatitis, AIDS and other infectious diseases. The parameters reported were obtained using the thermal lens aberrant model with the lasers arranged in the mismatched mode.

  14. [Serum resistance of Escherichia coli in chronic pyelonephritis. 1. Serum resistance in the human serum pool].

    PubMed

    Falkenhagen, U; Handschuck, I; Ulisko, I N; Ratiner YuA; Nimmich, W; Zingler, G; Naumann, G

    1984-07-01

    123 patients of the kidney department of the Clinic for Inner Medicine of Rostock University suffering from chronic pyelonephritis were taken into microbiological observation for between one and four years. 170 E. coli strains were bred from 59 patients with significant bacteriuria in the course of the disease and their serum resistence was determined with pooled human serum using Taylor's method. 78.24% of the strains examined were serum-sensitive, 11.18% intermediate and 10.59% serum-resistent. All strains were O-, K- and H-typed. 57.06% were successfully O-typed and were distributed over 40 O-serogroups. 24.12% were not typable and 18.82% were rough colonies. 86.50% of the resistent and intermediate strains strains were O-typable, 13.50% could not be typed. The significance of E. coli antigens (O, K, H) and serum resistence for the maintenance of a chronic infection is discussed. PMID:6385542

  15. A structural comparison of human serum transferrin and human lactoferrin.

    PubMed

    Wally, Jeremy; Buchanan, Susan K

    2007-06-01

    The transferrins are a family of proteins that bind free iron in the blood and bodily fluids. Serum transferrins function to deliver iron to cells via a receptor-mediated endocytotic process as well as to remove toxic free iron from the blood and to provide an anti-bacterial, low-iron environment. Lactoferrins (found in bodily secretions such as milk) are only known to have an anti-bacterial function, via their ability to tightly bind free iron even at low pH, and have no known transport function. Though these proteins keep the level of free iron low, pathogenic bacteria are able to thrive by obtaining iron from their host via expression of outer membrane proteins that can bind to and remove iron from host proteins, including both serum transferrin and lactoferrin. Furthermore, even though human serum transferrin and lactoferrin are quite similar in sequence and structure, and coordinate iron in the same manner, they differ in their affinities for iron as well as their receptor binding properties: the human transferrin receptor only binds serum transferrin, and two distinct bacterial transport systems are used to capture iron from serum transferrin and lactoferrin. Comparison of the recently solved crystal structure of iron-free human serum transferrin to that of human lactoferrin provides insight into these differences. PMID:17216400

  16. (PCG) Protein Crystal Growth Human Serum Albumin

    NASA Technical Reports Server (NTRS)

    1989-01-01

    (PCG) Protein Crystal Growth Human Serum Albumin. Contributes to many transport and regulatory processes and has multifunctional binding properties which range from various metals, to fatty acids, hormones, and a wide spectrum of therapeutic drugs. The most abundant protein of the circulatory system. It binds and transports an incredible variety of biological and pharmaceutical ligands throughout the blood stream. Principal Investigator on STS-26 was Larry DeLucas.

  17. Cultivation of Plasmodium falciparum using animal serum (horse, calf and bovine) as human serum substitute.

    PubMed

    Ramos, M I; Hermosura, M E; Nakabayashi, T

    1986-11-01

    Horse, calf and bovine serum were successfully used as human serum substitutes in the in vitro cultivation of Plasmodium falciparum. Positive results were obtained only after gradually adapting the parasites to the substitute serum. Adapted lines were established within 4-5 weeks. 10% horse serum was observed to be the best substitute with growth rates comparable or even surprising that obtained in human serum. Pure calf or bovine serum supported stable growths of 20-30% less which was enhanced to comparable levels after addition of 1% glucose-peptone to the medium. Direct transfers of adapted cultures to human serum showed enhanced growth rates. Lower growth rates of adapted cultures (i.e. horse serum-adapted cultures) in other substitute sera (i.e. calf or bovine sera) were improved in subsequent subcultures. Similarly, there were no adverse effects when they were returned back to the substitute serum they were originally adapted to. PMID:3541461

  18. Automated fudicial labeling on human body data

    NASA Astrophysics Data System (ADS)

    Lewark, Erick A.; Nurre, Joseph H.

    1998-03-01

    The Cyberware WB4 whole body scanner generates a high- resolution data set of the outer surface of the human body. The acquisition of anthropometric data from this data set is important for the development of custom sizing for the apparel industry. Software for locating anthropometric landmarks from a cloud of more than 200,000 three-dimensional data points, captured from a human subject, is presented. The first phase of identification is to locate externally placed fudicials on the human body using luminance information captured at scan time. The fudicials are then autonomously labeled and categorized according to their general position and anthropometric significance in the scan. Once registration of the landmarks is complete, body measurements may be extracted for apparel sizing.

  19. Interaction of Citrinin with Human Serum Albumin

    PubMed Central

    Poór, Miklós; Lemli, Beáta; Bálint, Mónika; Hetényi, Csaba; Sali, Nikolett; Kőszegi, Tamás; Kunsági-Máté, Sándor

    2015-01-01

    Citrinin (CIT) is a mycotoxin produced by several Aspergillus, Penicillium, and Monascus species. CIT occurs worldwide in different foods and drinks and causes health problems for humans and animals. Human serum albumin (HSA) is the most abundant plasma protein in human circulation. Albumin forms stable complexes with many drugs and xenobiotics; therefore, HSA commonly plays important role in the pharmacokinetics or toxicokinetics of numerous compounds. However, the interaction of CIT with HSA is poorly characterized yet. In this study, the complex formation of CIT with HSA was investigated using fluorescence spectroscopy and ultrafiltration techniques. For the deeper understanding of the interaction, thermodynamic, and molecular modeling studies were performed as well. Our results suggest that CIT forms stable complex with HSA (logK ~ 5.3) and its primary binding site is located in subdomain IIA (Sudlow’s Site I). In vitro cell experiments also recommend that CIT-HSA interaction may have biological relevance. Finally, the complex formations of CIT with bovine, porcine, and rat serum albumin were investigated, in order to test the potential species differences of CIT-albumin interactions. PMID:26633504

  20. Production of proteoglycans by human lung fibroblasts (IMR-90) maintained in a low concentration of serum.

    PubMed Central

    Vogel, K G; Sapién, R E

    1982-01-01

    Maintenance of fibroblasts in 0.5% serum results in viable but non-proliferative cells that may be analogous to fibroblasts in vivo. The synthesis of proteoglycans by human embryo lung fibroblasts in Eagle's minimal essential medium with 0.5% newborn-bovine serum or with 10% serum has been compared. A similar amount of [35S]sulphate-labelled glycosaminoglycan per cell was secreted by fibroblasts in 10% or 0.5% serum. 35SO42-incorporation into sulphated glycosaminoglycans was enhanced in 0.5% serum when expressed per mg of cell protein, but [3H]glucosamine incorporation was decreased. The charge density of these glycosaminoglycans was not changed as determined by ion-exchange chromatography. It was concluded that decreased protein/ cell resulted in an apparent increase in 35S-labelled glycosaminoglycan synthesis/mg of cell protein, whereas decreased uptake of [3H]glucosamine resulted in a decrease in their glucosamine labelling. The proteoglycans secreted by fibroblasts in 0.5% serum were similar in glycosaminoglycan composition, chain length and buoyant density to the dermatan sulphate proteoglycan, which is the major secreted component of cells in 10% serum. Larger heparan sulphate and chondroitin sulphate proteoglycans, which comprise about 40% of the total secreted proteoglycans of cultures in 10% serum, were greatly diminished in the medium of cultures in 0.5% serum. The proteoglycan profile of medium from density-inhibited cultures in 10% serum resembles that of proliferating cultures, indicating that lack of proliferation was not responsible for the alteration. The dermatan sulphate proteoglycan, participating in extracellular matrix structure, may be the primary tissue product of lung fibroblasts in vivo. Images Fig. 1. PMID:7165697

  1. The human serum paraoxonase/arylesterase polymorphism.

    PubMed Central

    Eckerson, H W; Wyte, C M; La Du, B N

    1983-01-01

    The heterozygous human serum paraoxonase phenotype can be clearly distinguished from both homozygous phenotypes on the basis of its distinctive ratio of paraoxonase to arylesterase activities. A trimodal distribution of the ratio values was found with 348 individual serum samples, measuring the ratio of paraoxonase activity (with 1 M NaCl in the assay) to arylesterase activity, using phenylacetate. The three modes corresponded to the three paraoxonase phenotypes, A, AB, and B (individual genotypes), and the expected Mendelian segregation of the trait was observed within families. The paraoxonase/arylesterase activity ratio showed codominant inheritance. We have defined the genetic locus determining the aromatic esterase (arylesterase) responsible for the polymorphic paraoxonase activity as esterase-A (ESA) and have designated the two common alleles at this locus by the symbols ESA*A and ESA*B. The frequency of the ESA*A allele was estimated to be .685, and that of the ESA*B allele, 0.315, in a sample population of unrelated Caucasians from the United States. We postulate that a single serum enzyme, with both paraoxonase and arylesterase activity, exists in two different isozymic forms with qualitatively different properties, and that paraoxon is a "discriminating" substrate (having a polymorphic distribution of activity) and phenylacetate is a "nondiscriminating" substrate for the two isozymes. Biochemical evidence for this interpretation includes the cosegregation of the degree of stimulation of paraoxonase activity by salt and paraoxonase/arylesterase activity ratio characteristics; the very high correlation between both the basal (non-salt stimulated) and salt-stimulated paraoxonase activities with arylesterase activity; and the finding that phenylacetate is an inhibitor for paraoxonase activities in both A and B types of enzyme. PMID:6316781

  2. Quantitative analysis of single amino acid variant peptides associated with pancreatic cancer in serum by an isobaric labeling quantitative method.

    PubMed

    Nie, Song; Yin, Haidi; Tan, Zhijing; Anderson, Michelle A; Ruffin, Mack T; Simeone, Diane M; Lubman, David M

    2014-12-01

    Single amino acid variations are highly associated with many human diseases. The direct detection of peptides containing single amino acid variants (SAAVs) derived from nonsynonymous single nucleotide polymorphisms (SNPs) in serum can provide unique opportunities for SAAV associated biomarker discovery. In the present study, an isobaric labeling quantitative strategy was applied to identify and quantify variant peptides in serum samples of pancreatic cancer patients and other benign controls. The largest number of SAAV peptides to date in serum including 96 unique variant peptides were quantified in this quantitative analysis, of which five variant peptides showed a statistically significant difference between pancreatic cancer and other controls (p-value < 0.05). Significant differences in the variant peptide SDNCEDTPEAGYFAVAVVK from serotransferrin were detected between pancreatic cancer and controls, which was further validated by selected reaction monitoring (SRM) analysis. The novel biomarker panel obtained by combining α-1-antichymotrypsin (AACT), Thrombospondin-1 (THBS1) and this variant peptide showed an excellent diagnostic performance in discriminating pancreatic cancer from healthy controls (AUC = 0.98) and chronic pancreatitis (AUC = 0.90). These results suggest that large-scale analysis of SAAV peptides in serum may provide a new direction for biomarker discovery research. PMID:25393578

  3. Farm Animal Serum Proteomics and Impact on Human Health

    PubMed Central

    Girolamo, Francesco Di; D’Amato, Alfonsina; Lante, Isabella; Signore, Fabrizio; Muraca, Marta; Putignani, Lorenza

    2014-01-01

    Due to the incompleteness of animal genome sequencing, the analysis and characterization of serum proteomes of most farm animals are still in their infancy, compared to the already well-documented human serum proteome. This review focuses on the implications of the farm animal serum proteomics in order to identify novel biomarkers for animal welfare, early diagnosis, prognosis and monitoring of infectious disease treatment, and develop new vaccines, aiming at determining the reciprocal benefits for humans and animals. PMID:25257521

  4. 101 Labeled Brain Images and a Consistent Human Cortical Labeling Protocol

    PubMed Central

    Klein, Arno; Tourville, Jason

    2012-01-01

    We introduce the Mindboggle-101 dataset, the largest and most complete set of free, publicly accessible, manually labeled human brain images. To manually label the macroscopic anatomy in magnetic resonance images of 101 healthy participants, we created a new cortical labeling protocol that relies on robust anatomical landmarks and minimal manual edits after initialization with automated labels. The “Desikan–Killiany–Tourville” (DKT) protocol is intended to improve the ease, consistency, and accuracy of labeling human cortical areas. Given how difficult it is to label brains, the Mindboggle-101 dataset is intended to serve as brain atlases for use in labeling other brains, as a normative dataset to establish morphometric variation in a healthy population for comparison against clinical populations, and contribute to the development, training, testing, and evaluation of automated registration and labeling algorithms. To this end, we also introduce benchmarks for the evaluation of such algorithms by comparing our manual labels with labels automatically generated by probabilistic and multi-atlas registration-based approaches. All data and related software and updated information are available on the http://mindboggle.info/data website. PMID:23227001

  5. Resistance of Gardnerella vaginalis to bactericidal activity of human serum.

    PubMed Central

    Boustouller, Y L; Johnson, A P

    1986-01-01

    To assess the sensitivity of Gardnerella vaginalis to the complement mediated bactericidal activity of serum, six laboratory strains were incubated with normal human serum and two strains freshly isolated from women with non-specific vaginitis (NSV) were each incubated with homologous patient serum. There was no significant difference between the number of organisms recovered from unheated or heat inactivated serum after incubation at 37 degrees C for one hour with any of the strains tested. A suspension of G vaginalis incubated at 37 degrees C for one hour in heat inactivated homologous mouse antiserum with unheated normal human serum as a source of complement did not show any less viability than the control mixture using heat inactivated human serum. In contrast, a serum resistant strain of Neisseria gonorrhoeae incubated in heat inactivated homologous mouse antiserum with unheated normal human serum showed noticeably less viability than the control. G vaginalis therefore seems to be resistant to the bactericidal activity of both normal and immune serum. PMID:3493201

  6. Label-Free Biomarker Sensing in Undiluted Serum with Suspended Microchannel Resonators

    PubMed Central

    von Muhlen, Marcio G.; Brault, Norman D.; Knudsen, Scott M.; Jiang, Shaoyi; Manalis, Scott R.

    2010-01-01

    Improved methods are needed for routine, inexpensive monitoring of biomarkers that could facilitate earlier detection and characterization of cancer. Suspended microchannel resonators (SMRs) are highly sensitive, batch-fabricated microcantilevers with embedded microchannels that can directly quantify adsorbed mass via changes in resonant frequency. As in other label-free detection methods, biomolecular measurements in complex media such as serum are challenging due to high background signals from non-specific binding. In this report, we demonstrate that carboxybetaine-derived polymers developed to adsorb directly onto SMR SiO2 surfaces act as ultra-low fouling and functionalizable surface coatings. Coupled with a reference microcantilever, this approach enables detection of activated leukocyte cell adhesion molecule (ALCAM), a model cancer biomarker, in undiluted serum with a limit of detection of 10 ng/mL. PMID:20148583

  7. Human serum metabolic profiles are age dependent.

    PubMed

    Yu, Zhonghao; Zhai, Guangju; Singmann, Paula; He, Ying; Xu, Tao; Prehn, Cornelia; Römisch-Margl, Werner; Lattka, Eva; Gieger, Christian; Soranzo, Nicole; Heinrich, Joachim; Standl, Marie; Thiering, Elisabeth; Mittelstraß, Kirstin; Wichmann, Heinz-Erich; Peters, Annette; Suhre, Karsten; Li, Yixue; Adamski, Jerzy; Spector, Tim D; Illig, Thomas; Wang-Sattler, Rui

    2012-12-01

    Understanding the complexity of aging is of utmost importance. This can now be addressed by the novel and powerful approach of metabolomics. However, to date, only a few metabolic studies based on large samples are available. Here, we provide novel and specific information on age-related metabolite concentration changes in human homeostasis. We report results from two population-based studies: the KORA F4 study from Germany as a discovery cohort, with 1038 female and 1124 male participants (32-81 years), and the TwinsUK study as replication, with 724 female participants. Targeted metabolomics of fasting serum samples quantified 131 metabolites by FIA-MS/MS. Among these, 71/34 metabolites were significantly associated with age in women/men (BMI adjusted). We further identified a set of 13 independent metabolites in women (with P values ranging from 4.6 × 10(-04) to 7.8 × 10(-42) , α(corr) = 0.004). Eleven of these 13 metabolites were replicated in the TwinsUK study, including seven metabolite concentrations that increased with age (C0, C10:1, C12:1, C18:1, SM C16:1, SM C18:1, and PC aa C28:1), while histidine decreased. These results indicate that metabolic profiles are age dependent and might reflect different aging processes, such as incomplete mitochondrial fatty acid oxidation. The use of metabolomics will increase our understanding of aging networks and may lead to discoveries that help enhance healthy aging. PMID:22834969

  8. Photothermal Measurements on Human Serum and Plasma

    NASA Astrophysics Data System (ADS)

    Bernal-Alvarado, J.; Sosa, M.; Hernández, L. C.; Hernández-Cabrera, F.; Mayén-Mondragón, R.; Yánez-Limón, J. M.; Flores-Farías, R.; Palomares, P.; Juárez, P.; Ramírez, R.

    2003-09-01

    Using a thermal lens experimental set up, the thermal diffusivity of serum and plasma was measured. Several samples were studied and the results are reported as the average with the standard deviation. The serum and plasma were obtained by aleatory sampling of healthy adult donors at the Guanajuato State Transfusion Center, Mexico; the donors were free of hepatitis and other diseases, clinically tested. The parameters reported were obtained using the thermal lens aberrant model with the lasers operating in the mismatched mode.

  9. Label-Free Kinetic Studies of Hemostasis-Related Biomarkers Including D-Dimer Using Autologous Serum Transfusion

    PubMed Central

    Winterhagen, Anna; Müller, Jens; Oldenburg, Johannes; Pötzsch, Bernd

    2015-01-01

    The objective of this study was to evaluate the elimination kinetics of hemostasis-related biomarkers including the prothrombin activation fragment F1+2, thrombin-antithrombin complex (TAT), plasmin-α2-antiplasmin complex (PAP), and D-dimer in humans. Autologous serum was used as a biomarker source and infused into 15 healthy volunteers. Serum was prepared from whole blood in the presence of recombinant tissue-type plasminogen activator (final concentration 20 μg/mL) to induce plasmin generation required for PAP and D-dimer formation. Serum transfusions (50 mL/30 min) were well tolerated by all subjects. Endogenous thrombin formation was not induced by serum infusions as measured using a highly sensitive oligonucleotide-based enzyme capture assay. Median peak levels (x-fold increase over baseline) of F1+2, TAT, PAP, and D-dimer of 3.7 nmol/L (28.9), 393 ng/mL (189.6), 3,829 ng/mL (7.0), and 13.4 mg/L (34.2) were achieved at the end of serum infusions. During a 48 h lasting follow-up period all biomarkers showed elimination kinetics of a two-compartment model. Median (interquartile range) terminal half-lives were 1.9 (1.3–3.6) h for F1+2, 0.7 (0.7–2.6) h for TAT, and 10.8 (8.8–11.4) h for PAP. With 15.8 (13.1–23.1) h the D-dimer half-life was about twice as long as previously estimated from radiolabeling studies in animals and small numbers of human subjects. The serum approach presented here allows label-free and simultaneous analysis of the elimination kinetics of various hemostasis-related biomarkers. Based on these data changes in biomarker levels could more precisely used to estimate the activity level of the hemostatic system. PMID:26658824

  10. Superhydrophobic Effect on the Adsorption of Human Serum Albumin

    PubMed Central

    Leibner, Evan S.; Barnthip, Naris; Chen, Weinan; Baumrucker, Craig R.; Badding, John V.; Pishko, Michael; Vogler, Erwin A.

    2009-01-01

    Analytical protocol greatly influences measurement of human-serum albumin (HSA) adsorption to commercial expanded polytetrafluororethylene (ePTFE) exhibiting superhydrophobic wetting properties. Degassing of buffer solutions and evacuation of ePTFE adsorbent to remove trapped air immediately prior to contact with protein solutions are shown to be essential. Results obtained with ePTFE as a prototypical superhydrophobic test material suggest that vacuum degassing should be applied in the measurement of protein adsorption to any surface exhibiting superhydrophobicity. Solution depletion quantified using radiometry (I-125 labeled HSA) or electrophoresis yield different measures of adsorption, with nearly four-fold higher surface concentrations of unlabeled HSA measured by the electrophoresis method. This outcome is attributed to the influence of the radiolabel on HSA hydrophilicity which decreases radiolabeled-HSA affinity for a hydrophobic adsorbent in comparison to unlabeled HSA. These results indicate that radiometry underestimates the actual amount of protein adsorbed to a particular material. Removal of radiolabeled HSA adsorbed to ePTFE by 3X serial buffer rinses also shows that the remaining “bound fraction” was about 35% lower than the amount measured by radiometric depletion. This observation implies that measurement of protein bound after surface rinsing significantly underestimates the actual amount of protein concentrated by adsorption into the surface region of a protein-contacting material. PMID:19135420

  11. Serum-free primary human fibroblast and keratinocyte coculture.

    PubMed

    Mujaj, Sally; Manton, Kerry; Upton, Zee; Richards, Sean

    2010-04-01

    Research has shown that the inclusion of a fibroblast cell support layer is required for the isolation and expansion of primary keratinocytes. Recent advances have provided keratinocyte culture with fibroblast-free alternatives. However, these technologies are often undefined and rely on the incorporation of purified proteins/components. To address this problem we developed a medium that used recombinant proteins to support the serum-free isolation and expansion of human dermal fibroblasts and keratinocytes. The human dermal fibroblasts were able to be isolated serum free by adding recombinant human albumin to a collagenase solution. These fibroblasts were then expanded using a serum-free medium containing recombinant proteins: epidermal growth factor, basic fibroblast growth factor, chimeric vitronectin:insulin-like growth factor-I protein, and recombinant human albumin. These fibroblasts maintained a typical morphology and expressed fibroblast markers during their serum-free isolation, expansion, and freezing. Moreover, these fibroblasts were able to support the serum-free isolation and expansion of primary keratinocytes using these recombinant proteins. Real-time polymerase chain reaction and immunofluorescence analysis confirmed that there were no differences in expression levels of p63 or keratins 1, 6, and 10 when keratinocytes were grown in either serum-supplemented or serum-free medium. Using a three-dimensional human skin equivalent model we demonstrated that these keratinocytes also maintained their ability to reform an epidermal layer. In summary, the techniques described provide a valuable alternative for culturing fibroblasts and keratinocytes using recombinant proteins. PMID:19929322

  12. A Survey of Membrane Proteins in Human Serum

    PubMed Central

    Dung, Nguyen Tien; Van Chi, Phan

    2012-01-01

    Serum and membrane proteins are two of the most attractive targets for proteomic analysis. Previous membrane protein studies tend to focus on tissue sample, while membrane protein studies in serum are still limited. In this study, an analysis of membrane proteins in normal human serum was carried out. Nano-liquid chromatography-electrospray ionization mass spectrometry (NanoLC-ESI-MS/MS) and bioinformatics tools were used to identify membrane proteins. Two hundred and seventeen membrane proteins were detected in the human serum, of which 129 membrane proteins have at least one transmembrane domain (TMD). Further characterizations of identified membrane proteins including their subcellular distributions, molecular weights, post translational modifications, transmembrane domains and average of hydrophobicity, were also implemented. Our results showed the potential of membrane proteins in serum for diagnosis and treatment of diseases. PMID:25288886

  13. Nanopore-based electrical and label-free sensing of enzyme activity in blood serum.

    PubMed

    Kukwikila, Mikiembo; Howorka, Stefan

    2015-09-15

    A generic strategy to expand the analytical scope of electrical nanopore sensing is presented. We specifically and electrically detect the activity of a diagnostically relevant hydrolytic enzyme and remove the analytically harmful interference from the biochemically complex sample matrix of blood serum. Our strategy is demonstrated at the example of the renin protease which is involved in regulation of blood pressure. The analysis scheme exploits a new approach to reduce sample complexity while generating a specific read-out signal. Within a single spin-column (i), the protease cleaves a resin-tethered peptide substrate (ii) which is affinity-purified using the same multifunctional resin to remove interfering blood serum components, followed by (iii) detecting the peptide via electrical nanopore recordings. Our approach is beneficial in several ways. First, by eliminating serum components, we overcome limitations of nanopore sensing when challenging samples lead to membrane instability and a poor signal-to-noise ratio. Second, the label-free sensing avoids drawbacks of currently used radiolabel-immunoassays for renin. Finally, the strategy of simultaneous generation and purification of a signal peptide within a multifunctional resin can very likely be expanded to other hydrolytic enzymes dissolved in any analyte matrix and exploited for analytical read-out methods other than nanopore sensing. PMID:26305576

  14. Atomic structure and chemistry of human serum albumin

    NASA Technical Reports Server (NTRS)

    He, Xiao M.; Carter, Daniel C.

    1992-01-01

    The three-dimensional structure of human serum albumin has been determined crystallographically to a resolution of 2.8 A. It comprises three homologous domains that assemble to form a heart-shaped molecule. Each domain is a product of two subdomains that possess common structural motifs. The principal regions of ligand binding to human serum albumin are located in hydrophobic cavities in subdomains IIA and ILIA, which exhibit similar chemistry. The structure explains numerous physical phenomena and should provide insight into future pharmacokinetic and genetically engineered therapeutic applications of serum albumin.

  15. Menaquinones content of human serum and feces

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Bacterially-synthesized menaquinones (MKn) may contribute to vitamin K (VK) nutriture. There are limited data on interindividual variability in endogenous MK synthesis and its relation to circulating forms of VK. Serum and fecal VK concentrations were assessed in 13 healthy adults (45-65 yr) consumi...

  16. Label-retaining assay enriches tumor-initiating cells in glioblastoma spheres cultivated in serum-free medium

    PubMed Central

    Zeng, Lingcheng; Zhao, Yiqing; Ouyang, Taohui; Zhao, Tianyuan; Zhang, Suojun; Chen, Jian; Yu, Jiasheng; Lei, Ting

    2016-01-01

    Label-retaining cells, which are characterized by dormancy or slow cycling, may be identified in a number of human normal and cancer tissues, and these cells demonstrate stem cell potential. In glioblastoma, label-retaining assays to enrich glioma stem cells remain to be fully investigated. In the present study, glioblastoma sphere cells cultured in serum-free medium were initially stained with the cell membrane fluorescent marker DiI. The fluorescence intensity during cell proliferation and sphere reformation was observed. At 2 weeks, the DiI-retaining cells were screened by fluorescence-activated cell sorting and compared phenotypically with the DiI-negative cells in terms of in vitro proliferation, clonogenicity and multipotency and for in vivo tumorigenicity, as well as sensitivity to irradiation and temozolomide treatment. It was observed that DiI-retaining cells accounted for a small proportion, <10%, within the glioblastoma spheres and that DiI-retaining cells proliferated significantly more slowly compared with DiI-negative cells (P=0.011, P=0.035 and P=0.023 in the of NCH421k, NCH441 and NCH644 glioblastoma sphere cell lines). Significantly increased clonogenicity (P=0.002, P=0.034 and P=0.016 in the NCH441, NCH644 and NCH421k glioblastoma sphere cell lines) and three-lineage multipotency were observed in DiI-retaining cells in vitro compared with DiI-negative cells. As few as 100 DiI-retaining cells were able to effectively generate tumors in the immunocompromised mouse brain, whereas the same number of DiI-negative cells possessed no such ability, indicating the increased tumorigenicity of DiI-retaining cells compared with DiI-negative cells. Furthermore, DiI-retaining cells demonstrated significant resistance following irradiation (P=0.012, P=0.024 and P=0.036) and temozolomide (P=0.003, P=0.005 and P=0.029) compared with DiI-negative cells in the NCH421k, NCH441 and NCH644 glioblastoma sphere cell lines, respectively. It was concluded that label

  17. Label-free detection of tobramycin in serum by transmission-localized surface plasmon resonance.

    PubMed

    Cappi, Giulia; Spiga, Fabio M; Moncada, Yessica; Ferretti, Anna; Beyeler, Michael; Bianchessi, Marco; Decosterd, Laurent; Buclin, Thierry; Guiducci, Carlotta

    2015-05-19

    In order to improve the efficacy and safety of treatments, drug dosage needs to be adjusted to the actual needs of each patient in a truly personalized medicine approach. Key for widespread dosage adjustment is the availability of point-of-care devices able to measure plasma drug concentration in a simple, automated, and cost-effective fashion. In the present work, we introduce and test a portable, palm-sized transmission-localized surface plasmon resonance (T-LSPR) setup, comprised of off-the-shelf components and coupled with DNA-based aptamers specific to the antibiotic tobramycin (467 Da). The core of the T-LSPR setup are aptamer-functionalized gold nanoislands (NIs) deposited on a glass slide covered with fluorine-doped tin oxide (FTO), which acts as a biosensor. The gold NIs exhibit localized plasmon resonance in the visible range matching the sensitivity of the complementary metal oxide semiconductor (CMOS) image sensor employed as a light detector. The combination of gold NIs on the FTO substrate, causing NIs size and pattern irregularity, might reduce the overall sensitivity but confers extremely high stability in high-ionic solutions, allowing it to withstand numerous regeneration cycles without sensing losses. With this rather simple T-LSPR setup, we show real-time label-free detection of tobramycin in buffer, measuring concentrations down to 0.5 μM. We determined an affinity constant of the aptamer-tobramycin pair consistent with the value obtained using a commercial propagating-wave based SPR. Moreover, our label-free system can detect tobramycin in filtered undiluted blood serum, measuring concentrations down to 10 μM with a theoretical detection limit of 3.4 μM. While the association signal of tobramycin onto the aptamer is masked by the serum injection, the quantification of the captured tobramycin is possible during the dissociation phase and leads to a linear calibration curve for the concentrations over the tested range (10-80 μM). The plasmon

  18. Serum-Induced Differentiation of Human Meibomian Gland Epithelial Cells

    PubMed Central

    Sullivan, David A.; Liu, Yang; Kam, Wendy R.; Ding, Juan; Green, Karin M.; Shaffer, Scott A.; Hatton, Mark P.; Liu, Shaohui

    2014-01-01

    Purpose. We hypothesize that culturing immortalized human meibomian gland epithelial cells in serum-containing medium will induce their differentiation. The purpose of this investigation was to begin to test our hypothesis, and explore the impact of serum on gene expression and lipid accumulation in human meibomian gland epithelial cells. Methods. Immortalized and primary human meibomian gland epithelial cells were cultured in the presence or absence of serum. Cells were evaluated for lysosome and lipid accumulation, polar and neutral lipid profiles, and gene expression. Results. Our results support our hypothesis that serum stimulates the differentiation of human meibomian gland epithelial cells. This serum-induced effect is associated with a significant increase in the expression of genes linked to cell differentiation, epithelium development, the endoplasmic reticulum, Golgi apparatus, vesicles, and lysosomes, and a significant decrease in gene activity related to the cell cycle, mitochondria, ribosomes, and translation. These cellular responses are accompanied by an accumulation of lipids within lysosomes, as well as alterations in the fatty acid content of polar and nonpolar lipids. Of particular importance, our results show that the molecular and biochemical changes of immortalized human meibomian gland epithelial cells during differentiation are analogous to those of primary cells. Conclusions. Overall, our findings indicate that immortalized human meibomian gland epithelial cells may serve as an ideal preclinical model to identify factors that control cellular differentiation in the meibomian gland. PMID:24867579

  19. A supervised patch-based approach for human brain labeling

    PubMed Central

    Rousseau, François; Habas, Piotr A.; Studholme, Colin

    2012-01-01

    We propose in this work a patch-based image labeling method relying on a label propagation framework. Based on image intensity similarities between the input image and an anatomy textbook, an original strategy which does not require any non-rigid registration is presented. Following recent developments in non-local image denoising, the similarity between images is represented by a weighted graph computed from an intensity-based distance between patches. Experiments on simulated and in-vivo MR images show that the proposed method is very successful in providing automated human brain labeling. PMID:21606021

  20. Quantification of active infliximab in human serum with liquid chromatography-tandem mass spectrometry using a tumor necrosis factor alpha -based pre-analytical sample purification and a stable isotopic labeled infliximab bio-similar as internal standard: A target-based, sensitive and cost-effective method.

    PubMed

    El Amrani, Mohsin; van den Broek, Marcel P H; Göbel, Camiel; van Maarseveen, Erik M

    2016-07-01

    The therapeutic monoclonal antibody Infliximab (IFX) is a widely used drug for the treatment of several inflammatory autoimmune diseases. However, approximately 10% of patients develop anti-infliximab antibodies (ATIs) rendering the treatment ineffective. Early detection of underexposure to unbound IFX would result in a timely switch of therapy which could aid in the treatment of this disease. Streptavidin coated 96 well plates were used to capture biotinylated-tumor necrosis factor -alpha (b-TNF-α), which in turn was used to selectively extract the active form of IFX in human serum. After elution, IFX was digested using trypsin and one signature peptide was selected for subsequent analysis on liquid chromatography-tandem mass spectrometry (LC-MS/MS). The internal standard used was a stable isotopic labeled IFX bio-similar. The assay was successfully validated according to European Medicines Agency (EMA) guidelines and was found to be linear in a range of 0.5-20μg/mL (r(2)=0.994). Lower limit of quantification for the assay (<20% CV) was 0.5μg/mL, requiring only 2μL of sample. Cross-validation against enzyme-linked immunosorbent assay (ELISA) resulted in a high correlation between methods (r(2)=0.95 with a ρc=0.83) and the accuracy was in line with previously published results. In conclusion, a sensitive, robust and cost-effective method was developed for the bio-analysis of IFX with LC-MS/MS by means of a target-based pre-analytical sample purification. Moreover, low volume and costs of consumables per sample promote its feasibility in (pre)clinical studies and in therapeutic drug monitoring. This method should be considered as first choice due to its accuracy and multiple degree of selectivity. PMID:27264745

  1. Interlobe communication in 13C-methionine-labeled human transferrin.

    PubMed

    Beatty, E J; Cox, M C; Frenkiel, T A; Tam, B M; Mason, A B; MacGillivray, R T; Sadler, P J; Woodworth, R C

    1996-06-18

    [1H, 13C] NMR investigations of metal-induced conformational changes in the blood serum protein transferrin (80 kDa) are reported. These are thought to play an important role in the recognition of this protein by its cellular receptors. [1H, 13C] NMR resonance assignments are presented for all nine methionine 13CH3 groups of recombinant deglycosylated human transferrin on the basis of studies of recombinant N-lobe (40 kDa, five Met residues), NOESY-relayed [1H, 13C] HMQC spectra, and structural considerations. The first specific assignments for C-lobe resonances of transferrin are presented. Using methionine 13CH3 resonances as probes, it is shown that, with oxalate as the synergistic anion, Ga3+ binds preferentially to the C-lobe and subsequently to the N-lobe. The NMR shifts of Met464, which is in the Trp460-centered hydrophobic patch of helix 5 in the C-lobe in contact with the anion and metal binding site, show that Ga3+ binding causes movement of side chains within this helix, as is also the case in the N-lobe. The C-lobe residue Met382, which contacts the N-lobe hinge region, is perturbed when Ga3+ binds to the N-lobe, indicative of interlobe communication, a feature which may control the recognition of fully-metallated transferrin by its receptor. These results demonstrate that selective 13C labeling is a powerful method for probing the structure and dynamics of high-molecular-mass proteins. PMID:8672464

  2. Serum Albumin Domain Structures in Human Blood Serum by Mass Spectrometry and Computational Biology*

    PubMed Central

    Belsom, Adam; Schneider, Michael; Fischer, Lutz; Brock, Oliver; Rappsilber, Juri

    2016-01-01

    Chemical cross-linking combined with mass spectrometry has proven useful for studying protein-protein interactions and protein structure, however the low density of cross-link data has so far precluded its use in determining structures de novo. Cross-linking density has been typically limited by the chemical selectivity of the standard cross-linking reagents that are commonly used for protein cross-linking. We have implemented the use of a heterobifunctional cross-linking reagent, sulfosuccinimidyl 4,4′-azipentanoate (sulfo-SDA), combining a traditional sulfo-N-hydroxysuccinimide (sulfo-NHS) ester and a UV photoactivatable diazirine group. This diazirine yields a highly reactive and promiscuous carbene species, the net result being a greatly increased number of cross-links compared with homobifunctional, NHS-based cross-linkers. We present a novel methodology that combines the use of this high density photo-cross-linking data with conformational space search to investigate the structure of human serum albumin domains, from purified samples, and in its native environment, human blood serum. Our approach is able to determine human serum albumin domain structures with good accuracy: root-mean-square deviation to crystal structure are 2.8/5.6/2.9 Å (purified samples) and 4.5/5.9/4.8Å (serum samples) for domains A/B/C for the first selected structure; 2.5/4.9/2.9 Å (purified samples) and 3.5/5.2/3.8 Å (serum samples) for the best out of top five selected structures. Our proof-of-concept study on human serum albumin demonstrates initial potential of our approach for determining the structures of more proteins in the complex biological contexts in which they function and which they may require for correct folding. Data are available via ProteomeXchange with identifier PXD001692. PMID:26385339

  3. Lung Cancer Serum Biomarker Discovery Using Label Free LC-MS/MS

    PubMed Central

    Zeng, Xuemei; Hood, Brian L.; Zhao, Ting; Conrads, Thomas P.; Sun, Mai; Gopalakrishnan, Vanathi; Grover, Himanshu; Day, Roger S.; Weissfeld, Joel L.; Wilson, David O.; Siegfried, Jill M.; Bigbee, William L.

    2011-01-01

    Introduction Lung cancer remains the leading cause of cancer-related death with poor survival due to the late stage at which lung cancer is typically diagnosed. Given the clinical burden from lung cancer, and the relatively favorable survival associated with early stage lung cancer, biomarkers for early detection of lung cancer are of important potential clinical benefit. Methods We performed a global lung cancer serum biomarker discovery study using liquid chromatography-tandem mass spectrometry (LC-MS/MS) in a set of pooled non-small cell lung cancer (NSCLC) case sera and matched controls. Immunoaffinity subtraction was used to deplete the top most abundant serum proteins; the remaining serum proteins were subjected to trypsin digestion and analyzed in triplicate by LC-MS/MS. The tandem mass spectrum data were searched against the human proteome database and the resultant spectral counting data were used to estimate the relative abundance of proteins across the case/control serum pools. The spectral counting derived abundances of some candidate biomarker proteins were confirmed with multiple reaction monitoring MS assays. Results A list of 49 differentially abundant candidate proteins was compiled by applying a negative binomial regression model to the spectral counting data (p<0.01). Functional analysis with Ingenuity Pathway Analysis tools showed significant enrichment of inflammatory response proteins, key molecules in cell-cell signaling and interaction network and differential physiological responses for the two common NSCLC subtypes. Conclusions We identified a set of candidate serum biomarkers with statistically significant differential abundance across the lung cancer case/control pools which, when validated, could improve lung cancer early detection. PMID:21304412

  4. Sequences Of Amino Acids For Human Serum Albumin

    NASA Technical Reports Server (NTRS)

    Carter, Daniel C.

    1992-01-01

    Sequences of amino acids defined for use in making polypeptides one-third to one-sixth as large as parent human serum albumin molecule. Smaller, chemically stable peptides have diverse applications including service as artificial human serum and as active components of biosensors and chromatographic matrices. In applications involving production of artificial sera from new sequences, little or no concern about viral contaminants. Smaller genetically engineered polypeptides more easily expressed and produced in large quantities, making commercial isolation and production more feasible and profitable.

  5. Detection and quantification of ATP in human blood serum.

    PubMed

    Akdeniz, Ali; Caglayan, Mehmet Gokhan; Polivina, Irina; Anzenbacher, Pavel

    2016-08-21

    Two fluorometric sensors based on the tri-serine tri-lactone scaffold and thiourea or sulfonamide moieties serving as hydrogen bond donors allowing for anion binding are described. The sensor utilizing thiourea as a recognition moiety shows fluorescence enhancement while the sensor with sulfonamide shows quenching upon addition of phosphates. Sensor arrays composed of two sensors are able to discriminate structurally similar organic phosphates in the presence of interferents in human blood serum. The quantitative analysis of ATP in human blood serum shows high accuracy (the root mean square error of prediction, 1.65%) without requiring any sample pretreatment. PMID:27454442

  6. Binding of temoporfin to the lipoprotein fractions of human serum.

    PubMed Central

    Michael-Titus, A T; Whelpton, R; Yaqub, Z

    1995-01-01

    The binding of a new photosensitizer, temoporfin, to human serum lipoproteins was investigated. [14C]-Temoporfin (0.1-10 micrograms ml-1) was incubated with human serum for 30 min at room temperature or for 20 h at 4 degrees C, prior to stepwise density flotation to separate the lipoprotein fractions. The distribution of the drug was independent of the initial concentration or time and temperature of the incubation. The proportion of temoporfin in each fraction was: very low density lipoprotein 6%, low density lipoprotein 22%, lipoprotein(a) 17%, high density lipoprotein 39% and lipoprotein deficient serum 16%. Autoradiography of agarose gels showed that the drug was associated with the lipoprotein in the fractions. Fractionation of plasma samples collected from a patient after an intravenous infusion of temoporfin revealed a binding profile similar to that obtained in the in vitro study. Images Figure 1 PMID:8703668

  7. Differential Gene Expression in Adipose Stem Cells Cultured in Allogeneic Human Serum Versus Fetal Bovine Serum

    PubMed Central

    Aho, Kaisa-Leena; Kuokkanen, Hannu; Räty, Sari; Huhtala, Heini; Lemponen, Riina; Yli-Harja, Olli; Suuronen, Riitta; Miettinen, Susanna

    2010-01-01

    In preclinical studies, human adipose stem cells (ASCs) have been shown to have therapeutic applicability, but standard expansion methods for clinical applications remain yet to be established. ASCs are typically expanded in the medium containing fetal bovine serum (FBS). However, sera and other animal-derived culture reagents stage safety issues in clinical therapy, including possible infections and severe immune reactions. By expanding ASCs in the medium containing human serum (HS), the problem can be eliminated. To define how allogeneic HS (alloHS) performs in ASC expansion compared to FBS, a comparative in vitro study in both serum supplements was performed. The choice of serum had a significant effect on ASCs. First, to reach cell proliferation levels comparable with 10% FBS, at least 15% alloHS was required. Second, while genes of the cell cycle pathway were overexpressed in alloHS, genes of the bone morphogenetic protein receptor–mediated signaling on the transforming growth factor beta signaling pathway regulating, for example, osteoblast differentiation, were overexpressed in FBS. The result was further supported by differentiation analysis, where early osteogenic differentiation was significantly enhanced in FBS. The data presented here underscore the importance of thorough investigation of ASCs for utilization in cell therapies. This study is a step forward in the understanding of these potential cells. PMID:20184435

  8. Agglutinating serum for distinguishing Staphylococcus aureus of human biotype.

    PubMed

    Live, I

    1975-08-01

    Antiserum to Staphylococcus aureus strain 17 was treated with S. aureus strain 61218 until the antibodies against thermostable agglutinogen were removed. The absorbed serum agglutinated phage-typable as well as phageuntypable staphylococci of human biotype, whether recovered from people or from dogs. PMID:125241

  9. [Determination of human serum galactosyltransferase using a kinetic spectrophotometric technic].

    PubMed

    Gauduchon, P; Baumann, J J; Bar, E; Le Talaër, J Y

    1985-01-01

    A kinetic spectrophotometric method in which galactose transfer is coupled to the production of NADH, has been adapted to the assay of galactosyltransferase activity in human serum. Under the described conditions, the rate of NADH production is linear with regard to enzyme concentration, and directly depends upon the various biochemical factors which control galactosyltransferase activity. PMID:3924359

  10. Improved protein labeling by stannous tartrate reduction of pertechnetate

    SciTech Connect

    Pettit, W.A.; DeLand, F.H.; Bennett, S.J.; Goldenberg, D.M.

    1980-01-01

    A procedure has been developed whereby small amounts of protein - specifically human serum albumin and immunoglobulin G - can be labeled with Tc-99m. Artifactual problems associated with electrolytic and stannous chloride labeling procedures are virtually eliminated. The procedure is satisfactory for labeling human serum albumin, normal goat immunoglobulin G, and goat anti-carcinoembryonic antigen immunoglobulin G.

  11. Isolation of Small Noncoding RNAs from Human Serum

    PubMed Central

    Khoury, Samantha; Ajuyah, Pamela; Tran, Nham

    2014-01-01

    The analysis of RNA and its expression is a common feature in many laboratories. Of significance is the emergence of small RNAs like microRNAs, which are found in mammalian cells. These small RNAs are potent gene regulators controlling vital pathways such as growth, development and death and much interest has been directed at their expression in bodily fluids. This is due to their dysregulation in human diseases such as cancer and their potential application as serum biomarkers. However, the analysis of miRNA expression in serum may be problematic. In most cases the amount of serum is limiting and serum contains low amounts of total RNA, of which small RNAs only constitute 0.4-0.5%1. Thus the isolation of sufficient amounts of quality RNA from serum is a major challenge to researchers today. In this technical paper, we demonstrate a method which uses only 400 µl of human serum to obtain sufficient RNA for either DNA arrays or qPCR analysis. The advantages of this method are its simplicity and ability to yield high quality RNA. It requires no specialized columns for purification of small RNAs and utilizes general reagents and hardware found in common laboratories. Our method utilizes a Phase Lock Gel to eliminate phenol contamination while at the same time yielding high quality RNA. We also introduce an additional step to further remove all contaminants during the isolation step. This protocol is very effective in isolating yields of total RNA of up to 100 ng/µl from serum but can also be adapted for other biological tissues. PMID:24998448

  12. Human erythrocytes inhibit complement-mediated solubilization of immune complexes by human serum

    SciTech Connect

    Dorval, B.L.

    1987-01-01

    The aim of this study was to develop an autologus human system to evaluate the effects of human erythrocytes on solubilization of immune complex precipitates (IC) by human serum. Incubation of IC with fresh human serum or guinea pig serum resulted in solubilization of IC. When packed erythrocytes were added to human serum or guinea pig serum binding of IC to the erythrocyte occurred and IC solubilization was inhibited significantly (p <.025). Sheep erythrocytes did not bind IC or inhibit IC solubilization. To evaluate the role of human erythrocyte complement receptor (CR1) on these findings, human erythrocytes were treated with trypsin or anti-CR1 antibodies. Both treatments abrogated IC binding to human erythrocytes but did not affect the ability of the human erythrocyte to inhibit IC solubilization. Radioimmunoassay was used to measure C3, C4 and C5 activation in human serum after incubation with IC, human erythrocytes, human erythrocytes plus IC, whole blood or in whole blood plus IC.

  13. Indirect electrochemical detection for total bile acids in human serum.

    PubMed

    Zhang, Xiaoqing; Zhu, Mingsong; Xu, Biao; Cui, Yue; Tian, Gang; Shi, Zhenghu; Ding, Min

    2016-11-15

    Bile acids level in serum is a useful index for screening and diagnosis of hepatobiliary diseases. As bile acids concentration is closely related to the degree of hepatobiliary diseases, detecting it is a vital factor to understand the stage of the diseases. The prevalent determination for bile acids is the enzymatic cycling method which has low sensitivity while reagent-consuming. It is desirable to develop a new method with lower cost and higher sensitivity. An indirect electrochemical detection (IED) for bile acids in human serum was established using the screen printed carbon electrode (SPCE). Since bile acids do not show electrochemical signals, they were converted to 3-ketosteroids by 3-α-hydroxysteroid dehydrogenase (3α-HSD) in the presence of nicotinamide adenine dinucleotide (NAD(+)), which was reduced to NADH. NADH could then be oxidized on the surface of SPCE, generating a signal that was used to calculate the total bile acids (TBA) concentration. A good linear calibration for TBA was obtained at the concentration range from 5.00μM to 400μM in human serum. Both the precisions and recoveries were sufficient to be used in a clinical setting. The TBA concentrations in 35 human serum samples by our IED method didn't show significant difference with the result by enzymatic cycling method, using the paired t-test. Moreover, our IED method is reagent-saving, sensitive and cost-effective. PMID:27236139

  14. A high-capacity hydrophobic adsorbent for human serum albumin.

    PubMed

    Belew, M; Peterson, E A; Porath, J

    1985-12-01

    A simple method, based on salting out hydrophobic interaction chromatography, for the efficient removal of trace amounts of serum albumin from partially purified protein preparations is described. The method is also successfully applied for the purification of albumin from Cohn fraction IV, a by-product obtained from the commercial fractionation of human serum proteins by the ethanol precipitation procedure. About 70% of the adsorbed albumin can be eluted by buffer of low ionic strength and can thus be lyophilized directly, if required. The adsorbent can be used for several cycles of adsorption and desorption without affecting its selectivity or capacity. Its adsorption properties and capacity for serum albumin are compared with those of the commercially available adsorbent Blue Sepharose CL-6B. PMID:3879424

  15. Proliferation and responsiveness to estrogen of human endometrial cancer cells under serum-free culture conditions.

    PubMed

    Holinka, C F; Anzai, Y; Hata, H; Kimmel, N; Kuramoto, H; Gurpide, E

    1989-06-15

    Studies of hormonal growth regulation in cultured human endometrial cancer cells are limited by the requirement of exogenous growth factors, usually supplied by addition of serum. The present report provides evidence that estradiol can stimulate proliferation of endometrial cancer cells of the Ishikawa line in the absence of serum or added growth factors. Mitogenic effects of estrogen were demonstrated in two different experimental systems, in cells attached to the substratum of mammalian tissue culture dishes, and in cells forming colonies in soft agar under anchorage-independent conditions. Addition of estradiol to a mixture of serum-free, phenol red-free Dulbecco's minimal essential medium and Ham's F-12 medium, supplemented with L-glutamine and 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid [basal medium: (BM)] significantly increased the proliferation of cells attached to culture dishes. Dose-response experiments revealed maximal estradiol stimulation at 10 nM; significant responses were also observed at 1 nM and at 100 nM concentrations. The mitogenic effect of 10 nM estradiol was comparable to that of 1% charcoal-treated fetal bovine serum and the two effects were additive. The presence of estradiol in serum-free BM resulted in a shortening of the doubling time of exponentially proliferating cells from 38 to 29 h. From the labeling index, measured after exposure to a pulse of [3H]thymidine, and from the mitotic index, both determined in exponentially proliferating cells, the lengths of the S and M phases were calculated to be 11 and 1 h, respectively. From these data it was estimated that estradiol shortened the G1 phase by approximately 40%, from 22 to 13 h. Estradiol doubled the colony formation efficiency of cells plated in BM containing 0.3% agar in the absence of serum as well as in the presence of 1% charcoal-treated fetal bovine serum. The stimulation of colony formation by estradiol was influenced by medium components, since no effects were

  16. A study on human serum albumin influence on glycation of fibrinogen

    SciTech Connect

    Kielmas, Martyna; Szewczuk, Zbigniew; Stefanowicz, Piotr

    2013-09-13

    Highlights: •The glycation of fibrinogen was investigated by isotopic labeling method. •The potential glycation sites in fibrinogen were identified. •Human serum albumin (HSA) inhibits the glycation of fibrinogen. •The effect of HSA on fibrinogen glycation is sequence-dependent. -- Abstract: Although in vivo glycation proceeds in complex mixture of proteins, previous studies did not take in consideration the influence of protein–protein interaction on Maillard reaction. The aim of our study was to test the influence of human serum albumin (HSA) on glycation of fibrinogen. The isotopic labeling using [{sup 13}C{sub 6}] glucose combined with LC-MS were applied as tool for identification possible glycation sites in fibrinogen and for evaluation the effect of HSA on the glycation level of selected amino acids in fibrinogen. The obtained data indicate that the addition of HSA protects the fibrinogen from glycation. The level of glycation in presence of HSA is reduced by 30–60% and depends on the location of glycated residue in sequence of protein.

  17. Binding of dapsone and its analogues to human serum albumin.

    PubMed

    Karp, W B; Subramanyam, S B; Robertson, A F

    1985-06-01

    The binding of dapsone, 4,4'-sulfonylbis(aniline)(1), and its diacetylated derivative, 4,4"'-sulfonylbis(acetanilide)(2), to human serum albumin is reported. To assess the ability of these compounds to displace 4'-[(4-aminophenyl)sulfonyl]acetanilide (3) from albumin, a dialysis rate technique was used. Competition for the bilirubin binding site on albumin was measured with the peroxidase assay. Compounds 1 and 2 strongly displaced both 3 and bilirubin from human serum albumin. The association constants for 1 and 2 with respect to bilirubin binding were 1.29 X 10(3) and 1.15 X 10(4) M-1, respectively. These results suggest that the binding site for 3 and the bilirubin binding site are similar with respect to 1 and 2 and that the binding of dapsone and its derivatives probably does not involve the amino function. PMID:4020658

  18. Human Serum Versus Human Serum Albumin Supplementation in Human Islet Pretransplantation Culture: In Vitro and In Vivo Assessment.

    PubMed

    Nacher, Montserrat; Estil Les, Elisabet; Garcia, Ainhoa; Nadal, Belen; Pairó, Mar; Garcia, Cristofer; Secanella, Lluís; Novials, Anna; Montanya, Eduard

    2016-01-01

    There is conflicting evidence favoring both the use of human serum (HS) and of human serum albumin (HSA) in human islet culture. We evaluated the effects of HS versus HSA supplementation on 1) in vitro β-cell viability and function and 2) in vivo islet graft revascularization, islet viability, β-cell death, and metabolic outcome after transplantation. Islets isolated from 14 cadaveric organ donors were cultured for 3 days in CMRL 1066 medium supplemented with HS or HSA. After 3 days in culture, β-cell apoptosis was lower in HS group (1.41 ± 0.27 vs. 2.38 ± 0.39%, p = 0.029), and the recovery of islets was 77 ± 11% and 54 ± 1% in HS- and HSA-cultured groups, respectively. Glucose-stimulated insulin secretion (GSIS) was higher in HS group (29.4, range 10.4-99.9, vs. 22.3, range 8.7-70.6, p = 0.031). In vivo viability and revascularization was determined in HS- and HSA-cultured islets transplanted into the anterior chamber of the eye of Balb/c mice (n = 14), and β-cell apoptosis in paraffin-embedded mouse eyes. Islet viability and β-cell apoptosis were similar in both groups. Revascularization was observed in one graft (HS group) on day 10 after transplantation. Islet function was determined in streptozotocin (STZ)-diabetic nude mice (n = 33) transplanted with 2,000 IEQs cultured with HS or HSA that showed similar blood glucose levels and percentage of normoglycemic animals over time. In conclusion, human islets cultured in medium supplemented with HS showed higher survival in vitro, as well as islet viability and function. The higher in vitro survival increased the number of islets available for transplantation. However, the beneficial effect on viability and function did not translate into an improved metabolic evolution when a similar number of HSA- and HS-cultured islets was transplanted. PMID:25955150

  19. Mechanism of Trypanosoma brucei gambiense resistance to human serum.

    PubMed

    Uzureau, Pierrick; Uzureau, Sophie; Lecordier, Laurence; Fontaine, Frédéric; Tebabi, Patricia; Homblé, Fabrice; Grélard, Axelle; Zhendre, Vanessa; Nolan, Derek P; Lins, Laurence; Crowet, Jean-Marc; Pays, Annette; Felu, Cécile; Poelvoorde, Philippe; Vanhollebeke, Benoit; Moestrup, Soren K; Lyngsø, Jeppe; Pedersen, Jan Skov; Mottram, Jeremy C; Dufourc, Erick J; Pérez-Morga, David; Pays, Etienne

    2013-09-19

    The African parasite Trypanosoma brucei gambiense accounts for 97% of human sleeping sickness cases. T. b. gambiense resists the specific human innate immunity acting against several other tsetse-fly-transmitted trypanosome species such as T. b. brucei, the causative agent of nagana disease in cattle. Human immunity to some African trypanosomes is due to two serum complexes designated trypanolytic factors (TLF-1 and -2), which both contain haptoglobin-related protein (HPR) and apolipoprotein LI (APOL1). Whereas HPR association with haemoglobin (Hb) allows TLF-1 binding and uptake via the trypanosome receptor TbHpHbR (ref. 5), TLF-2 enters trypanosomes independently of TbHpHbR (refs 4, 5). APOL1 kills trypanosomes after insertion into endosomal/lysosomal membranes. Here we report that T. b. gambiense resists TLFs via a hydrophobic β-sheet of the T. b. gambiense-specific glycoprotein (TgsGP), which prevents APOL1 toxicity and induces stiffening of membranes upon interaction with lipids. Two additional features contribute to resistance to TLFs: reduction of sensitivity to APOL1 requiring cysteine protease activity, and TbHpHbR inactivation due to a L210S substitution. According to such a multifactorial defence mechanism, transgenic expression of T. b. brucei TbHpHbR in T. b. gambiense did not cause parasite lysis in normal human serum. However, these transgenic parasites were killed in hypohaptoglobinaemic serum, after high TLF-1 uptake in the absence of haptoglobin (Hp) that competes for Hb and receptor binding. TbHpHbR inactivation preventing high APOL1 loading in hypohaptoglobinaemic serum may have evolved because of the overlapping endemic area of T. b. gambiense infection and malaria, the main cause of haemolysis-induced hypohaptoglobinaemia in western and central Africa. PMID:23965626

  20. Hyaluronan-binding properties of human serum hemopexin.

    PubMed

    Hrkal, Z; Kuzelová, K; Muller-Eberhard, U; Stern, R

    1996-03-25

    Hemopexin, the heme-binding serum glycoprotein, exhibits a complex electrophoretic pattern on two-dimensional immunoelectrophoresis on agarose gels into which hyaluronic acid is incorporated in the first and monospecific anti-hemopexin in the second dimension. This heterogeneity reflects a range of interactions of hemopexin isoforms with hyaluronic acid. Electrophoretic patterns of individual human sera greatly differ in their contents of hyaluronan-interacting hemopexin species. Hemopexin itself has no hyaluronidase activity. PMID:8612795

  1. Immunoproteomic Analysis of Potential Serum Biomarker Candidates in Human Glaucoma

    PubMed Central

    Tezel, Gülgün; Thornton, Ivey L.; Tong, Melissa G.; Luo, Cheng; Yang, Xiangjun; Cai, Jian; Powell, David W.; Soltau, Joern B.; Liebmann, Jeffrey M.; Ritch, Robert

    2012-01-01

    Purpose. Evidence supporting the immune system involvement in glaucoma includes increased titers of serum antibodies to retina and optic nerve proteins, although their pathogenic importance remains unclear. This study using an antibody-based proteomics approach aimed to identify disease-related antigens as candidate biomarkers of glaucoma. Methods. Serum samples were collected from 111 patients with primary open-angle glaucoma and an age-matched control group of 49 healthy subjects without glaucoma. For high-throughput characterization of antigens, serum IgG was eluted from five randomly selected glaucomatous samples and analyzed by linear ion trap mass spectrometry (LC-MS/MS). Serum titers of selected biomarker candidates were then measured by specific ELISAs in the whole sample pool (including an additional control group of diabetic retinopathy). Results. LC-MS/MS analysis of IgG elutes revealed a complex panel of proteins, including those detectable only in glaucomatous samples. Interestingly, many of these antigens corresponded to upregulated retinal proteins previously identified in glaucomatous donors (or that exhibited increased methionine oxidation). Moreover, additional analysis detected a greater immunoreactivity of the patient sera to glaucomatous retinal proteins (or to oxidatively stressed cell culture proteins), thereby suggesting the importance of disease-related protein modifications in autoantibody production/reactivity. As a narrowing-down strategy for selection of initial biomarker candidates, we determined the serum proteins overlapping with the retinal proteins known to be up-regulated in glaucoma. Four of the selected 10 candidates (AIF, cyclic AMP-responsive element binding protein, ephrin type-A receptor, and huntingtin) exhibited higher ELISA titers in the glaucomatous sera. Conclusions. A number of serum proteins identified by this immunoproteomic study of human glaucoma may represent diseased tissue-related antigens and serve as candidate

  2. A glycoprotein secreted by lung cancer cells is present in human serum as an immunoglobulin-binding protein.

    PubMed

    Nonaka, N; Kobayashi, K; Hirai, H

    1994-01-01

    The 6B3-Ag recognized by a monoclonal antibody 6B3 to human large cell lung carcinoma cell line (HLC-2) is a high-molecular-weight glycoprotein of 1,000,000. Its serum level is increased in various adenocarcinoma patients. When a patient's serum with a high concentration of 6B3.Ag (54 micrograms/ml) or concentrated 6B3.Ag from normal human serum was analyzed by immunoelectrophoresis, 6B3.Ag showed a long bimodal precipitin line extending from the per-beta to beta globulin region. However, the precipitin line of 6B3.Ag in the HLC-2 culture medium was formed only in the pre-beta globulin region. The 6B3.Ag was purified from pooled patients' serum by salting out, precipitation by acidification at pH 4.5 and Sepharose 4B and immunoaffinity chromatographies. Western blotting indicated that the 6B3.Ag from human serum contained IgG and/or IgM. The 6B3.Ag from human serum showed a dose-dependent reaction in a sandwich enzyme-linked immunosorbent assay with anti-6B3.Ag antibody as a solid-phase antibody and anti-human IgG or anti-human IgM antibody labeled with alkaline phosphatase. The 6B3.Ag was concluded to be partly present as a complex with IgG and/or IgM in human serum, and this complex showed a precipitin line in the beta globulin region on immunoelectrophoresis. PMID:7508905

  3. 21 CFR 866.5700 - Whole human plasma or serum immunological test system.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Whole human plasma or serum immunological test... Systems § 866.5700 Whole human plasma or serum immunological test system. (a) Identification. A whole human plasma or serum immunological test system is a device that consists of reagents used to measure...

  4. 21 CFR 866.5700 - Whole human plasma or serum immunological test system.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 21 Food and Drugs 8 2011-04-01 2011-04-01 false Whole human plasma or serum immunological test... Systems § 866.5700 Whole human plasma or serum immunological test system. (a) Identification. A whole human plasma or serum immunological test system is a device that consists of reagents used to measure...

  5. 21 CFR 866.5700 - Whole human plasma or serum immunological test system.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 21 Food and Drugs 8 2012-04-01 2012-04-01 false Whole human plasma or serum immunological test... Systems § 866.5700 Whole human plasma or serum immunological test system. (a) Identification. A whole human plasma or serum immunological test system is a device that consists of reagents used to measure...

  6. 21 CFR 866.5700 - Whole human plasma or serum immunological test system.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 21 Food and Drugs 8 2013-04-01 2013-04-01 false Whole human plasma or serum immunological test... Systems § 866.5700 Whole human plasma or serum immunological test system. (a) Identification. A whole human plasma or serum immunological test system is a device that consists of reagents used to measure...

  7. 21 CFR 866.5700 - Whole human plasma or serum immunological test system.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 21 Food and Drugs 8 2014-04-01 2014-04-01 false Whole human plasma or serum immunological test... Systems § 866.5700 Whole human plasma or serum immunological test system. (a) Identification. A whole human plasma or serum immunological test system is a device that consists of reagents used to measure...

  8. Lowering of serum cholesterol in hypercholesterolemic humans by tocotrienols (palmvitee).

    PubMed

    Qureshi, A A; Qureshi, N; Wright, J J; Shen, Z; Kramer, G; Gapor, A; Chong, Y H; DeWitt, G; Ong, A; Peterson, D M

    1991-04-01

    A double-blind, crossover, 8-wk study was conducted to compare effects of the tocotrienol-enriched fraction of palm oil (200 mg palmvitee capsules/day) with those of 300 mg corn oil/d on serum lipids of hypercholesterolemic human subjects (serum cholesterol 6.21-8.02 mmol/L). Concentrations of serum total cholesterol (-15%), LDL cholesterol (-8%), Apo B (-10%), thromboxane (-25%), platelet factor 4 (-16%), and glucose (-12%) decreased significantly only in the 15 subjects given palmvitee during the initial 4 wk. The crossover confirmed these actions of palmvitee. There was a carry over effect of palmvitee. Serum cholesterol concentrations of seven hypercholesterolemic subjects (greater than 7.84 mmol/L) decreased 31% during a 4-wk period in which they were given 200 mg gamma-tocotrienol/d. This indicates that gamma-tocotrienol may be the most potent cholesterol inhibitor in palmvitee capsules. The results of this pilot study are very encouraging. PMID:2012010

  9. Evaluation of human platelet lysate versus fetal bovine serum for culture of mesenchymal stromal cells.

    PubMed

    Hemeda, Hatim; Giebel, Bernd; Wagner, Wolfgang

    2014-02-01

    Culture media for therapeutic cell preparations-such as mesenchymal stromal cells (MSCs)-usually comprise serum additives. Traditionally, fetal bovine serum is supplemented in basic research and in most clinical trials. Within the past years, many laboratories adapted their culture conditions to human platelet lysate (hPL), which further stimulates proliferation and expansion of MSCs. Particularly with regard to clinical application, human alternatives for fetal bovine serum are clearly to be preferred. hPL is generated from human platelet units by disruption of the platelet membrane, which is commonly performed by repeated freeze and thaw cycles. Such culture supplements are notoriously ill-defined, and many parameters contribute to batch-to-batch variation in hPL such as different amounts of plasma, a broad range of growth factors and donor-specific effects. The plasma components of hPL necessitate addition of anticoagulants such as heparins to prevent gelatinization of hPL medium, and their concentration must be standardized. Labels for description of hPL-such as "xenogen-free," "animal-free" and "serum free"-are not used consistently in the literature and may be misleading if not critically assessed. Further analysis of the precise composition of relevant growth factors, attachment factors, microRNAs and exosomes will pave the way for optimized and defined culture conditions. The use of hPL has several advantages and disadvantages: they must be taken into account because the choice of cell culture additive has major impact on cell preparations. PMID:24438898

  10. The significance of using pooled human serum in human articular cartilage tissue engineering.

    PubMed

    Azmi, B; Aminuddin, B S; Sharaf, I; Samsudin, O C; Munirah, S; Chua, K H; Ruszymah, B H I

    2004-05-01

    Animal serum is commonly used in chondrocytes culture expansion to promote cell proliferation and shorten the time lag before new tissue reconstruction is possible. However, animal serum is not suitable for regeneration of clinical tissue because it has potential risk of viral and prion related disease transmission particularly mad cow disease and foreign protein contamination that can stimulate immune reaction leading to graft rejection. In this context, human serum as homologous supplement has a greater potential as growth promoting agents for human chondrocytes culture. PMID:15468795

  11. A Homogeneous Fluorescent Sensor for Human Serum Albumin

    PubMed Central

    Wang, Rongsheng E.; Tian, Ling; Chang, Yie-Hwa

    2012-01-01

    Human serum albumin is the most abundant protein in the body and is an important biomarker used for disease-related diagnosis. Although the traditional enzyme-linked immunosorbent assay (ELISA) approach can precisely measure the concentration of human serum albumin, the multi-step procedure and time-consuming preparations of ELISA limit its diagnostic applications, preventing accurate point-of-care testing, for example. Herein, we report the recent development of an antibody-based albumin sensor that allows for a homogeneous measurement of albumin concentrations in saliva, urine and serum, in which this type of sensor is validated for the first time. The assay only requires simple mixing, and relies on time-resolved (TR) fluorescence resonance energy transfer (FRET) to produce robust, sensitive signals. The whole process, from sample preparation to final read-out, is expected to take less than one hour and requires only a standard plate-reader, thus making the sensor a convenient and cost-effective tool for albumin analysis. PMID:22326845

  12. Fructosylation generates neo-epitopes on human serum albumin.

    PubMed

    Allarakha, Shaziya; Ahmad, Parvez; Ishtikhar, Mohd; Zaheer, Mohammad Shoaib; Siddiqi, Sheelu Shafiq; Moinuddin; Ali, Asif

    2015-05-01

    Hyperglycemia is the defining feature of diabetes mellitus. The persistently high levels of reducing sugars like glucose and fructose cause glycation of various macromolecules in the body. Human serum albumin (HSA), the most abundant serum protein with a myriad of functions, is prone to glycation and consequent alteration in its structural and biological properties. This study aimed to assess the role of fructose-modified human serum albumin as a marker of diabetic pathophysiology. We carried out modification of HSA with fructose and the changes induced were studied by various physicochemical studies. Fructose modified-HSA showed hyperchromicity in UV spectrum and increased AGE-specific fluorescence as well as quenching of tryptophan fluorescence. In SDS-PAGE protein aggregation was seen. Amadori products were detected by NBT. The fructose modified HSA had higher content of carbonyls along with perturbations in secondary structure as revealed by CD and FT-IR. A greater hydrodynamic radius of fructose-modified HSA was evident by DLS measurement. The fructose-modified HSA induced high titre antibodies in experimental animals exhibiting high specificity towards the immunogen. PMID:25914162

  13. Human platelet vasopressin receptor identification by direct ultraviolet photoaffinity labeling

    SciTech Connect

    Thibonnier, M.

    1987-08-15

    Tritiated vasopressin ((/sup 3/H)AVP) was directly crosslinked to its human platelet receptor by using an ultraviolet irradiation procedure. After preincubation with (/sup 3/H)AVP, the hydrodynamic parameters of the hormone-receptor complexes solubilized with 3-((3-cholamidopropyl)dimethylammonio)-1-propane sulfonate were derived from Sephacryl S-300 superfine gel filtration and from sucrose density gradient ultracentrifugation experiments. The following values were obtained: Stoke's radius = 5.48 +/- 0.1 nm, apparent sedimentation coefficient = 5.55 +/- 0.1 S, and calculated molecular weight = 132,000. On sodium dodecyl sulfate-8% polyacrylamide slab gel electrophoresis under reducing conditions, (/sup 3/H)AVP preferentially and specifically labeled a 125,000-dalton protein. The labeling of this protein was suppressed by addition of excess cold vasopressin, whereas angiotensin II did not inhibit incorporation of tritiated vasopressin in this protein. These results suggest that direct UV-photoaffinity labelling with (/sup 3/H)AVP is a suitable tool for the purification of the human platelet vasopressin receptor.

  14. Human gut microbes impact host serum metabolome and insulin sensitivity.

    PubMed

    Pedersen, Helle Krogh; Gudmundsdottir, Valborg; Nielsen, Henrik Bjørn; Hyotylainen, Tuulia; Nielsen, Trine; Jensen, Benjamin A H; Forslund, Kristoffer; Hildebrand, Falk; Prifti, Edi; Falony, Gwen; Le Chatelier, Emmanuelle; Levenez, Florence; Doré, Joel; Mattila, Ismo; Plichta, Damian R; Pöhö, Päivi; Hellgren, Lars I; Arumugam, Manimozhiyan; Sunagawa, Shinichi; Vieira-Silva, Sara; Jørgensen, Torben; Holm, Jacob Bak; Trošt, Kajetan; Kristiansen, Karsten; Brix, Susanne; Raes, Jeroen; Wang, Jun; Hansen, Torben; Bork, Peer; Brunak, Søren; Oresic, Matej; Ehrlich, S Dusko; Pedersen, Oluf

    2016-07-21

    Insulin resistance is a forerunner state of ischaemic cardiovascular disease and type 2 diabetes. Here we show how the human gut microbiome impacts the serum metabolome and associates with insulin resistance in 277 non-diabetic Danish individuals. The serum metabolome of insulin-resistant individuals is characterized by increased levels of branched-chain amino acids (BCAAs), which correlate with a gut microbiome that has an enriched biosynthetic potential for BCAAs and is deprived of genes encoding bacterial inward transporters for these amino acids. Prevotella copri and Bacteroides vulgatus are identified as the main species driving the association between biosynthesis of BCAAs and insulin resistance, and in mice we demonstrate that P. copri can induce insulin resistance, aggravate glucose intolerance and augment circulating levels of BCAAs. Our findings suggest that microbial targets may have the potential to diminish insulin resistance and reduce the incidence of common metabolic and cardiovascular disorders. PMID:27409811

  15. Heparin-binding properties of human serum spreading factor.

    PubMed

    Barnes, D W; Reing, J E; Amos, B

    1985-08-01

    Human serum spreading factor (SF) is a blood glycoprotein that promotes attachment and spreading and influences growth, migration, and differentiation of a variety of animal cells in culture. SF purified from human plasma or serum by chromatographic methods reported previously (Barnes, D. W., and Silnutzer, J. (1983) J. Biol. Chem. 258, 12548-12552) does not bind to heparin-Sepharose under conditions of physiological ionic strength and pH. In a further examination of the heparin-binding properties of human serum SF, we found that exposure of purified SF to 8 M urea altered several properties of the protein, including heparin affinity, and these alterations remained after removal of the urea from SF solutions. Urea-treated SF bound to heparin under physiological conditions, and salt concentrations of 0.4 M or higher were required for elution of urea-treated SF from heparin-Sepharose at pH 7.0. The alteration of heparin-binding properties of SF also was observed upon exposure of the protein to heat or acid. Treatment of SF with urea, heat, or acid resulted additionally in greatly decreased cell spreading-promoting activity of the molecule. The decreased biological activity was associated with a reduced ability of the treated SF to bind to the cell culture substratum, a prerequisite for the attachment-promoting activity of the molecule. Experiments examining the heparin-binding properties of native SF in unfractionated human plasma indicated that the major portion of SF in blood did not bind to heparin under conditions of physiological ionic strength and pH. PMID:2410408

  16. Label-free quantitative proteomic analysis of benzo(a)pyrene-transformed 16HBE cells serum-free culture supernatant and xenografted nude mice sera.

    PubMed

    Zhao, Peng; Fu, Juanling; Yao, Biyun; Jia, Yongrui; Zhang, Hongtao; Li, Xuehui; Dong, Lisha; Gao, Ya; Liu, Wenli; Chen, Wen; Zhou, Zongcan

    2016-02-01

    To screen potential biomarkers of benzo(a)pyrene (BaP)-induced lung cancer, the proteomic profiles of BaP-transformed 16HBE cell line T-16HBE-C1 cells serum-free culture supernatant and xenografted nude mice sera were compared with those of 16HBE group by utilizing label-free quantitative proteomic strategy. By employing nano-LC-MS/MS technology followed by MaxQuant and Perseus processing, 489 differentially expressed proteins were identified between T-16HBE-C1 and 16HBE cells serum-free culture supernatant, and 49 significantly up-regulated proteins were identified in T-16HBE-C1 xenografted nude mice sera. Three proteins neuropilin-2 (NRP2), clusterin (CLU) and A-kinase anchor protein 12 (AKAP12) were up-regulated in the serum-free culture supernatant of T-16HBE-C1 cells. These 3 human proteins were present in the sera of nude mice xenografted with T-16HBE-C1 cells, but were undetectable in mice xenografted with 16HBE cells. The proteomic results of NRP2 and AKAP12 were confirmed by Western blotting and enzyme-linked immunosorbent assays, respectively. Moreover, the serum NRP2 levels were significantly elevated at the 4th day after tumor cell implantation and showed good positive correlation with tumor growth characterized by tumor volume. In conclusion, serum NRP2, CLU and AKAP12 could be potential biomarkers of BaP-induced lung cancer. The proteomic results will gain deeper insights into the mechanisms of BaP-induced carcinogenesis. PMID:26748308

  17. The Binding Constant of Estradiol to Bovine Serum Albumin: An Upper-Level Experiment Utilizing Tritium-Labeled Estradiol and Liquid Scintillation Counting

    ERIC Educational Resources Information Center

    Peihong Liang; Adhyaru, Bhavin; Pearson, Wright L.; Williams, Kathryn R.

    2006-01-01

    The experiment used [to the third power]H-labeled estradiol to determine the binding constant of estradiol to bovine serum albumin. Estradiol must complex with serum proteins for the transport in the blood stream because of its low solubility in aqueous systems and estradiol-protein binding constant, where K[subscript B] is important to understand…

  18. Performance of human mass balance studies with stable isotope-labeled drug and continuous flow-isotope ratio mass spectrometry: a progress report.

    PubMed

    Browne, T R; Szabo, G K; Ajami, A; Browne, D G

    1998-04-01

    We propose performing human mass balance studies by administering stable isotope labeled (13C or 15N) drug and quantitating excess (above background) 13C or 15N in urine, serum, and feces by continuous flow-isotope ratio mass spectrometry (CF-IRMS). Theoretical calculations and empirical data (dynamic range, linearity, sensitivity, precision, accuracy) are presented to establish that commercially available CF-IRMS instruments can quantitate stable isotope labeled (one or two 15N or 13C labels) drug concentrations of 1.0 microg/mL or greater in urine, serum (15N), or feces. More than two 13C labels may be necessary to quantitate 1.0 microg/mL of drug in serum. Three volunteers received 650 mg of 15N13C2-acetaminophen, and urine was collected for 72 hours. Percent of administered label recovered in urine from the three subjects was 97.4, 78.9, and 95.4 for 13C and 90.3, 77.0, and 90.6 for 15N. Fecal recovery of label for one subject was 0.9% (13C2) and 1.1% (15N). Serum pharmacokinetic values obtained by counting 13C or 15N in one subject were as expected for acetaminophen. This method appears to be promising, and further validation is ongoing. PMID:9590457

  19. Comparative Analysis of Human Growth Hormone in Serum Using SPRi, Nano-SPRi and ELISA Assays

    PubMed Central

    Henrich, Vincent C.; Sandros, Marinella G.

    2016-01-01

    Sensitive and selective methods for the detection of human growth hormone (hGH) over a wide range of concentrations (high levels of 50-100 ng ml−1 and minimum levels of 0.03 ng ml−1) in circulating blood are essential as variable levels may indicate altered physiology. For example, growth disorders occurring in childhood can be diagnosed by measuring levels of hGH in blood. Also, the misuse of recombinant hGH in sports not only poses an ethical issue it also presents serious health threats to the abuser. One popular strategy for measuring hGH misuse, relies on the detection of the ratio of 22 kDa hGH to total hGH, as non-22 kDa endogenous levels drop after exogenous recombinant hGH (rhGH) administration.Surface plasmon resonance imaging (SPRi) is an analytical tool that allows direct (label-free) monitoring and visualization of biomolecular interactions by recording changes of the refractive index adjacent to the sensor surface in real time. In contrast, the most frequently used colorimetric method, enzyme-linked immunosorbent assay (ELISA) uses enzyme labeled detection antibodies to indirectly measure analyte concentration after the addition of a substrate that induces a color change. To increase detection sensitivity, amplified SPRi uses a sandwich assay format and near infrared quantum dots (QDs) to increase signal strength. After direct SPRi detection of recombinant rhGH in spiked human serum, the SPRi signal is amplified by the sequential injection of detection antibody coated with near-infrared QDs (Nano-SPRi). In this study, the diagnostic potential of direct and amplified SPRi was assessed for measuring rhGH spiked in human serum and compared directly with the capabilities of a commercially available ELISA kit. PMID:26780354

  20. Comparative Analysis of Human Growth Hormone in Serum Using SPRi, Nano-SPRi and ELISA Assays.

    PubMed

    Vance, Stephen; Zeidan, Effat; Henrich, Vincent C; Sandros, Marinella G

    2016-01-01

    Sensitive and selective methods for the detection of human growth hormone (hGH) over a wide range of concentrations (high levels of 50-100 ng ml(-) (1) and minimum levels of 0.03 ng ml(-) (1)) in circulating blood are essential as variable levels may indicate altered physiology. For example, growth disorders occurring in childhood can be diagnosed by measuring levels of hGH in blood. Also, the misuse of recombinant hGH in sports not only poses an ethical issue it also presents serious health threats to the abuser. One popular strategy for measuring hGH misuse, relies on the detection of the ratio of 22 kDa hGH to total hGH, as non-22 kDa endogenous levels drop after exogenous recombinant hGH (rhGH) administration. Surface plasmon resonance imaging (SPRi) is an analytical tool that allows direct (label-free) monitoring and visualization of biomolecular interactions by recording changes of the refractive index adjacent to the sensor surface in real time. In contrast, the most frequently used colorimetric method, enzyme-linked immunosorbent assay (ELISA) uses enzyme labeled detection antibodies to indirectly measure analyte concentration after the addition of a substrate that induces a color change. To increase detection sensitivity, amplified SPRi uses a sandwich assay format and near infrared quantum dots (QDs) to increase signal strength. After direct SPRi detection of recombinant rhGH in spiked human serum, the SPRi signal is amplified by the sequential injection of detection antibody coated with near-infrared QDs (Nano-SPRi). In this study, the diagnostic potential of direct and amplified SPRi was assessed for measuring rhGH spiked in human serum and compared directly with the capabilities of a commercially available ELISA kit. PMID:26780354

  1. 78 FR 57394 - Draft Guidance for Industry on Patient Counseling Information Section of Labeling for Human...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2013-09-18

    ... products. In the Federal Register of January 24, 2006 (71 FR 3922), FDA published a final rule on labeling... Information Section of Labeling for Human Prescription Drug and Biological Products--Content and Format... Counseling Information Section of Labeling for Human Prescription Drug and Biological Products--Content...

  2. Influence of honeybee sting on peptidome profile in human serum.

    PubMed

    Matysiak, Jan; Światły, Agata; Hajduk, Joanna; Matysiak, Joanna; Kokot, Zenon J

    2015-05-01

    The aim of this study was to explore the serum peptide profiles from honeybee stung and non-stung individuals. Two groups of serum samples obtained from 27 beekeepers were included in our study. The first group of samples was collected within 3 h after a bee sting (stung beekeepers), and the samples were collected from the same person a second time after at least six weeks after the last bee sting (non-stung beekeepers). Peptide profile spectra were determined using MALDI-TOF mass spectrometry combined with Omix, ZipTips and magnetic beads based on weak-cation exchange (MB-WCX) enrichment strategies in the mass range of 1-10 kDa. The samples were classified, and discriminative models were established by using the quick classifier, genetic algorithm and supervised neural network algorithms. All of the statistical algorithms used in this study allow distinguishing analyzed groups with high statistical significance, which confirms the influence of honeybee sting on the serum peptidome profile. The results of this study may broaden the understanding of the human organism's response to honeybee venom. Due to the fact that our pilot study was carried out on relatively small datasets, it is necessary to conduct further proteomic research of the response to honeybee sting on a larger group of samples. PMID:26008235

  3. Influence of Honeybee Sting on Peptidome Profile in Human Serum

    PubMed Central

    Matysiak, Jan; Światły, Agata; Hajduk, Joanna; Matysiak, Joanna; Kokot, Zenon J.

    2015-01-01

    The aim of this study was to explore the serum peptide profiles from honeybee stung and non-stung individuals. Two groups of serum samples obtained from 27 beekeepers were included in our study. The first group of samples was collected within 3 h after a bee sting (stung beekeepers), and the samples were collected from the same person a second time after at least six weeks after the last bee sting (non-stung beekeepers). Peptide profile spectra were determined using MALDI-TOF mass spectrometry combined with Omix, ZipTips and magnetic beads based on weak-cation exchange (MB-WCX) enrichment strategies in the mass range of 1–10 kDa. The samples were classified, and discriminative models were established by using the quick classifier, genetic algorithm and supervised neural network algorithms. All of the statistical algorithms used in this study allow distinguishing analyzed groups with high statistical significance, which confirms the influence of honeybee sting on the serum peptidome profile. The results of this study may broaden the understanding of the human organism’s response to honeybee venom. Due to the fact that our pilot study was carried out on relatively small datasets, it is necessary to conduct further proteomic research of the response to honeybee sting on a larger group of samples. PMID:26008235

  4. Evaluation of nanoparticle aggregation in human blood serum.

    PubMed

    Rausch, Kristin; Reuter, Anika; Fischer, Karl; Schmidt, Manfred

    2010-11-01

    In a certain stage of development, the performance of nanoparticle- or polymer-drug conjugates is tested "in vivo", that is, in mice or rats. Besides pharmaceutical and chemical characterization, the structural characterization of such drug carrier systems in terms of size, size distribution, and shape is typically performed in physiological salt solution prior to animal tests. The present work introduces a simple method based on dynamic light scattering to monitor the particle size in blood serum. Utilizing a model system of pegylated poly-l-lysines (PLL-g-PEOx) of various degrees of pegylation, x, it is demonstrated that large aggregates may form in human serum solution that are not observed in isotonic salt solution. Aggregates of a few hundred nanometers in size were found in mixtures of serum solution and PLL-g-PEOx with degrees of pegylation <10%, whereas no aggregates are being observed if the degree of pegylation exceeds 20%. The described method may have the potential to become an easy and routine test for drug carrier systems prior to animal applications. PMID:20961117

  5. Three-dimensional structure of human serum albumin

    NASA Technical Reports Server (NTRS)

    Carter, Daniel C.; He, Xiao-Min; Munson, Sibyl H.; Twigg, Pamela D.; Gernert, Kim M.; Broom, M. Beth; Miller, Teresa Y.

    1989-01-01

    The three-dimensional structure of human serum albumin has been solved at 6.0 A resolution by the method of multiple isomorphous replacement. Crystals were grown from solutions of polyethylene glycol in the infrequently observed space group P42(1)2 and diffracted X-rays to lattice d-spacings of less than 2.9 A. The electron density maps are of high quality and revealed the structure as a predominantly alpha-helical globin protein in which the course of the polypeptide can be traced. The binding loci of several organic compounds have been determined.

  6. Simple and rapid determination of myristicin in human serum.

    PubMed

    Dawidowicz, Andrzej L; Dybowski, Michal P

    2013-01-01

    Myristicin (5-allyl-1-methoxy-2,3-methylenodioxybenzene) is the main component of nutmeg (Myristica fragrans Houtt.) essential oil. The increasing use of myristicin as a cheap hallucinogenic intoxicant, frequently causing fatal cases of myristicin poisoning, requires new methods for determination of this compound in blood. This report describes the rapid, simple, and useful procedure for myristicin analysis in human serum, involving myristicin-protein complex degradation before chromatographic analysis. The developed method is characterized by a high recovery (above 99 %), a low detection limit (6.0 ng/g) and good repeatability (average RDS of 2.01 %). PMID:23440626

  7. Review: modifications of human serum albumin and their binding effect.

    PubMed

    Lee, Philbert; Wu, Xiaoyang

    2015-01-01

    Human serum albumin (HSA) regulates the transport and availability of numerous chemical compounds and molecules in the blood vascular system. While previous HSA research has found that HSA interacts with specific varieties of ligands, new research efforts aim to expand HSA's ability to interact with more different drugs in order to improve the delivery of various pharmacological drugs. This review will cover fatty acid chain and posttranslational modifications of HSA that potentially modulate how HSA interacts with various pharmacological drugs, including glycation, cysteinylation, S-nitrosylation, S-transnitrosation and S-guanylation. PMID:25732553

  8. Review: Modifications of Human Serum Albumin and Their Binding Effect

    PubMed Central

    Lee, Philbert; Wu, Xiaoyang

    2015-01-01

    Human serum albumin (HSA) regulates the transport and availability of numerous chemical compounds and molecules in the blood vascular system. While previous HSA research has found that HSA interacts with specific varieties of ligands, new research efforts aim to expand HSA’s ability to interact with more different drugs in order to improve the delivery of various pharmacological drugs. This review will cover fatty acid chain and post-translational modifications of HSA that potentially modulate how HSA interacts with various pharmacological drugs, including glycation, cysteinylation, S-nitrosylation, S-transnitrosation and S-guanylation. PMID:25732553

  9. Interaction of ergosterol with bovine serum albumin and human serum albumin by spectroscopic analysis.

    PubMed

    Cheng, Zhengjun

    2012-10-01

    This study was designed to examine the interactions of ergosterol with bovine serum albumin (BSA) and human serum albumin (HSA) under physiological conditions with the drug concentrations in the range of 2.99-105.88 μM and the concentration of proteins was fixed at 5.0 μM. The analysis of emission spectra quenching at different temperatures revealed that the quenching mechanism of HSA/BSA by ergosterol was the static quenching. The number of binding sites n and the binding constants K were obtained at various temperatures. The distance r between ergosterol and HSA/BSA was evaluated according to Föster non-radioactive energy transfer theory. The results of synchronous fluorescence, 3D fluorescence, FT-IR, CD and UV-Vis absorption spectra showed that the conformations of HSA/BSA altered in the presence of ergosterol. The thermodynamic parameters, free energy change (ΔG), enthalpy change (ΔH) and entropy change (ΔS) for BSA-ergosterol and HSA-ergosterol systems were calculated by the van't Hoff equation and discussed. Besides, with the aid of three site markers (for example, phenylbutazone, ibuprofen and digitoxin), we have reported that ergosterol primarily binds to the tryptophan residues of BSA/HSA within site I (subdomain II A). PMID:22733490

  10. Fluorescence lifetime measurements of native and glycated human serum albumin and bovine serum albumin

    NASA Astrophysics Data System (ADS)

    Joshi, Narahari V.; Joshi, Virgina O. d.; Contreras, Silvia; Gil, Herminia; Medina, Honorio; Siemiarczuk, Aleksander

    1999-05-01

    Nonenzymatic glycation, also known as Maillard reaction, plays an important role in the secondary complications of the diabetic pathology and aging, therefore, human serum albumin (HSA) and bovine serum albumin (BSA) were glycated by a conventional method in our laboratory using glucose as the glycating agent. Fluorescence lifetime measurements were carried out with a laser strobe fluorometer equipped with a nitrogen/dye laser and a frequency doubler as a pulsed excitation source. The samples were excited at 295 nm and the emission spectra were recorded at 345 nm. The obtained decay curves were tried for double and triple exponential functions. It has been found that the shorter lifetime increases for glycated proteins as compared with that of the native ones. For example, in the case of glycated BSA the lifetime increased from 1.36 ns to 2.30 ns. Similarly, for HSA, the lifetime increases from 1.58 ns to 2.26 ns. Meanwhile, the longer lifetime changed very slightly for both proteins (from 6.52 ns to 6.72 ns). The increase in the lifetime can be associated with the environmental effect; originated from the attachment of glucose to some lysine residues. A good example is Trp 214 which is in the cage of Lys 225, Lys 212, Lys 233, Lys 205, Lys 500, Lys 199 and Lys 195. If fluorescence lifetime technique is calibrated and properly used it could be employed for assessing glycation of proteins.

  11. Direct detection of C-reactive proteins in human serum using nanoparticle-enhanced surface plasmon resonance biosensing

    NASA Astrophysics Data System (ADS)

    Lin, H.-Y.; Tsang, K. Y.; Hu, W. P.; Hsu, H.-Y.; Chiou, A.; Chang, G.-L.; Chen, S.-J.

    2006-08-01

    C-reactive protein (CRP) produced by the liver is one of the most characteristic acute-phase proteins. It has been suggested that the level of CRP in human serum may be a significant tool of detecting risks of developing cardiovascular disease and atherosclerosis. Here we propose an advanced plasmonic surface plasmon resonance (SPR) bioassay with Au nanoparticles embedded in the dielectric film that demonstrates a 10X improvement in resolution compared to the conventional SPR biosensor. The co-sputtered film was modified with (3-Aminopropyl)triethoxysilane to sequentially immobilize protein G, monoclonal anti-CRP antibody (C8), and human serum albumins (HSA). After blocked by ethanolamine, the sensor was used to detect CRP. Using this extremely sensitive biochip, the lowest reliable concentration of CRP without any exterior labeling is simplified to human physiological level. The novel assay has the latent capability of not only eliminating the disturbances coming from serum proteins resulting in false signals, but is also able to be applied in rapid and label-free clinical detections of CRP with large improved sensitivity.

  12. Human serum albumin and its relation with oxidative stress.

    PubMed

    Sitar, Mustafa Erinç; Aydin, Seval; Cakatay, Ufuk

    2013-01-01

    Human serum albumin, a negative acute phase reactant and marker of nutritive status, presents at high concentrations in plasma. Albumin has always been used in many clinical states especially to improve circulatory failure. It has been showed that albumin is involved in many bioactive functions such as regulation of plasma osmotic pressure, binding and transport of various endogenous or exogenous compounds, and finally extracellular antioxidant defenses. Molecules like transferrin, caeruloplasmin, haptoglobin, uric acid, bilirubin, alpha-tocopherol, glucose, and albumin constitute extracellular antioxidant defenses in blood plasma but albumin is the most potent one. Most of the antioxidant properties of albumin can be attributed to its unique biochemical structure. The protein possesses antioxidant properties such as binding copper tightly and iron weakly, scavenging free radicals, e.g., hypochlorous acid (HOCl) and Peroxynitrite (ONOOH) and providing thiol group (-SH). Whether it is chronic or acute, during many pathological conditions, biomarkers of oxidative protein damage increase and this observation continues with considerable oxidation of human serum albumin. There is an important necessity to specify its interactions with Reactive Oxygen Species. Generally, it may lower the availability of pro-oxidants and be preferentially oxidized to protect other macromolecules but all these findings make it necessary that researchers give a more detailed explanation of albumin and its relations with oxidative stress. PMID:24273915

  13. A microarray platform for detecting disease-specific circulating miRNA in human serum.

    PubMed

    Roy, Somenath; Soh, Jun Hui; Ying, Jackie Y

    2016-01-15

    Circulating microRNAs (miRNAs) are emerging as potential blood-based biomarkers for cancer and other critical diseases. To profile the expression levels of these tiny molecules, especially in a point-of-care setting, it is imperative to quantify them directly in complex biological fluids. Herein, we report the development of a microarray platform with carboxyl-polyethylene glycol (PEG) as a functional layer and aminated hairpin nucleic acid molecules as target-specific capture probes (CPs). Due to the anti-fouling effect conferred by the carboxyl-PEG layer, we could directly detect as little as 10fM of miRNA targets in 20µl of unprocessed human serum. In contrast to the conventional miRNA microarrays, our platform does not require RNA extraction, labeling and target amplification, thus significantly reducing both the sample preparation steps as well as the total assay duration. The use of specially designed hairpin CPs entails reliable discrimination of miRNA sequences with high sequence homology. A nanoparticle-based detection technique, with the help of differential interference contrast (DIC) microscopy, offers excellent resolution down to a single molecule. With the capability of detecting disease-specific miRNA targets directly in human serum, our microarray platform has potential applications in rapid, minimally invasive clinical diagnostic assays. PMID:26319167

  14. Renal targeted delivery of triptolide by conjugation to the fragment peptide of human serum albumin.

    PubMed

    Yuan, Zhi-xiang; Wu, Xiao-juan; Mo, Jingxin; Wang, Yan-li; Xu, Chao-qun; Lim, Lee Yong

    2015-08-01

    We have previously demonstrated that peptide fragments (PFs) of the human serum albumin could be developed as potential renal targeting carriers, in particular, the peptide fragment, PF-A299-585 (A299-585 representing the amino acid sequence of the human serum albumin). In this paper, we conjugated triptolide (TP), the anti-inflammatory Chinese traditional medicine, to PF-A299-585 via a succinic acid spacer to give TPS-PF-A299-585 (TP loading 2.2% w/w). Compared with the free TP, TPS-PF-A299-585 exhibited comparable anti-inflammatory activity in the lipopolysaccharide stimulated MDCK cells, but was significantly less cytotoxic than the free drug. Accumulation of TPS-PF-A299-585 in the MDCK cells in vitro and in rodent kidneys in vivo was demonstrated using FITC-labeled TPS-PF-A299-585. Renal targeting was confirmed in vivo in a membranous nephropathic (MN) rodent model, where optical imaging and analyses of biochemical markers were combined to show that TPS-PF-A299-585 was capable of alleviating the characteristic symptoms of MN. The collective data affirm PF-A299-585 to be a useful carrier for targeting TP to the kidney. PMID:26117184

  15. DNA-mediated strand displacement facilitates sensitive electronic detection of antibodies in human serums.

    PubMed

    Dou, Baoting; Yang, Jianmei; Shi, Kai; Yuan, Ruo; Xiang, Yun

    2016-09-15

    We describe here the development of a sensitive and convenient electronic sensor for the detection of antibodies in human serums. The sensor is constructed by self-assembly formation of a mixed monolayer containing the small molecule epitope conjugated double stranded DNA probes on gold electrode. The target antibody binds the epitope on the dsDNA probe and lowers the melting temperature of the duplex, which facilitates the displacement of the antibody-linked strand of the duplex probe by an invading methylene blue-tagged single stranded DNA (MB-ssDNA) through the strand displacement reaction and leads to the capture of many MB-ssDNA on the sensor surface. Subsequent electrochemical oxidation of the methylene blue labels results in amplified current response for sensitive monitoring of the antibodies. The antibody assay conditions are optimized and the sensor exhibits a linear range between 1.0 and 25.0nM with a detection limit of 0.67nM for the target antibody. The sensor is also selective and can be employed to detect the target antibodies in human serum samples. With the advantages of using small molecule epitope as the antibody recognition element over traditional antigen, the versatile manipulability of the DNA probes and the unique properties of the electrochemical transduction technique, the developed sensor thus hold great potential for simple and sensitive detection of different antibodies and other proteins in real samples. PMID:27111124

  16. Zwitterionic polymer-modified silicon microring resonators for label-free biosensing in undiluted human plasma

    PubMed Central

    Kirk, James T.; Brault, Norman D.; Baehr-Jones, Tom; Hochberg, Michael; Jiang, Shaoyi; Ratner, Daniel M.

    2013-01-01

    A widely acknowledged goal in personalized medicine is to radically reduce the costs of highly parallelized, small fluid volume, point-of-care and home-based diagnostics. Recently, there has been a surge of interest in using complementary metal-oxide-semiconductor (CMOS)-compatible silicon photonic circuits for biosensing, with the promise of producing chip-scale integrated devices containing thousands of orthogonal sensors, at minimal cost on a per-chip basis. A central challenge in biosensor translation is to engineer devices that are both sensitive and specific to a target analyte within unprocessed biological fluids. Despite advances in the sensitivity of silicon photonic biosensors, poor biological specificity at the sensor surface remains a significant factor limiting assay performance in complex media (i.e. whole blood, plasma, serum) due to the non-specific adsorption of proteins and other biomolecules. Here, we chemically modify the surface of silicon microring resonator biosensors for the label-free detection of an analyte in undiluted human plasma. This work highlights the first application of a non-fouling zwitterionic surface coating to enable silicon photonic-based label-free detection of a protein analyte at clinically relevant sensitivities in undiluted human plasma. PMID:23202337

  17. Optical quantification of hemolysis, icterus, and lipemia in human serum

    NASA Astrophysics Data System (ADS)

    Kasagani, Vimal Kumar

    In order to increase the automation and efficiency for a national reference laboratory, the ability to quantify interferences like Hemolysis, Icterus, and Lipemia in serum samples is investigated. The system is intended as a screening step prior to clinical analysis of medical samples to prevent false results caused by the interferences. The system is based on selective absorption of transmitted light by the interferences that cause loss of light at specific wavelengths. The absorption spectra of interferences are analyzed to identify the appropriate wavelengths, resulting in a mathematical formulation between the absorbance and concentrations. An absorption wavelength is selected so that the transmitted power of light through a tube with the sample significantly decreased due to the presence of condition of interest, while the reference wavelength is selected so that the transmitted light varies mostly due to the presence of tube material and labels and does not vary due to the presence of interference. A computational model is formulated using a commercial software package, ANSYS FLUENT, in order to understand the absorption and scattering effects, the thermal effects of higher power irradiation on the biological samples, as well as to determine the radiant power of transmitted light through the sample for different power levels. The Discrete Ordinates Method is used to model the radiation through a participating medium. The temperature distribution and spectral power of transmitted radiation are determined for water in a tube for different wavelengths used in the current system.

  18. Plant-derived recombinant human serum transferrin demonstrates multiple functions.

    PubMed

    Brandsma, Martin E; Diao, Hong; Wang, Xiaofeng; Kohalmi, Susanne E; Jevnikar, Anthony M; Ma, Shengwu

    2010-05-01

    Human serum transferrin (hTf) is the major iron-binding protein in human plasma, having a vital role in iron transport. Additionally, hTf has many other uses including antimicrobial functions and growth factor effects on mammalian cell proliferation and differentiation. The multitask nature of hTf makes it highly valuable for different therapeutic and commercial applications. However, the success of hTf in these applications is critically dependent on the availability of high-quality hTf in large amounts. In this study, we have developed plants as a novel platform for the production of recombinant (r)hTf. We show here that transgenic plants are an efficient system for rhTf production, with a maximum accumulation of 0.25% total soluble protein (TSP) (or up to 33.5 microg/g fresh leaf weight). Furthermore, plant-derived rhTf retains many of the biological activities synonymous with native hTf. In particular, rhTf reversibly binds iron in vitro, exhibits bacteriostatic activity, supports cell proliferation in serum-free medium and can be internalized into mammalian cells in vitro. The success of this study validates the future application of plant rhTf in a variety of fields. Of particular interest is the use of plant rhTf as a novel carrier for cell-specific or oral delivery of protein/peptide drugs for the treatment of human diseases such as diabetes.To demonstrate this hypothesis, we have additionally expressed an hTf fusion protein containing glucagon-like peptide 1 (GLP-1) or its derivative in plants. Here, we show that plant-derived hTf-GLP-1 fusion proteins retain the ability to be internalized by mammalian cells when added to culture medium in vitro. PMID:20432512

  19. Optimization of the radioimmunoassays for measuring fentanyl and alfentanil in human serum

    SciTech Connect

    Schuettler, J.; White, P.F.

    1984-09-01

    Measurement of serum fentanyl and alfentanil concentrations by radioimmunoassay (RIA) may result in significant errors and high variability when the technique described in the available fentanyl and alfentanil RIA kits is used. The authors found a 29-94% overestimation of measured fentanyl and alfentanil serum levels when 3H-fentanyl or 3H-alfentanil was added lastly to the mixture of antiserum and sample. This finding is related to a reduction in binding sites for the labeled compounds after preincubation of sample and antiserum. If this sequence is used, it becomes necessary to extend the incubation period up to 6 h for fentanyl and up to 10 h for alfentanil in order to achieve equilibration between unlabeled and labeled drug with respect to antiserum binding. However, when antiserum is added lastly to the mixture of sample and labeled drug, measurement accuracy and precision for fentanyl and alfentanil serum concentrations are enhanced markedly. In addition, it is important to perform the calibration curves and sample measurements using the same medium (i.e., serum alone or a serum/buffer dilution). In summary, to optimize the RIA for fentanyl and alfentanil, the authors recommend the following: 1) adding the antiserum lastly to the mixture of sample and labeled drug; 2) performing calibration curves using patient's blank serum when possible; 3) carefully examining and standardizing each step of the RIA procedure to reduce variability, and, finally; 4) comparing results with those of other established RIA laboratories.

  20. Photoaffinity labeling of the progesterone receptor from human endometrial carcinoma

    SciTech Connect

    Clarke, C.L.; Satyaswaroop, P.G.

    1985-11-01

    A nude mouse model for the growth of human endometrial carcinoma and hormonal modulation of the progesterone receptor (PR) was established previously. This study describes the effect of 17 beta-estradiol and tamoxifen (TAM) on growth rate and PR concentration in a hormonally responsive human endometrial tumor (EnCa 101) grown in this experimental system and presents the first characterization of human endometrial carcinoma PR. EnCa 101 was transplanted subcutaneously into ovariectomized, BALB/c, nu/nu athymic mice and grown under 17 beta-estradiol-stimulated, TAM-stimulated, and control conditions. Both 17 beta-estradiol and TAM increased the growth rate of EnCa 101 in nude mice, and a parallel increase in the cytosol PR concentration was observed. PR was partially purified by phosphocellulose and DEAE cellulose chromatography, and the DEAE eluate was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and photoaffinity labeling with (17 alpha-methyl-TH)promegestone ((TH)R5020). Two PR-negative tumors (EnCa K and EnCa V) were also examined in parallel. Photolabeling and sodium dodecyl sulfate-polyacrylamide gel electrophoresis of EnCa 101 grown in the presence of 17 beta-estradiol or TAM revealed incorporation of (3H)R5020 into proteins of molecular weight approximately 116,000 and 85,000. Labeled proteins of molecular weight 66,000, 45,000, and 35,000 were also observed. No incorporation of (TH)R5020 was observed in EnCa 101 grown in the absence of estrogen, nor was any observed in EnCa K or EnCa V.

  1. Mycovirus-like DNA virus sequences from cattle serum and human brain and serum samples from multiple sclerosis patients.

    PubMed

    Lamberto, Iranzu; Gunst, Karin; Müller, Hermann; Zur Hausen, Harald; de Villiers, Ethel-Michele

    2014-01-01

    Myco-like viruses have been isolated from fungi, feces of various animals, and plant leaves. We report here the isolation of 3 complete genome sequences of gemycircularvirus-related viruses from healthy bovine serum and human brain and serum samples from patients with multiple sclerosis (MS). Their putative capsid proteins share similarity to Torque teno virus (TTV) open reading frame 1 (ORF1) proteins. PMID:25169858

  2. Mycovirus-Like DNA Virus Sequences from Cattle Serum and Human Brain and Serum Samples from Multiple Sclerosis Patients

    PubMed Central

    Lamberto, Iranzu; Gunst, Karin; Müller, Hermann; zur Hausen, Harald

    2014-01-01

    Myco-like viruses have been isolated from fungi, feces of various animals, and plant leaves. We report here the isolation of 3 complete genome sequences of gemycircularvirus-related viruses from healthy bovine serum and human brain and serum samples from patients with multiple sclerosis (MS). Their putative capsid proteins share similarity to Torque teno virus (TTV) open reading frame 1 (ORF1) proteins. PMID:25169858

  3. Preparation of carbon quantum dots with a high quantum yield and the application in labeling bovine serum albumin

    NASA Astrophysics Data System (ADS)

    Liu, Pengpeng; Zhang, Changchang; Liu, Xiang; Cui, Ping

    2016-04-01

    An economic and green approach of manufacturing carbon quantum dots (CQDs) with a high quantum yield (denoted with HQY-CQDs) and the application in labeling bovine serum albumin (BSA) were described in detail in this work. Firstly, the cheap resources of citric acid and glycine were pyrolysed in drying oven for preparing the CQDs. Then the product was immersed in tetrahydrofuran for 8 h. HQY-CQDs were obtained by removing tetrahydrofuran from the supernate and were evaluated that they possessed a much higher quantum yield compared with that without dealing with tetrahydrofuran and a wonderful photo-bleaching resistance. Such HQY-CQDs could be functionalized by N-hydroxysuccinimide and successively combined with BSA covalently. Thus fluorescent labeling on BSA was realized. The HQY-CQDs were demonstrated with transmission electron microscopy and the chemical modification with N-hydroxysuccinimide was proved by infrared and X-ray photoelectron spectra. Labeling BSA with the HQY-CQDs was confirmed by gel electrophoresis and fluorescence imaging.

  4. Determination of phosphodiesterase I activity in human blood serum.

    PubMed

    Hynie, I; Meuffels, M; Poznanski, W J

    1975-09-01

    Phosphodiesterase I (EC 3.1.4.1) activity was detected in normal human blood serum. The enzyme is stable at laboratory temperature for three days, but is inactivated at pH less than 7. The pH for optimum activity increases with the substrate concentration (under the conditions used, from pH 9.0 to 10.2) and, conversely, the Km increases with pH and buffer concentration. The enzyme is inhibited by ethylenediaminetetraacetate but not by phosphate (0.1 mol/liter). We developed a simple quantitative method for its determination, based on hydrolysis of the p-nitrophenyl ester of thymidine 5'-monophosphate and subsequent measurement of the liberated p-nitrophenol at 400 nm in NaOH (0.1 mol/liter). Normal values (mean +/- 2 SD) were determined to be 33 +/- 6.4 U/liter. Preliminary studies indicate that phosphodiesterase I activity is greater than normal in serum of patients with necrotic changes in the liver or kidney or in cases of breast cancer, but not in that of patients with myocardial infarction, bone cancer, lung cancer, or chronic liver cirrhosis. PMID:168991

  5. Human platelet lysate: Replacing fetal bovine serum as a gold standard for human cell propagation?

    PubMed

    Burnouf, Thierry; Strunk, Dirk; Koh, Mickey B C; Schallmoser, Katharina

    2016-01-01

    The essential physiological role of platelets in wound healing and tissue repair builds the rationale for the use of human platelet derivatives in regenerative medicine. Abundant growth factors and cytokines stored in platelet granules can be naturally released by thrombin activation and clotting or artificially by freeze/thaw-mediated platelet lysis, sonication or chemical treatment. Human platelet lysate prepared by the various release strategies has been established as a suitable alternative to fetal bovine serum as culture medium supplement, enabling efficient propagation of human cells under animal serum-free conditions for a multiplicity of applications in advanced somatic cell therapy and tissue engineering. The rapidly increasing number of studies using platelet derived products for inducing human cell proliferation and differentiation has also uncovered a considerable variability of human platelet lysate preparations which limits comparability of results. The main variations discussed herein encompass aspects of donor selection, preparation of the starting material, the possibility for pooling in plasma or additive solution, the implementation of pathogen inactivation and consideration of ABO blood groups, all of which can influence applicability. This review outlines the current knowledge about human platelet lysate as a powerful additive for human cell propagation and highlights its role as a prevailing supplement for human cell culture capable to replace animal serum in a growing spectrum of applications. PMID:26561934

  6. Binding of labeled thyroxin analog to serum proteins evaluated after radioimmunoassay of free thyroxin

    SciTech Connect

    Arevalo, G.

    1989-03-01

    In ambulatory patients, assay of free thyroxin (FT4) in serum correlates well with thyroid status and with results obtained by equilibrium dialysis. The validity of FT4 results has been questioned mainly in euthyroid patients with altered concentrations of thyroid hormone-binding proteins, as in nonthyroidal illness, hereditary analbuminemia, familial dysalbuminemic hyperthyroxinemia (FDH), and the presence of iodothyronine-binding antibodies. I present here a study of the binding of (/sup 125/I)T4-derivative to serum proteins in the supernate, which is ordinarily discarded after determination of FT4 by one-step radioimmunoassay with dextran-coated charcoal used to separate the free and bound fractions. The results are expressed as a ratio, with results for a normal serum pool as reference. The average ratio was high in hyperthyroid subjects, 1.26 (SD 0.12, n = 25), and in hypoalbuminemia, 1.20 (SD 0.10, n = 15), and low in FDH, 0.62 (SD 0.11, n = 9), and hypothyroid subjects, 0.90 (SD 0.06, n = 20). In normal individuals it was 0.98 (SD 0.05, n = 30). Determination of the analog-binding rate complements the FT4 result and allows for the recognition of cases with abnormal binding by serum proteins, without recourse to other tests recommended for thyroid-function studies.

  7. Human serum albumin crystals and method of preparation

    NASA Technical Reports Server (NTRS)

    Carter, Daniel C. (Inventor)

    1989-01-01

    Human serum albumin (HSA) crystals are provided in the form of tetragonal plates having the space groups P42(sub 1)2, the crystals being grown to sizes in excess of 0.5 mm in two dimensions and a thickness of 0.1 mm. Growth of the crystals is carried out by a hanging drop method wherein a precipitant solution containing polyethylene glycol (PEG) and a phosphate buffer is mixed with an HSA solution, and a droplet of mixed solution is suspended over a well of precipitant solution. Crystals grow to the desired size in 3 to 7 days. Concentration of reagents, pH and other parameters are controlled within prescribed limits. The resulting crystals exhibit a size and quality such as to allow performance of x ray diffraction studies and enable the conduct of drug binding studies as well as genetic engineering studies.

  8. Hydrophobic conjugated microporous polymers for sorption of human serum albumin

    NASA Astrophysics Data System (ADS)

    Zheng, Chunli; Du, Miaomiao; Feng, Shanshan; Sun, Hanxue; Li, An; He, Chi; Zhang, TianCheng; Wang, Qiaorui; Wei, Wei

    2016-02-01

    This paper investigated the sorption of human serum albumin (HSA) from water by three kinds of conjugated microporous polymers (CMPs) with surface hydrophobicity and intrinsic porosity. It was found that the three CMPs captured HSA with fast sorption kinetics and good working capacity. Equilibrium was obtained at 80 min for all the tests, and the maximum sorption quantity (qm) ranged from 0.07 to 0.14 mg/mg. With the increase in the particle external surface area of the CMPs, a greater extent of HSA sorption was achieved. Moreover, promoting the dispersion of CMPs in HSA aqueous solution was also beneficial to the extraction. Attenuated Total Reflection Fourier Transform Infrared spectroscopy verified the interactions between the CMPs and the Nsbnd H, Cdbnd O, and Csbnd N groups of HSA. This paper might provide fundamental guidance for the practical application of CMPs to proteins separation and recovery.

  9. In vitro interaction between ceruloplasmin and human serum transferrin.

    PubMed

    Ha-Duong, Nguyêt-Thanh; Eid, Chantal; Hémadi, Miryana; El Hage Chahine, Jean-Michel

    2010-12-01

    The thermodynamics of the interactions of serum apotransferrin (T) and holotransferrin (TFe(2)) with ceruloplasmin (Cp), as well as those of human lactoferrin (Lf), were assessed by fluorescence emission spectroscopy. Cp interacts with two Lf molecules. The first interaction depends on pH and μ, whereas the second does not. Dissociation constants were as follows: K(11Lf) = 1.5 ± 0.2 μM, and K(12Lf) = 11 ± 2 μM. Two slightly different interactions of T or TFe(2) with Cp are detected for the first time. They are both independent of pH and μ and occur with 1:1 stoichiometry: K(1T) = 19 ± 7 μM, and K(1TFe2) = 12 ± 4 μM. These results can improve our understanding of the probable process of the transfer of iron from Cp to T in iron and copper transport and homeostasis. PMID:21049900

  10. Three-dimensional structure of human serum albumin

    NASA Technical Reports Server (NTRS)

    Carter, Daniel C.; He, Xiao-Min; Twigg, Pamela D.; Casale, Elena

    1991-01-01

    The binding locations to human serum albumin (HSA) of several drug molecules were determined at low resolution using crystallographic methods. The principal binding sites are located within subdomains IIA and IIIA. Preliminary studies suggest that an approach to increasing the in vivo efficacy of drugs which are rendered less effective or ineffective by virtue of their interaction with HSA, would be the use of competitive displacement in drug therapies and/or the development of a general inhibitor to the site within subdomain IIIA. These findings also suggest that the facilitated transfer of various ligands across organ/circulatory interfaces such as liver, kidney, and brain may be associated with binding to the IIIA subdomain.

  11. Heme-based catalytic properties of human serum albumin

    PubMed Central

    Ascenzi, P; di Masi, A; Fanali, G; Fasano, M

    2015-01-01

    Human serum albumin (HSA): (i) controls the plasma oncotic pressure, (ii) modulates fluid distribution between the body compartments, (iii) represents the depot and carrier of endogenous and exogenous compounds, (iv) increases the apparent solubility and lifetime of hydrophobic compounds, (v) affects pharmacokinetics of many drugs, (vi) inactivates toxic compounds, (vii) induces chemical modifications of some ligands, (viii) displays antioxidant properties, and (ix) shows enzymatic properties. Under physiological and pathological conditions, HSA has a pivotal role in heme scavenging transferring the metal-macrocycle from high- and low-density lipoproteins to hemopexin, thus acquiring globin-like reactivity. Here, the heme-based catalytic properties of HSA are reviewed and the structural bases of drug-dependent allosteric regulation are highlighted.

  12. Serum Immunoglobulin A Cross-Strain Blockade of Human Noroviruses

    PubMed Central

    Lindesmith, Lisa C.; Beltramello, Martina; Swanstrom, Jesica; Jones, Taylor A.; Corti, Davide; Lanzavecchia, Antonio; Baric, Ralph S.

    2015-01-01

    Background. Human noroviruses are the leading cause of acute viral gastroenteritis, justifying vaccine development despite a limited understanding of strain immunity. After genogroup I (GI).1 norovirus infection and immunization, blockade antibody titers to multiple virus-like particles (VLPs) increase, suggesting that GI cross-protection may occur. Methods. Immunoglobulin (Ig)A was purified from sera collected from GI.1-infected participants, and potential neutralization activity was measured using a surrogate neutralization assay based on antibody blockade of ligand binding. Human and mouse monoclonal antibodies (mAbs) were produced to multiple GI VLPs to characterize GI epitopes. Results. Immunoglobulin A purified from day 14 post-GI.1 challenge sera blocked binding of GI.1, GI.3, and GI.4 to carbohydrate ligands. In some subjects, purified IgA preferentially blocked binding of other GI VLPs compared with GI.1, supporting observations that the immune response to GI.1 infection may be influenced by pre-exposure history. For other subjects, IgA equivalently blocked multiple GI VLPs. Only strain-specific mAbs recognized blockade epitopes, whereas strain cross-reactive mAbs recognized nonblockade epitopes. Conclusions. These studies are the first to describe a functional role for serum IgA in norovirus immunity and the first to characterize human monoclonal antibodies to GI strains, expanding our understanding of norovirus immunobiology. PMID:26180833

  13. (99m)Tc-human serum albumin nanocolloids: particle sizing and radioactivity distribution.

    PubMed

    Persico, Marco G; Lodola, Lorenzo; Buroni, Federica E; Morandotti, Marco; Pallavicini, Piersandro; Aprile, Carlo

    2015-07-01

    Several parameters affect the biodistribution of administered nanocolloids (NC) for Sentinel Lymph Node (SLN) detection: particle size distribution, number of Tc atoms per particle and specific activity (SA). Relatively few data are available with frequently conflicting results. (99m)Tc-NC-human serum albumin (HSA) Nanocoll®, Nanoalbumon® and Nanotop® were analysed for particles' dimensional and radioactivity distribution, and a mathematical model was elaborated to estimate the number of particles involved. Commercially available kits were reconstituted at maximal SA of 11 MBq/µg HSA. Particles size distribution was evaluated by Dynamic Light Scattering. These data were related to the radioactivity distribution analysis passing labelled NC through three polycarbonate filters (15-30-50-nm pore size) under vacuum. Highest radioactivity was carried by 30-50 nm particles. The smallest ones, even though most numerous, carried only the 10% of (99m)Tc atoms. Nanocoll and Nanotop are not significantly different, while Nanoalbumon is characterized by largest particles (>30 nm) that carried the most of radioactivity (80%). Smallest particles could saturate the clearing capacity of macrophages; therefore, if the tracer is used for SLN detection, more node tiers could be visualized, reducing accuracy of SLN mapping. Manufacturers could implement technical leaflets with particle size distribution and could improve the labelling protocol to provide clinicians useful information. PMID:26198778

  14. Adhesion of human platelets to serum amyloid A.

    PubMed

    Urieli-Shoval, Simcha; Shubinsky, George; Linke, Reinhold P; Fridkin, Mati; Tabi, Israel; Matzner, Yaacov

    2002-02-15

    Serum amyloid A (SAA) is an acute phase reactant, and its level in the blood is elevated to 1000-fold in response of the body to trauma, infection, inflammation, and neoplasia. SAA was reported to inhibit platelet aggregation and to induce adhesion of leukocytes. This study looked at adhesion of human platelets to SAA. Immobilized SAA supported the adhesion of human washed platelets; level of adhesion to SAA was comparable to fibronectin and lower than to fibrinogen. Adhesion to SAA was further enhanced by Mn(2+) and the physiological agonist, thrombin. Platelet adhesion to SAA was completely abolished by anti-SAA antibody. SAA-induced adhesion was inhibited by antibodies against the integrin receptor alphaIIbbeta3, by the peptide GRGDSP and by SAA-derived peptide containing YIGSR-like and RGD-like adhesion motifs (amino acids 29 to 42). Adhesion was not inhibited by control immunoglobulin G, by antibody against the integrin receptor alphaVbeta3, by the peptide GRGESP, and by SAA-derived peptide that includes incomplete RGD motif. SAA-derived peptide 29 to 42 also inhibited platelet adhesion to fibronectin. Transfected human melanoma cells expressing alphaIIbbeta3 adhered to SAA, whereas transfected cells expressing alphaVbeta3 did not. By using flow cytometry, the alphaIIbbeta3 cells displayed significantly higher levels of binding of soluble SAA than the alphaVbeta3 cells. These data indicate that human platelets specifically adhere to SAA in an RGD- and alphaIIbbeta3-dependent manner. Thus, SAA may play a role in modulating platelet adhesion at vascular injury sites by sharing platelet receptors with other platelet-adhesive proteins. PMID:11830469

  15. Posaconazole in Human Serum: a Greater Pharmacodynamic Effect than Predicted by the Non-Protein-Bound Serum Concentration ▿

    PubMed Central

    Lignell, Anders; Löwdin, Elisabeth; Cars, Otto; Chryssanthou, Erja; Sjölin, Jan

    2011-01-01

    It is generally accepted that only the unbound fraction of a drug is pharmacologically active. Posaconazole is an antifungal agent with a protein binding of 98 to 99%. Taking into account the degree of protein binding, plasma levels in patients, and MIC levels of susceptible strains, it can be assumed that the free concentration of posaconazole sometimes will be too low to exert the expected antifungal effect. The aim was therefore to test the activity of posaconazole in serum in comparison with that of the calculated unbound concentrations in protein-free media. Significant differences (P < 0.05) from the serum control were found at serum concentrations of posaconazole of 1.0 and 0.10 mg/liter, with calculated free concentrations corresponding to 1× MIC and 0.1× MIC, respectively, against one Candida lusitaniae strain selected for proof of principle. In RPMI 1640, the corresponding calculated unbound concentration of 0.015 mg/liter resulted in a significant effect, whereas that of 0.0015 mg/liter did not. Also, against seven additional Candida strains tested, there was an effect of the low posaconazole concentration in serum, in contrast to the results in RPMI 1640. Fluconazole, a low-grade-protein-bound antifungal, was used for comparison at corresponding concentrations in serum and RPMI 1640. No effect was observed at the serum concentration, resulting in a calculated unbound concentration of 0.1× MIC. In summary, there was a substantially greater pharmacodynamic effect of posaconazole in human serum than could be predicted by the non-protein-bound serum concentration. A flux from serum protein-bound to fungal lanosterol 14α-demethylase-bound posaconazole is suggested. PMID:21502622

  16. Human serum albumin-polyethylenimine nanoparticles for gene delivery.

    PubMed

    Rhaese, Stephanie; von Briesen, Hagen; Rübsamen-Waigmann, Helga; Kreuter, Jörg; Langer, Klaus

    2003-09-19

    Nanoparticles consisting of DNA, human serum albumin (HSA) and polyethylenimine (PEI) were formed and tested for transfection efficiency in vitro with the aim of generating a nonviral gene delivery vehicle. HSA-PEI-DNA nanoparticles containing the pGL3 vector coding for luciferase as reporter gene were formed by charge neutralization. The particles were characterized by gel retardation assay, dynamic light scattering (size) and electrophoretic mobility measurements (charge). Stability was determined by spectrophotometric analysis and transfection efficiency was evaluated in cell culture using human embryonic epithelial kidney 293 cells. HSA-PEI-DNA nanoparticles were prepared by co-encapsulation of PEI as a lysosomotropic agent at varying nitrogen to phosphate (N/P) ratios. An optimum transfection efficiency was achieved when the particles were prepared at N/P ratios between 4.8 and 8.4. Furthermore, they displayed a low cytotoxicity when tested in cell culture. Our results show that HSA-PEI-DNA nanoparticles are a versatile carrier for DNA that may be suitable for i.v. administration. PMID:14499197

  17. Human Serum Albumin Complexed with Myristate and AZT

    SciTech Connect

    Zhu, Lili; Yang, Feng; Chen, Liqing; Meehan, Edward J.; Huang, Mingdong

    2008-06-16

    3'-Azido-3'-deoxythymidine (AZT) is the first clinically effective drug for the treatment of human immunodeficiency virus infection. The drug interaction with human serum albumin (HSA) has been an important component in understanding its mechanism of action, especially in drug distribution and in drug-drug interaction on HSA in the case of multi-drug therapy. We present here crystal structures of a ternary HSA-Myr-AZT complex and a quaternary HSA-Myr-AZT-SAL complex (Myr, myristate; SAL, salicylic acid). From this study, a new drug binding subsite on HSA Sudlow site 1 was identified. The presence of fatty acid is needed for the creation of this subsite due to fatty acid induced conformational changes of HSA. Thus, the Sudlow site 1 of HSA can be divided into three non-overlapped subsites: a SAL subsite, an indomethacin subsite and an AZT subsite. Binding of a drug to HSA often influences simultaneous binding of other drugs. From the HSA-Myr-AZT-SAL complex structure, we observed the coexistence of two drugs (AZT and SAL) in Sudlow site 1 and the competition between these two drugs in subdomain IB. These results provide new structural information on HSA-drug interaction and drug-drug interaction on HSA.

  18. Increased serum clearance of oligomannose species present on a human IgG1 molecule

    PubMed Central

    Alessandri, Leslie; Ouellette, David; Acquah, Aima; Rieser, Mathew; LeBlond, David; Saltarelli, Mary; Radziejewski, Czeslaw; Fujimori, Taro; Correia, Ivan

    2012-01-01

    The role of Fc glycans on clearance of IgG molecule has been examined by various groups in experiments where specific glycans have been enriched or the entire spectrum of glycans was studied after administration in pre-clinical or clinical pharmacokinetic (PK) studies. The overall conclusions from these studies are inconsistent, which may result from differences in antibody structure or experimental design. In the present study a well-characterized recombinant monoclonal IgG1 molecule (mAb-1) was analyzed from serum samples obtained from a human PK study. mAb-1 was recovered from serum using its ligand cross-linked to Sepharose beads. The overall purity and recovery of all isoforms were carefully evaluated using a variety of methods. Glycans were then enzymatically cleaved, labeled using 2-aminobenzamide and analyzed by normal phase high performance liquid chromatography. The assays for recovering mAb-1 from serum and subsequent glycan analysis were rigorously qualified at a lower limit of quantitation of 15 μg/mL, thus permitting analysis to day 14 of the clinical PK study. Eight glycans were monitored and classified into two groups: (1) the oligomannose type structures (M5, M6 and M7) and (2) fucosylated biantennary oligosaccharides (FBO) structures (NGA2F, NA1F, NA2F, NA1F-GlcNAc and NGA2F-GlcNAc). We observed that the oligomannose species were cleared at a much faster rate (40%) than FBOs and conclude that high mannose species should be carefully monitored and controlled as they may affect PK of the therapeutic; they should thus be considered an important quality attribute. These observations were only possible through the application of rigorous analytical methods that we believe will need to be employed when comparing innovator and biosimilar molecules. PMID:22669558

  19. Upgrading pretransplant human islet culture technology requires human serum combined with media renewal.

    PubMed

    Kerr-Conte, Julie; Vandewalle, Brigitte; Moerman, Ericka; Lukowiak, Bruno; Gmyr, Valery; Arnalsteen, Laurent; Caiazzo, Robert; Sterkers, Adrien; Hubert, Thomas; Vantyghem, Marie Christine; Pattou, François

    2010-05-15

    BACKGROUND.: The original Edmonton protocol used fresh islets, but for obvious logistic advantages most transplant centers have implemented pretransplant culture in human albumin. The aim of this study was to improve current pretransplant human islet culture techniques. METHODS.: Clinical-grade purified human islets from a total of 24 donors were directly resuspended after isolation in CMRL 1066-based media at 37 degrees C, and media additions and renewal were tested. At days 1 and 5 of culture, in vitro quality controls included islet viability, insulin content and function, apoptosis, and in vivo islet potency assay in nude mice. RESULTS.: Replacing human albumin with human AB serum improved 1- and 5-day preservation of islet function and viability which was further enhanced with antioxidant Stem Ease, leading to the iCulture medium (enriched CMRL: pyruvate, zinc sulfate, insulin, transferrin, selenium, 2.5% human AB serum and Stem Ease). Major damage occurs in the first day of culture and frequent media renewal (25% vol/hr) in this period further improved viability, apoptosis, islet recovery, and function in vitro and in vivo, compared with only changing medium after overnight culture. CONCLUSIONS.: The described human islet culture technique (iCulture medium+renewal) seems to be the best choice for clinical human islet culture when short (1 day) or long (5 days) periods are used. Media choice and dilution play a major role in the function and survival of human islets in culture. PMID:20098354

  20. Photoaffinity labeling of serum vitamin D binding protein by 3-deoxy-3-azido-25-hydroxyvitamin D3

    SciTech Connect

    Link, R.P.; Kutner, A.; Schnoes, H.K.; DeLuca, H.F.

    1987-06-30

    3-Deoxy-3-azido-25-hydroxyvitamin D3 was covalently incorporated in the 25-hydroxyvitamin D3 binding site of purified human plasma vitamin D binding protein. Competition experiments showed that 3-deoxy-3-azido-25-hydroxyvitamin D3 and 25-hydroxyvitamin D3 bind at the same site on the protein. Tritiated 3-deoxy-3-azido-25-hydroxyvitamin D3 was synthesized from tritiated 25-hydroxyvitamin D3, retaining the high specific activity of the parent compound. The tritiated azido label bound reversibly to human vitamin D binding protein in the dark and covalently to human vitamin D binding protein after exposure to ultraviolet light. Reversible binding of tritiated 3-deoxy-3-azido-25-hydroxyvitamin D3 was compared to tritiated 25-hydroxyvitamin D3 binding to human vitamin D binding protein. Scatchard analysis of the data indicated equivalent maximum density binding sites with a KD,app of 0.21 nM for 25-hydroxyvitamin D3 and a KD,app of 1.3 nM for the azido derivative. Covalent binding was observed only after exposure to ultraviolet irradiation, with an average of 3% of the reversibly bound label becoming covalently bound to vitamin D binding protein. The covalent binding was reduced 70-80% when 25-hydroxyvitamin D3 was present, indicating strong covalent binding at the vitamin D binding site of the protein. When tritiated 3-deoxy-3-azido-25-hydroxyvitamin D3 was incubated with human plasma in the absence and presence of 25-hydroxyvitamin D3, 12% of the azido derivative was reversibly bound to vitamin D binding protein. After ultraviolet irradiation, four plasma proteins covalently bound the azido label, but vitamin D binding protein was the only protein of the four that was unlabeled in the presence of 25-hydroxyvitamin D3.

  1. Use of peroxidase-labelled antigen for the detection of antibodies to Borrelia burgdorferi in human and animal sera.

    PubMed

    Eiffert, H; Lotter, H; Thomssen, R

    1991-01-01

    We have developed a modified ELISA for the detection of anti-Borrelia burgdorferi (Bb) antibodies based on a peroxidase enzyme labelled antigen (ELAT). Microtiter plates were coated with antigen of Bb. The immunoglobulins of the serum samples were bound to the antigen and specific antibodies were detected by an enzyme labelled antigen. The test principle facilitates the recognition of specific antibodies in different collectives of human and animal sera. We performed epidemiological studies with the ELAT on 231 sera from mothers in maternity wards (9.5% positive), 219 patient sera sent to the Bb routine diagnostics (15% positive) and 230 sera from forestry workers (21.3% positive). We further investigated sera from red deer from South Lower Saxony which remained 55% Bb-antibody positive; deer were 37% and fallow deer were 29% positive. PMID:2028231

  2. Transient serum exposure regimes to support dual differentiation of human mesenchymal stem cells.

    PubMed

    France, L A; Scotchford, C A; Grant, D M; Rashidi, H; Popov, A A; Sottile, V

    2014-08-01

    Human mesenchymal stem cells (MSCs), which can generate both osteoblasts and chondrocytes, represent an ideal resource for orthopaedic repair using tissue-engineering approaches. One major difficulty for the development of osteochondral constructs using undifferentiated MSCs is that serum is typically used in culture protocols to promote differentiation of the osteogenic component, whereas existing chondrogenic differentiation protocols rely on the use of serum-free conditions. In order to define conditions which could be compatible with both chondrogenic and osteogenic differentiation in a single bioreactor, we have analysed the efficiency of new biphasic differentiation regimes based on transient serum exposure followed by serum-free treatment. MSC differentiation was assessed either in serum-free medium or with a range of transient exposure to serum, and compared to continuous serum-containing treatment. Although osteogenic differentation was not supported in the complete absence of serum, marker expression and extensive mineralization analyses established that 5 days of transient exposure triggered a level of differentiation comparable to that observed when serum was present throughout. This initial phase of serum exposure was further shown to support the successful chondrogenic differentiation of MSCs, comparable to controls maintained in serum-free conditions throughout. This study indicates that a culture based on temporal serum exposure followed by serum-free treatment is compatible with both osteogenic and chondrogenic differentiation of MSCs. These results will allow the development of novel strategies for osteochondral tissue engineering approaches using MSCs for regenerative medicine. PMID:23161724

  3. Open Label Trial of Naltrexone Implants: Measuring Blood Serum Levels of Naltrexone

    PubMed Central

    Colquhoun, Ross M.

    2013-01-01

    The usefulness of oral naltrexone has been limited by compliance. Sub-cutaneous implants would seem to offer a solution to this problem and improve long-term outcomes. The aim of the present study was to compare levels of blood serum naltrexone of patients who had received a naltrexone implant after detoxification to a number of dependent variables of interest. These dependent variables included drug use including urine screens of each patient, any adverse response to the implant, subjective evaluation of self-esteem, quality of relationships, and changes in social functioning. Sixty six patients received an implant and were surveyed; urine and blood samples were taken at about 1, 3, and 6 months after implantation. Naltrexone levels were on average above 1 ng/mL at 6 months after insertion and patients showed significant improvements on all dependent variables. The preliminary evidence indicates that implants can improve compliance rates and outcomes. PMID:23761973

  4. Use of Lanthanide-Containing Polyoxometalates to Sensitise the Emission of Fluorescent Labelled Serum Albumin.

    PubMed

    Holmes-Smith, A Sheila; Crisp, Jacob; Hussain, Firasat; Patzke, Greta R; Hungerford, Graham

    2016-02-01

    Monitoring the interaction of biomolecules is important, and the use of energy transfer is a principal technique in elucidating nanoscale interactions. Lanthanide compounds are promising luminescent probes for biological samples as their emission is longer-lived than any native autofluorescence. Polyoxometalates (POMs) are interesting structural motifs to incorporate lanthanides, offering low toxicity and a size pertinent for biological applications. Here, we employ iso-structured POMs containing either terbium or europium and assess their interaction with serum albumin by sensitisation of a fluorescent tag on the protein via LRET (luminescence resonance energy transfer) by exciting the lanthanide. Time-resolved measurements showed energy transfer with an efficiency of over 90% for the POM-protein systems. The Tb-POM results were relatively straightforward, while those with the iso-structured Eu-POM were complicated by the effect of protein shielding from the aqueous environment. PMID:26642428

  5. Absolute quantification of DcR3 and GDF15 from human serum by LC-ESI MS

    PubMed Central

    Lancrajan, Ioana; Schneider-Stock, Regine; Naschberger, Elisabeth; Schellerer, Vera S; Stürzl, Michael; Enz, Ralf

    2015-01-01

    Biomarkers are widely used in clinical diagnosis, prognosis and therapy monitoring. Here, we developed a protocol for the efficient and selective enrichment of small and low concentrated biomarkers from human serum, involving a 95% effective depletion of high-abundant serum proteins by partial denaturation and enrichment of low-abundant biomarkers by size exclusion chromatography. The recovery of low-abundance biomarkers was above 97%. Using this protocol, we quantified the tumour markers DcR3 and growth/differentiation factor (GDF)15 from 100 μl human serum by isotope dilution mass spectrometry, using 15N metabolically labelled and concatamerized fingerprint peptides for the both proteins. Analysis of three different fingerprint peptides for each protein by liquid chromatography electrospray ionization mass spectrometry resulted in comparable concentrations in three healthy human serum samples (DcR3: 27.23 ± 2.49 fmol/ml; GDF15: 98.11 ± 0.49 fmol/ml). In contrast, serum levels were significantly elevated in tumour patients for DcR3 (116.94 ± 57.37 fmol/ml) and GDF15 (164.44 ± 79.31 fmol/ml). Obtained data were in good agreement with ELISA and qPCR measurements, as well as with literature data. In summary, our protocol allows the reliable quantification of biomarkers, shows a higher resolution at low biomarker concentrations than antibody-based strategies, and offers the possibility of multiplexing. Our proof-of-principle studies in patient sera encourage the future analysis of the prognostic value of DcR3 and GDF15 for colon cancer patients in larger patient cohorts. PMID:25823874

  6. Effect of HSA coated iron oxide labeling on human umbilical cord derived mesenchymal stem cells

    NASA Astrophysics Data System (ADS)

    Sanganeria, Purva; Chandra, Sudeshna; Bahadur, Dhirendra; Khanna, Aparna

    2015-03-01

    Human umbilical cord derived mesenchymal stem cells (hUC-MSCs) are known for self-renewal and differentiation into cells of various lineages like bone, cartilage and fat. They have been used in biomedical applications to treat degenerative disorders. However, to exploit the therapeutic potential of stem cells, there is a requirement of sensitive non-invasive imaging techniques which will offer the ability to track transplanted cells, bio-distribution, proliferation and differentiation. In this study, we have analyzed the efficacy of human serum albumin coated iron oxide nanoparticles (HSA-IONPs) on the differentiation of hUC-MSCs. The colloidal stability of the HSA-IONPs was tested over a long period of time (≥20 months) and the optimized concentration of HSA-IONPs for labeling the stem cells was 60 μg ml-1. Detailed in vitro assays have been performed to ascertain the effect of the nanoparticles (NPs) on stem cells. Lactate dehydrogenase (LDH) assay showed minimum release of LDH depicting the least disruptions in cellular membrane. At the same time, mitochondrial impairment of the cells was also not observed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Flow cytometry analysis revealed lesser generation of reactive oxygen species in HSA-IONPs labeled hUC-MSCs in comparison to bare and commercial IONPs. Transmission electron microscopy showed endocytic engulfment of the NPs by the hUC-MSCs. During the process, the gross morphologies of the actin cytoskeleton were found to be intact as shown by immunofluorescence microscopy. Also, the engulfment of the HSA-IONPs did not show any detrimental effect on the differentiation potential of the stem cells into adipocytes, osteocytes and chondrocytes, thereby confirming that the inherent properties of stem cells were maintained.

  7. Spectroscopic study on binding of rutin to human serum albumin

    NASA Astrophysics Data System (ADS)

    Pastukhov, Alexander V.; Levchenko, Lidiya A.; Sadkov, Anatoli P.

    2007-10-01

    Steady-state and time-resolved fluorescence spectroscopy techniques were used to study the interaction of the flavonoid rutin with human serum albumin (HSA) as well as spectral properties of the protein-bound flavonoid. Both quenching of the intrinsic fluorescence of the protein (Trp214) and the ligand fluorescence, appearing upon complexation with HSA, were used to determine binding parameters. The binding constant determined from the quenching of the Trp214 fluorescence by rutin is equal to 6.87 ± 0.22 × 10 4 M -1 and that obtained from the fluorescence of HSA-bound rutin is 3.8 ± 0.4 × 10 4 M -1. Based on the Job plot analysis, the 1:1 binding stoichiometry for the HSA-rutin complex was determined. The efficient quenching of the Trp214 fluorescence by rutin, fluorescence resonance energy transfer from excited Trp214 to rutin, and competitive binding of warfarin indicate that the binding site for the flavonoid is situated within subdomain IIA of HSA. The presence of the sugar moiety in the flavonoid molecule reduces affinity of rutin for binding to HSA but does not affect the binding stoichiometry and location of the binding site compared with aglycone analogues.

  8. Cooperative binding of drugs on human serum albumin

    NASA Astrophysics Data System (ADS)

    Varela, L. M.; Pérez-Rodríguez, M.; García, M.

    In order to explain the adsorption isotherms of the amphiphilic penicillins nafcillin and cloxacillin onto human serum albumin (HSA), a cooperative multilayer adsorption model is introduced, combining the Brunauer-Emmet-Teller (BET) adsorption isotherm with an amphiphilic ionic adsorbate, whose chemical potential is derived from Guggenheim's theory. The non-cooperative model has been previously proved to qualitatively predict the measured adsorption maxima of these drugs [Varela, L. M., García, M., Pérez-Rodríguez, M., Taboada, P., Ruso, J. M., and Mosquera, V., 2001, J. chem. Phys., 114, 7682]. The surface interactions among adsorbed drug molecules are modelled in a mean-field fashion, so the chemical potential of the adsorbate is assumed to include a term proportional to the surface coverage, the constant of proportionality being the lateral interaction energy between bound molecules. The interaction energies obtained from the empirical binding isotherms are of the order of tenths of the thermal energy, therefore suggesting the principal role of van der Waals forces in the binding process.

  9. Heterogeneity of serum low density lipoproteins in normal human subjects

    SciTech Connect

    Shen, M.M.S.; Krauss, R.M.; Lindgren, F.T.; Forte, T.M.

    1981-01-01

    Equilibrium density gradient ultracentrifugation of serum low density lipoprotein (LDL) from twelve healthy human subjects was used to separate six subfractions with mean dinsity ranging from 1.0268 to 1.0597 g/ml. Mean corrected peak flotation rate (S/sup o//sub f/) measured by analytic ultracentrifugation, and mean particle diameter determined by negative staining electron microscopy, both declined significantly with increasing density of the subfractions. Major differences in chemical composition of the subfractions were noted, including a singnificantly lower triglyceride content and higher ratio of cholesteryl ester to triglyceride in the middle fractions compared with those of highest and lowest density. Concentration of fraction 2 correlated positively with HDL (P < 0.01) and negatively with VLDL (P < 0.001); concentration of fraction 4 correlated negatively with HDL (P < 0.05) and positively with VLDL (P < 0.001) and IDL (P < 0.01). LDL may thus include subspecies of differing structure and composition which might also have different metabolic and atherogenic roles.

  10. Binding of amifostine to human serum albumin: a biophysical study.

    PubMed

    Sun, Yifu; Wu, Han; Zhao, Guoqing; Shi, Ying

    2015-02-01

    The aim of this present work is to investigate the interaction between amifostine and human serum albumin (HSA) in simulated physiological conditions by spectroscopic methods to reveal potential toxic effects of the drug. The results reflected that amifostine caused fluorescence quenching of HSA through a static quenching process, which was further confirmed by the electrochemical experiments. The binding constants at 290, 297 and 304 K were obtained as 2.53 × 10(5) /M, 8.13 × 10(4) /M and 3.59 × 10(4) /M, respectively. There may be one binding site of amifostine on HSA. The thermodynamic parameters indicated that the interaction between amifostine and HSA was driven mainly by hydrogen bonding and electrostatic forces. Synchronous fluorescence spectra, circular dichroism and Fourier transform infrared spectroscopy results showed amifostine binding slightly changed the conformation of HSA with secondary structural content changes. Förster resonance energy transfer study revealed high possibility of energy transfer with amifostine-Trp-214 distance of 3.48 nm. The results of the present study may provide valuable information for studying the distribution, toxicological and pharmacological mechanisms of amifostine in vivo. PMID:24962599

  11. Interaction of Human Serum Albumin with Metal Protoporphyrins

    NASA Astrophysics Data System (ADS)

    Hu, Jie; Brancaleon, Lorenzo

    2015-03-01

    Fluorescence spectroscopy is widely used in biotechnology, nanotechnology, and molecular biophysics, since it can provide information on a wide range of molecular processes, e.g. the interactions of solvent molecules with fluorophores, conformational changes, and binding interactions etc. In this study, we present the photophysical properties of the interaction of human serum albumin (HSA) with a series of metal compound of Protoporphyrin IX (PPIX), including ZnPPIX, FePPIX, MgPPIX, MnPPIX and SnPPIX respectively, as well as the free base PPIX. Binding constants were retrieved independently using the Benesi-Hildebrand analysis of the porphyrin emission or absorption spectra and the fluorescence quenching (i.e. Stern-Volmer analysis) and reveal that the two methods yield a difference of approximately one order or magnitude between the two. Fluorescence lifetimes was used to probe whether binding of the porphyrin changes the conformation of the protein or if the interaction places the porphyrin at a location that can prompt resonance energy transfer with the lone Tryptophan residue. In recent years it has been discovered that HSA provides a specific binding site for metal-chelated protoporphyrins in subdomain IA. This has opened a novel field of study over the importance of this site for biomedical applications but it has also created the potential for a series of biotechnological applications of the HSA/protoporphyrin complexes. Our study provides a preliminary investigation of the interaction with metal-chelated protoporphyrins that had not been previously investigated.

  12. Investigation of the interaction between naringin and human serum albumin

    NASA Astrophysics Data System (ADS)

    Zhang, Yaheng; Li, Ying; Dong, Lijun; Li, Jiazhong; He, Wenying; Chen, Xingguo; Hu, Zhide

    2008-03-01

    The interaction between naringin and human serum albumin (HSA) has been thoroughly studied by fluorescence quenching technique in combination with UV absorption spectroscopy, Fourier transform infrared (FT-IR) spectroscopy, circular dichroism (CD) spectroscopy and molecular modeling method. Under the simulative physiological conditions, fluorescence data revealed the presence of the binding site on HSA and its binding constants ( K) are 1.62 × 10 4, 1.68 × 10 4, 1.72 × 10 4, and 1.79 × 10 4 M -1 at 289, 296, 303, and 310 K, respectively. The alterations of protein secondary structure in the presence of naringin aqueous solution were qualitative and quantitative calculated by the evidence from CD and FT-IR spectroscopes. In addition, according to the Van't Hoff equation, the thermodynamic functions standard enthalpy (Δ H0) and standard entropy (Δ S0) for the reaction were calculated to be 3.45 kJ mol -1 and 92.52 J mol -1 K -1. These results indicated that naringin binds to HSA mainly by a hydrophobic interaction. Furthermore, the displacement experiments confirmed that naringin could bind to the site I of HSA, which was also in agreement with the result of the molecular modeling study.

  13. Thermodynamic analysis of hydration in human serum heme-albumin

    SciTech Connect

    Baroni, Simona; Pariani, Giorgio; Fanali, Gabriella; Longo, Dario; Ascenzi, Paolo; Aime, Silvio; Fasano, Mauro

    2009-07-31

    Ferric human serum heme-albumin (heme-HSA) shows a peculiar nuclear magnetic relaxation dispersion (NMRD) behavior that allows to investigate structural and functional properties. Here, we report a thermodynamic analysis of NMRD profiles of heme-HSA between 20 and 60 {sup o}C to characterize its hydration. NMRD profiles, all showing two Lorentzian dispersions at 0.3 and 60 MHz, were analyzed in terms of modulation of the zero field splitting tensor for the S = {sup 5}/{sub 2} manifold. Values of correlation times for tensor fluctuation ({tau}{sub v}) and chemical exchange of water molecules ({tau}{sub M}) show the expected temperature dependence, with activation enthalpies of -1.94 and -2.46 {+-} 0.2 kJ mol{sup -1}, respectively. The cluster of water molecules located in the close proximity of the heme is progressively reduced in size by increasing the temperature, with {Delta}H = 68 {+-} 28 kJ mol{sup -1} and {Delta}S = 200 {+-} 80 J mol{sup -1} K{sup -1}. These results highlight the role of the water solvent in heme-HSA structure-function relationships.

  14. Effects of glycation on meloxicam binding to human serum albumin

    NASA Astrophysics Data System (ADS)

    Trynda-Lemiesz, Lilianna; Wiglusz, Katarzyna

    2011-05-01

    The current study reports a binding of meloxicam a pharmacologically important new generation, non-steroidal anti-inflammatory drug to glycated form of the human serum albumin (HSA). The interaction of the meloxicam with nonglycated and glycated albumin has been studied at pH 7.4 in 0.05 M sodium phosphate buffer with 0.1 M NaCl, using fluorescence quenching technique and circular dichroism spectroscopy. Results of the present study have shown that the meloxicam could bind both forms of albumin glycated and nonglycated at a site, which was close to the tryptophan residues. Similarly, how for native albumin glycated form has had one high affinity site for the drug with association constants of the order of 10 5 M -1. The glycation process of the HSA significantly has affected the impact of the meloxicam on the binding of other ligands such as warfarin and bilirubin. The affinity of the glycated albumin for bilirubin as for native albumin has been reduced by meloxicam but observed effect was weaker by half (about 20%) compared with nonglycated albumin. In contrast to the native albumin meloxicam binding to glycated form of the protein only slightly affected the binding of warfarin. It seemed possible that the effects on warfarin binding might be entirely attributable to the Lys 199 modification which was in site I.

  15. Label-free electrochemical detection of human methyltransferase from tumors.

    PubMed

    Furst, Ariel L; Muren, Natalie B; Hill, Michael G; Barton, Jacqueline K

    2014-10-21

    The role of abnormal DNA methyltransferase activity in the development and progression of cancer is an essential and rapidly growing area of research, both for improved diagnosis and treatment. However, current technologies for the assessment of methyltransferase activity, particularly from crude tumor samples, limit this work because they rely on radioactivity or fluorescence and require bulky instrumentation. Here, we report an electrochemical platform that overcomes these limitations for the label-free detection of human DNA(cytosine-5)-methyltransferase1 (DNMT1) methyltransferase activity, enabling measurements from crude cultured colorectal cancer cell lysates (HCT116) and biopsied tumor tissues. Our multiplexed detection system involving patterning and detection from a secondary electrode array combines low-density DNA monolayer patterning and electrocatalytically amplified DNA charge transport chemistry to measure selectively and sensitively DNMT1 activity within these complex and congested cellular samples. Based on differences in DNMT1 activity measured with this assay, we distinguish colorectal tumor tissue from healthy adjacent tissue, illustrating the effectiveness of this two-electrode platform for clinical applications. PMID:25288757

  16. Strategies for Optimizing the Serum Persistence of Engineered Human Arginase I for Cancer Therapy

    PubMed Central

    Stone, Everett; Chantranupong, Lynne; Gonzalez, Candice; O'Neal, Jamye; Rani, Mridula; VanDenBerg, Carla; Georgiou, George

    2011-01-01

    Systemic l-Arginine depletion following intravenous administration of l-Arginine hydrolyzing enzymes has been shown to selectively impact tumors displaying urea-cycle defects including a large fraction of hepatocellular carcinomas, metastatic melanomas and small cell lung carcinomas. However, the human arginases display poor serum stability (t1/2 =4.8 hrs) whereas a bacterial arginine deiminase evaluated in phase II clinical trials was reported to be immunogenic, eliciting strong neutralizing antibody responses. Recently, we showed that substitution of the Mn2+ metal center in human Arginase I with Co2+ (Co-hArgI) results in an enzyme that displays 10-fold higher catalytic efficiency for l-Arg hydrolysis, 12–15 fold reduction in the IC50 towards a variety of malignant cell lines and, importantly a t1/2= 22 hrs in serum. To investigate the utility of Co-hArgI for l-Arg depletion therapy in cancer we systematically investigated three strategies for enhancing the persistence of the enzyme in circulation: (i) site specific conjugation of Co-hArgI engineered with an accessible N-terminal Cys residue to 20 KDa PEG-maleimide (Co-hArgI-CPEG-20K); (ii) engineering of the homotrimeric Co-hArgI into a linked, monomeric 110 KDa polypeptide (Co-hArgI ×3) and (iii) lysyl conjugation of 5 KDa PEG-N-hydroxysuccinimide (NHS) ester (Co-hArgI-KPEG-5K). Surprisingly, even though all three formulations resulted in proteins with a predicted hydrodynamic radius larger than the cut-off for renal filtration, only CohArgI amine conjugated to 5 KDa PEG remained in circulation for sufficiently long durations. Using CohArgI-KPEG-5K labeled with an end-terminal fluorescein for easy detection, we demonstrated that following intraperitoneal administration at 6 mg/kg weight, a well tolerated dose, the circulation t1/2 of the protein in Balb/c mice is 63 ± 10 hrs. Very low levels of serum l-Arg (<5 μM) could be sustained for over 75 hrs after injection, representing a 9-fold increase in

  17. Inactivation of phosphoglycerate mutase and creatine kinase isoenzymes in human serum

    PubMed Central

    Durany, N; Carreras, J; Valentí, M; Cámara, J; Carreras, J

    2002-01-01

    Aims/Background: Total phosphoglycerate mutase (PGM) activity in serum has been shown to be increased in acute myocardial infarction with the same time course as creatine kinase (CK) activity. However, the increase in the muscle (MM) and in the cardiac (MB) PGM isoenzymes was not as high as expected. The present study was undertaken to characterise PGM inactivation by serum and to compare it with serum CK inactivation. Methods: The PGM and the CK activities of extracts of human heart, skeletal muscle, and brain were determined spectrophotometrically after incubation with different media, namely: plasma, whole serum, dialysed serum, heated serum, serum ultrafiltrate, urate solution, and buffer solution. Results: Type MM PGM was inactivated by plasma, whole serum, heated serum, dialysed serum, and serum ultrafiltrate. Inactivation in dialysed serum was reduced by EDTA and largely reversed by thiol agents. Inactivation in serum ultrafiltrate was not prevented by EDTA and only partially reversed by dithiothreitol. The muscle and type BB CK isoenzymes were inactivated in all the tested media. The incubation of human and rabbit skeletal muscle PGM and CK in urate solution showed that urate does not affect mutase activity under conditions that inactivate CK. Conclusions: These results confirm the mechanisms of CK inactivation proposed by others and show that the type M PGM subunit is inactivated by two different mechanisms, which appear to involve the thiol groups of the enzyme. One mechanism is caused by either a protein component or a protein bound serum component and involves calcium ions and/or another chelatable metal ion. The other mechanism is caused by a lower molecular weight serum component and is metal ion independent. PMID:12147715

  18. Membrane changes induced by exposure of Escherichia coli to human serum.

    PubMed Central

    Kroll, H P; Bhakdi, S; Taylor, P W

    1983-01-01

    The effect of bactericidal concentrations of lysozyme-free human serum on parameters of membrane integrity has been studied in serum-susceptible and serum-resistant Escherichia coli strains. Serum treatment released all of the alkaline phosphatase from the periplasmic space of two rapidly serum-susceptible strains but did so at different rates. In contrast, no periplasmic enzyme was released from two serum-resistant strains or from one moderately susceptible smooth strain. Lysozyme-free serum and heat-inactivated serum released comparable amounts of 86Rb+ from preloaded cells at comparable rates, regardless of serum susceptibility. Serum decreased the rate of phospholipid biosynthesis in both serum-susceptible and serum-resistant strains. In susceptible but not in resistant strains, intracellular ATP pools were depleted after serum exposure. Outer membranes and cytoplasmic membranes were prepared from serum-treated E. coli, and assays for C3 and C5b-9(m) were performed. With rapidly susceptible strains, C3 deposition on the outer membrane without attachment of C5b-9(m) occurred during the short prekilling phase. Subsequent bacterial killing was accompanied by deposition of C5b-9(m), which was recovered with C3 exclusively in outer membrane fractions with increased density and by eventual total loss of recoverable cytoplasmic membranes. Minimal deposition of complement components, without accompanying cytoplasmic membrane loss, occurred with serum-resistant strains. Loss of recoverable cytoplasmic membrane was not due to the action of either serum or bacterial phospholipase A. The results raise the possibilities that C5b-9(m) primarily damages the outer membrane and that the bacteria themselves actively participate in the ensuing, as yet unclarified, metabolic reactions that finally lead to their death. Images PMID:6358036

  19. Optimization of a colorimetric assay for glycosylated human serum albumin

    SciTech Connect

    Bohney, J.P.; Feldhoff, R.C.

    1986-05-01

    The thiobarbituric acid (TBA) assay has been used for several years to quantitate the amount of glucose which has been non-enzymatically linked to hemoglobin and other proteins. The ketoamine-protein adduct is converted to 5-hydroxymethylfurfural (HMF) by mild hydrolysis with oxalic acid. Reaction of HMF with TBA yields a colored product which has an absorbance maximum at 443 nm. Several modifications of the original procedure has been published, but none permit the unambiguous quantitation of glycosylated human serum albumin (glc-HSA). Problems relate to reagent preparation and stability, the time and temperature of hydrolysis, the choice of standards, and background color corrections. The authors have found that maximum color yield occurs after hydrolysis in an autoclave for 2 h. This increases the sensitivity 3-fold and cuts the assay time in half relative to hydrolysis for 4.5 h at 100/sup 0/C. A NaBH/sub 4/ reduction of a parallel protein sample must be performed to correct for variable background color associated with different sample sources and amounts. HMF can be used as a standard, however, corrections must be made for HMF degradation. Fructose is a better standard, but HMF formation from fructose is faster than formation from glc-HSA. This may result in an underestimate of percent glycosylation. The best standard appears to be glc-HSA prepared with (/sup 3/H)glucose. It appears that with proper controls and standards the TBA assay can be used to determine actual rather than relative percent glycosylation.

  20. Structural basis of transport of lysophospholipids by human serum albumin

    SciTech Connect

    Guo, Shihui; Shi, Xiaoli; Yang, Feng; Chen, Liqing; Meehan, Edward J.; Bian, Chuanbing; Huang, Mingdong

    2010-10-08

    Lysophospholipids play important roles in cellular signal transduction and are implicated in many biological processes, including tumorigenesis, angiogenesis, immunity, atherosclerosis, arteriosclerosis, cancer and neuronal survival. The intracellular transport of lysophospholipids is through FA (fatty acid)-binding protein. Lysophospholipids are also found in the extracellular space. However, the transport mechanism of lysophospholipids in the extracellular space is unknown. HSA (human serum albumin) is the most abundant carrier protein in blood plasma and plays an important role in determining the absorption, distribution, metabolism and excretion of drugs. In the present study, LPE (lysophosphatidylethanolamine) was used as the ligand to analyse the interaction of lysophospholipids with HSA by fluorescence quenching and crystallography. Fluorescence measurement showed that LPE binds to HSA with a K{sub d} (dissociation constant) of 5.6 {micro}M. The presence of FA (myristate) decreases this binding affinity (K{sub d} of 12.9 {micro}M). Moreover, we determined the crystal structure of HSA in complex with both myristate and LPE and showed that LPE binds at Sudlow site I located in subdomain IIA. LPE occupies two of the three subsites in Sudlow site I, with the LPE acyl chain occupying the hydrophobic bottom of Sudlow site I and the polar head group located at Sudlow site I entrance region pointing to the solvent. This orientation of LPE in HSA suggests that HSA is capable of accommodating other lysophospholipids and phospholipids. The study provides structural information on HSA-lysophospholipid interaction and may facilitate our understanding of the transport and distribution of lysophospholipids.

  1. Effects of Beryllium on Human Serum Immunoglobulin and Lymphocyte Subpopulation

    PubMed Central

    Kim, DaeSeong; Won, Yong Lim; Kang, Seong-Kyu

    2013-01-01

    To investigate the effects of short-term exposure of beryllium on the human immune system, the proportion of T-lymphocytes such as CD3+, CD4+, CD8+, CD95, and NK cells, andthe proportion of B cells and TNFα level in peripheral blood and immunoglobulins in the serum of 43 exposed workers and 34 healthy control subjects were studied. External exposure to beryllium was measured by atomic absorption spectrometer as recommended by the NIOSH analytical method 7300. T lymphocyte subpopulation analysis was carried out with flow cytometer. The working duration of exposed workers was less than 3 months and the mean ambient beryllium level was 3.4 μg/m3, 112.3 μg/m3, and 2.3 μg/m3 in molding (furnace), deforming (grinding), and sorting processes, respectively (cited from Kim et al., 2008). However, ambient beryllium level after process change was non-detectable (< 0.1 μg/m3). The number of T lymphocytes and the amount of immunoglobulins in the beryllium-exposed workers and control subjects were not significantly different, except for the total number of lymphocytes and CD95 (APO1/FAS). The total number of lymphocytes was higher in the beryllium-exposed individuals than in the healthy control subjects. Multiple logistic regression analysis showed lymphocytes to be affected by beryllium exposure (odd ratio = 7.293; p < 0.001). These results show that short-term exposure to beryllium does not induce immune dysfunction but is probably associated with lymphocytes proliferation. PMID:24278637

  2. HPLC-DAD protein kinase inhibitor analysis in human serum.

    PubMed

    Dziadosz, Marek; Lessig, Rüdiger; Bartels, Heidemarie

    2012-04-15

    We here describe an HPLC-DAD method to analyse different protein kinase inhibitors. Potential applications of this method are pharmacokinetic studies and therapeutic drug monitoring. Optimised chromatography conditions resulted in a very good separation of seven inhibitors (vatalanib, bosutinib, canertinib, tandutinib, pazopanib, dasatinib - internal standard and erlotinib). The good sensitivity makes this method competitive with LC/MS/MS. The separation was performed with a Lichrospher 100-5 RP8, 250 mm × 4 mm column maintained at 30 ± 1 °C, and with a mobile phase of 0.05 M H(3)PO(4)/KH(2)PO(4) (pH=2.3)-acetonitrile (7:3, v/v) at a flow rate of 0.7 mL/min. A simple and fast sample preparation sequence with liquid-liquid extraction led to good recoveries (73-90%) of all analytes. The recovery hardly reached 50% only for pazopanib. This method can also be used for targeted protein kinase inhibitor quantification. A perfect linearity in the validated range (20-10,000 ng/mL) and an LOQ of 20 ng/mL were achieved. The relative standard deviations and accuracies of all examined drug concentrations gave values much lower than 15% both for between- and within-batch calculations. All analysed PKIs were stable for 6 months in a 1mg/mL dimethyl sulfoxide stock solution. Vatalanib, bosutinib and erlotinib were also stable in human serum in the whole examined concentration range. PMID:22425385

  3. Photoaffinity labeling of the human erythrocyte glucose transporter with /sup 4/H-labelled forskolin

    SciTech Connect

    Shanahan, M.F.; Edwards, B.M.; Morris, D.P.

    1986-05-01

    Forskolin, a potent activator of adenylate cyclase, is also known to inhibit glucose transport in a number of cells. The authors have investigated photoincorporation of (/sup 3/H)forskolin into erythrocyte membrane proteins using a technique they previously developed for photolabeling the erythrocyte glucose transporter with cytochalasin B (CB). A 30-40s irradiation of erythrocyte ghosts in the presence of (/sup 3/H)forskolin resulted in a concentration-dependent, covalent incorporation of radiolabel into all of the major membrane protein bands. However, most of the incorporation occurred in only three regions of the gel. Peak 1 was a sharp peak near the top of the gel in the region corresponding to spectrin, peak 2 appeared to be associated with band 3 (approx. 90kDa), and the third region labeled was between 41-60 kDa which corresponds to the region of the glucose transporter. This region appeared to contain several overlapping peaks with the largest incorporation of label occurring around 45 kDa in the area of red cell actin. When photolabeling was performed in the presence of 400 ..mu..M cytochalasin B (8.0 ..mu..M forskolin) the labeling in the 41-60 kDa region was totally inhibited while labeling of the 90 kDa peak was partially blocked. CB had no effect on the photolabeling of peak 1 by forskolin.

  4. Probing thyroglobulin in undiluted human serum based on pattern recognition and competitive adsorption of proteins

    NASA Astrophysics Data System (ADS)

    Wang, Ran; Huang, Shuai; Li, Jing; Chae, Junseok

    2014-10-01

    Thyroglobulin (Tg) is a sensitive indicator of persistent or recurrent differentiated thyroid cancer of follicular cell origin. Detection of Tg in human serum is challenging as bio-receptors, such as anti-Tg, used in immunoassay have relatively weak binding affinity. We engineer sensing surfaces using the competitive adsorption of proteins, termed the Vroman Effect. Coupled with Surface Plasmon Resonance, the "cross-responsive" interactions of Tg on the engineered surfaces produce uniquely distinguishable multiple signature patterns, which are discriminated using Linear Discriminant Analysis. Tg-spiked samples, down to 2 ng/ml Tg in undiluted human serum, are sensitively and selectively discriminated from the control (undiluted human serum).

  5. Evaluation of Serum Creatinine Changes With Integrase Inhibitor Use in Human Immunodeficiency Virus-1 Infected Adults

    PubMed Central

    Lindeman, Tara A.; Duggan, Joan M.; Sahloff, Eric G.

    2016-01-01

    This retrospective chart review evaluated changes in serum creatinine and creatinine clearance (CrCl) after initiation of an integrase inhibitor (INSTI)-based regimen as initial treatment in human immunodeficiency virus-infected adults. Serum creatinine and CrCl changes were similar to those seen in clinical trials for INSTIs. No renal-related serious adverse events or discontinuations occurred. PMID:27092314

  6. The Reactivity of Human Serum Albumin towards trans-4-Hydroxy-2-nonenal

    PubMed Central

    Liu, Qingyuan; Simpson, David C.; Gronert, Scott

    2012-01-01

    Mass spectrometry was used to probe the preferred locations of trans-4-hydroxy-2-nonenal (HNE) addition to the cysteine, histidine, and lysine residues of human serum albumin (HSA). Considering only those modified peptides supported by high mass accuracy Orbitrap precursor ion measurements (high confidence hits), with HNE:HSA ratios of 1:1 and 10:1, 3 and 15 addition sites, respectively, were identified. Using less stringent criteria, a total of 34 modifications were identified at the higher concentration. To gain quantitative data, iTRAQ labeling studies were completed. Previous work had identified Cys34, the only free cysteine, as the most reactive residue in HSA and we have found that Lys199, His242/7, and His288 are the next most reactive residues. Although the kinetic data indicate the lysines and histidines can react at relatively similar rates, the results show that lysine addition is much less favorable thermodynamically; under our reaction conditions, lysine addition generally does not go to completion. This suggests that under physiological conditions, HNE addition to lysine is only relevant in situations where unusually high HNE concentrations or access to irreversible secondary reactions are found. PMID:22689617

  7. A xenograft mouse model coupled with in-depth plasma proteome analysis facilitates identification of novel serum biomarkers for human ovarian cancer.

    PubMed

    Tang, Hsin-Yao; Beer, Lynn A; Chang-Wong, Tony; Hammond, Rachel; Gimotty, Phyllis; Coukos, George; Speicher, David W

    2012-02-01

    Proteomics discovery of novel cancer serum biomarkers is hindered by the great complexity of serum, patient-to-patient variability, and triggering by the tumor of an acute-phase inflammatory reaction. This host response alters many serum protein levels in cancer patients, but these changes have low specificity as they can be triggered by diverse causes. We addressed these hurdles by utilizing a xenograft mouse model coupled with an in-depth 4-D protein profiling method to identify human proteins in the mouse serum. This strategy ensures that identified putative biomarkers are shed by the tumor, and detection of low-abundance proteins shed by the tumor is enhanced because the mouse blood volume is more than a thousand times smaller than that of a human. Using TOV-112D ovarian tumors, more than 200 human proteins were identified in the mouse serum, including novel candidate biomarkers and proteins previously reported to be elevated in either ovarian tumors or the blood of ovarian cancer patients. Subsequent quantitation of selected putative biomarkers in human sera using label-free multiple reaction monitoring (MRM) mass spectrometry (MS) showed that chloride intracellular channel 1, the mature form of cathepsin D, and peroxiredoxin 6 were elevated significantly in sera from ovarian carcinoma patients. PMID:22032327

  8. Fluid Secretion in Isolated Proximal Straight Renal Tubules EFFECT OF HUMAN UREMIC SERUM

    PubMed Central

    Grantham, Jared J.; Irwin, Richard L.; Qualizza, Patti B.; Tucker, Donald R.; Whittier, Frederick C.

    1973-01-01

    We have examined the effect of normal and uremic human sera on the transtubular flow of fluid in isolated perfused segments of rabbit proximal convoluted and straight renal tubules. Proximal convoluted and straight tubules absorbed fluid from the lumen when the external bath was normal rabbit serum. Normal human sera in the bath depressed net fluid absorption in both tubular segments, but more importantly, uremic human serum caused proximal straight tubules to secrete fluid into the lumen. Fluid secretion was also demonstrated indirectly by observing in nonperfused proximal straight, but not proximal convoluted tubules, that the normally collapsed lumens opened widely in uremic serum. Nonperfused proximal straight tubules developed expanded lumens even after a 25-fold dilution of human uremic serum with normal rabbit serum, whereas lumen expansion occurred only in undiluted normal human serum, on the average. Serum from acutely uremic rabbits possessed secretory activity but normal rabbit serum did not. The secretory effect of uremic sera in proximal straight tubules was inhibited by cooling and ouabain and probenecid. The secretory activity of uremic sera was removed by dialysis, but not by freezing or boiling. Para-aminohippurate and benzoate caused fluid secretion in proximal straight tubules but urea, creatinine, guanidinosuccinate, and urate did not. On the basis of these results, we suggest that the secretory factor in serum may be a substance or group of substances possibly related to the hippurate class of organic molecules that are accumulated to relatively high concentrations in renal failure. The secretory material in the serum of uremic patients may significantly influence the transport of salt and water in relatively intact residual nephrons. Images PMID:4738063

  9. Development and validation of an LC-MS/MS method for determination of phencyclidine in human serum and its application to human drug abuse cases

    PubMed Central

    Chimalakonda, Krishna C.; Hailey, Chris; Black, Ryan; Beekman, Allison; Carlisle, Rebecca; Lowman-Smith, Elizabeth; Singletary, Heathe; Owens, S. Michael; Hendrickson, Howard

    2010-01-01

    A new analytical method was developed and validated for the rapid determination of phencyclidine (PCP) in human blood and serum. Rapid chromatographic separation decreased the analysis time relative to standard gas chromatography (GC)-based methodologies. The method involved the use of solid-phase extraction for sample preparation and cleanup followed by liquid chromatography tandem spectrometric (LC-MS/MS) analysis and an electrospray-ionization (ESI) interface. PCP was quantified using multiple-reaction-monitoring with deuterium labeled PCP (PCP-d5) as an internal standard. The method was validated for accuracy, precision, linearity, and recovery. The method was accurate with error <14% and precision with coefficient of variation (CV) <5.0%. The assay was linear over the entire range of calibration standards (r2 > 0.997). The recovery of PCP after solid-phase extraction was greater than 90% with the lower limit of detection (LLOD) for PCP in 500 µl of human serum after solid-phase extraction at 0.06 ng ml−1. This method was used to determine the levels of PCP in postmortem human blood samples. The LLOD in blood was 1 ng ml−1. Blood PCP concentrations were also determined separately using GC and flame ionization detection (FID). Blood calibration standards and serum calibration standards yielded similar concentrations when used to quantitate authentic human blood samples that tested positive for PCP under the GC-FID method. Extraction of PCP from serum required fewer steps and therefore could be used as a calibration matrix in place of blood. The LC-MS/MS methodology shown here was higher throughput compared with GC-based methods because of very short chromatographic run times. This was accomplished without sacrificing analytical sensitivity. PMID:20959870

  10. Elevated serum melatonin levels during human late pregnancy and labour.

    PubMed

    Wierrani, F; Grin, W; Hlawka, B; Kroiss, A; Grünberger, W

    1997-09-01

    Melatonin (MLT) shows an influence on gonadal steroid genesis, and has soporific effects. Serum MLT levels were examined during late pregnancy and 4 days after delivery in 25 women. Circulating levels of melatonin were analysed as integrated values (areas under the curve [AUC]) over 24 hours, 5 to 2 days before and 4 days after delivery. Antepartum AUCs were significantly increased compared with postpartum AUCs. Additionally, MLT levels were measured every 2 hours in a subgroup of 11 women during spontaneous labour between 08.00 and 12.00 h at a time when physiological serum MLT levels were low. Increased MLT levels were determined and compared to MLT levels measured in a previous evaluation of the antepartum AUCs. Elevated serum MLT levels during late-pregnancy and labour may influence the concentration of receptors of gonadal steroids in the gravid uterus at term and the psychic perception of painful uterine contractions during labour. PMID:15511919

  11. Endocytosis of a mannose-terminated glycoprotein and formaldehyde-treated human serum albumin in liver and kidney cells from fish (Salmo alpinus L.).

    PubMed

    Smedsrud, T; Dannevig, B H; Tolleshaug, H; Berg, T

    1984-01-01

    The uptake and degradation of a mannose-terminated glycoprotein, yeast invertase, in char (Salmo alpinus L.) tissue was studied after intravenously injection of the 125I-labelled protein. 125I-labelled formaldehyde-treated human serum albumin (fHSA) and native HSA was also injected for comparison. Labelled invertase was rapidly cleared from blood and at about the same rate as labelled fHSA (at 8 degrees C). Approximately 50% of the initial concentration remained in blood 15 min after the injection of the ligands. Acid soluble degradation products appeared in the circulation about 60 min after the injection of the proteins. 125I-labelled invertase was recovered in the liver, pronephros and kidney. The clearance of labelled invertase from blood and the uptake in the organs were inhibited by co-injection of excess unlabelled invertase. fHSA was taken up in the pronephros and kidney tissue, while HSA was not taken up in any organs. In vitro degradation of the labelled ligands was studied in isolated pronephros cells, which had taken up the proteins in vivo. The degradation of invertase in isolated cells was partly inhibited by ammonium chloride. Ammonium chloride and chloroquine inhibited degradation of fHSA, but not leupeptin. These results together suggest that invertase and fHSA were taken up in the organs described by the receptor-mediated endocytosis. The degradation was partly or wholly lysosomal. PMID:6500136

  12. The conversion of 16β hydroxyldehydroepiandrosterone in human serum.

    PubMed

    Ke, Yuyong; Gonthier, Renaud; Labrie, Fernand

    2016-05-01

    The circulating levels of 16β hydroxydehydroepiandrosterone (16β OH-DHEA) are at the limit of detection (less than 10pg/mL), unlike the serum concentrations of 16α-hydroxydehydroepiandrosterone (16α OH-DHEA, 10-300pg/mL) in premenopausal, postmenopausal and male serum. A major reason could be the rapid conversion of 16β OH-DHEA to 5-androstene-3beta, 17beta-diol 16 one (3β, 17β-diol 16-oxo) in serum due to the stereospecific structure of 16β OH-DHEA. In ultrapure H2O, there is no apparent conversion observed while 16β OH-DHEA (10ng/mL) spiked in stripped or unstripped serum is quickly converted to 3β, 17β-diol 16-oxo at room temperature. During this conversion, a further converted product was observed with a difference in molecular weight of 16Da from that of 16β OH-DHEA and 3β, 17β-diol 16-oxo, which could be their hydroxylation product, i.e. triol-ketone. Under basic conditions, further conversion occurs. The present data can explain the practically undetectable concentration of serum 16β OH-DHEA while 3β, 17β-diol 16-oxo is at the level of less than 50pg/mL. Serum concentrations of (0.0-9.9pg/mL for 16β OH-DHEA, 8.9-50.7pg/mL for 3β, 17β-diol 16-oxo and 10.0-285.0pg/mL for 16α OH-DHEA are measured in sera of premenopausal, postmenopausal women and men over 50years of age. PMID:26896786

  13. Neutralization of Human Serum β-Lysin by Sodium Polyanetholsulfonate and Sodium Amylosulfate

    PubMed Central

    Traub, Walter H.; Ima, Paula I. Fukushi

    1979-01-01

    Normal fresh and heat-inactivated (56°C, 30 min) human sera (80 vol%, i.e., 80% [vol/vol] of a 2-ml assay volume) killed Bacillus subtilis ATCC 6633 cell inocula of 1.5 × 104 colony-forming units per ml within 1 to 2 h after exposure. The B. subtilis assay strain proved slightly and reversibly susceptible to 5 μg of egg white lysozyme per ml. Seitz filtration of fresh human serum completely removed β-lysin activity; significant amounts of serum lysozyme were removed as well, as determined with the bioassay strain Micrococcus lysodeikticus ATCC 4698. However, bactericidal activity of human serum via classical or alternative complement pathway activation remained intact. Addition of 0.01 M dithiothreitol to fresh human serum abolished β-lysin activity, but not that of serum lysozyme. Chelation of fresh and heat-inactivated human serum with 0.01 M MgCl2 + 0.01 M ethylene glycol tetraacetic acid, but not with 0.01 M ethylenediaminetetraacetic acid, markedly retarded β-lysin activity; however, lysozyme activity remained unaffected. Chelation of serum with 0.01 M MgCl2 + 0.01 M ethylene glycol tetraacetic acid + 0.01 M CaCl2 completely abrogated β-lysin activity, but not that of lysozyme. Absorption of human serum with 10 mg of bentonite per ml (10 min, 37°C) completely removed β-lysin and lysozyme activity, but failed to affect serum bactericidal activity against Escherichia coli control strain C. Reconstitution of 50 vol% of bentonite-absorbed serum with 40 vol% of heat-inactivated human serum restored both β-lysin and lysozyme activity. Addition of either 63 to 500 μg of sodium polyanetholsulfonate per ml or 63 to 500 μg of sodium amylosulfate per ml to 80 vol% of fresh human serum completely neutralized β-lysin activity for the entire observation period of 22 h. PMID:227918

  14. NAA For Human Serum Analysis: Comparison With Conventional Analyses

    SciTech Connect

    Oliveira, Laura C.; Zamboni, Cibele B.; Medeiros, Jose A. G.; Azevedo, Maria R.

    2010-08-04

    Instrumental and Comparator methods of Neutron Activation Analysis (NAA) were applied to determine elements of clinical relevancy in serum samples of adult population (Sao Paulo city, Brazil). A comparison with the conventional analyses, Colorimetric for calcium, Titrymetric for chlorine and Ion Specific Electrode for sodium and potassium determination were also performed permitting a discussion about the performance of NAA methods for clinical chemistry research.

  15. Antiplasmin activity of electrophoretically separated human serum fractions

    PubMed Central

    Mann, R. D.; Cotton, Susan; Jackson, D.

    1966-01-01

    The antiplasmin which migrates electrophoretically with the alpha2 globulins preponderates in effect over that of the alpha1 migrating antiplasmin. This preponderance persists at physiological pH value in vitro and the significance of this finding is discussed. No evidence has been obtained of the existence of anti-urokinase activity in antiplasmin-free serum fractions. PMID:4160096

  16. Etofenamate levels in human serum and synovial fluid following iontophoresis.

    PubMed

    Bender, T; Bariska, J; Rojkovich, B; Bálint, G

    2001-01-01

    The absorption of etofenamate (CAS 30544-47-9, Rheumon gel) by iontophoresis in 11 patients with low back pain and in 13 patients with synovitis of the knee was evaluated. During the 5-day treatment period, the test gel in a quantity corresponding to 100 mg etofenamate was applied to affected body regions every day by 20-min iontophoresis sessions. Two hours after the fifth application, the concentration of etofenamate in serum and synovial fluid (in patients who had knee joint iontophoresis) were measured by HPLC. Iontophoresis of etofenamate into the lumbar region as well as to the knee joint resulted in consistent serum levels: 219 +/- 136.3 micrograms/l and 191 +/- 84.6 micrograms/l, respectively. In patients with synovitis of the knee, the synovial level of etofenamate (368 +/- 109.2 micrograms/l) was almost twice as high than the serum concentration. The authors conclude that with topical application of etofenamate by iontophoresis the drug appears not only in the serum but also--with higher levels--in the synovial fluid. PMID:11455681

  17. Aberrant sialylation of a prostate-specific antigen: Electrochemical label-free glycoprofiling in prostate cancer serum samples.

    PubMed

    Pihikova, Dominika; Kasak, Peter; Kubanikova, Petra; Sokol, Roman; Tkac, Jan

    2016-08-31

    Electrochemical detection method allowing to detect prostate-specific antigen (PSA), a biomarker of prostate cancer (PCa), with PSA glycoprofiling was applied in an analysis of PCa serum samples for the first time. Electrochemical impedance spectroscopy (EIS) as a label-free method with immobilized anti-PSA was applied for PSA detection and lectins to glycoprofile captured PSA on the same surface. A proper choice of blocking agent providing high selectivity of biosensor detection with the immobilized anti-PSA antibody was done. The biosensor could detect PSA down to 100 ag/mL with a linear concentration working range from 100 ag/mL up to 1 μg/mL, i.e. 10 orders of concentration magnitude and the sensitivity of (5.5 ± 0.2)%/decade. The results showed that a commercial carbo-free blocking solution was the best one, reducing non-specific binding 55-fold when compared to the immunosensor surface without any blocking agent applied, while allowing to detect PSA. The biosensor response obtained after addition of lectin (i.e. proportional to the amount of a particular glycan on PSA) divided by the biosensor response obtained after incubation with a sample (i.e. proportional to the PSA level in the sample) was applied to distinguish serum samples of PCa patients from those of healthy individuals. The results showed that Maackia amurensis agglutinin (MAA) recognizing α-2,3-terminal sialic acid can be applied to distinguish between these two sets of samples since the MAA/PSA response obtained from the analysis of the PCa samples was significantly higher (5.3-fold) compared to the MAA/PSA response obtained by the analysis of samples from healthy individuals. Thus, combined analysis of serological PSA levels together with PSA glycoprofiling of aberrant glycosylation of PSA (i.e. increase in the level of α-2,3-terminal sialic acid) has a potential to improve detection of PCa. PMID:27506346

  18. Comparative Analysis of KnockOut™ Serum with Fetal Bovine Serum for the In Vitro Long-Term Culture of Human Limbal Epithelial Cells

    PubMed Central

    Liu, Zaoxia

    2016-01-01

    The limbal epithelial cells can be maintained on 3T3 feeder layer with fetal bovine serum supplemented culture medium, and these cells have been used to successfully treat limbal stem cell deficiency. However, fetal bovine serum contains unknown components and displays quantitative and qualitative lot-to-lot variations. To improve the culture condition, the defined KnockOut serum replacement was investigated to replace fetal bovine serum for culturing human limbal epithelial cell. Human primary limbal epithelial cells were cultured in KnockOut serum and fetal bovine serum supplemented medium, respectively. The cell growth rate, gene expression, and maintenance of limbal epithelial stem cells were studied and compared between these two groups. Human primary limbal epithelial cells were isolated and successfully serially cultivated in this novel KnockOut serum supplemented medium; the cell proliferation and stem cell maintenance were similar to those of cells grown in fetal bovine serum supplemented medium. These data suggests that this KnockOut serum supplemented medium is an efficient replacement to traditional fetal bovine serum supplemented medium for limbal epithelial cell culture, and this medium has great potential for long term maintenance of limbal epithelial cells, limbal epithelial stem cells transplantation, and tissue regeneration. PMID:27446607

  19. Label-Free LC-MSe in Tissue and Serum Reveals Protein Networks Underlying Differences between Benign and Malignant Serous Ovarian Tumors

    PubMed Central

    Wegdam, Wouter; Argmann, Carmen A.; Kramer, Gertjan; Vissers, Johannes P.; Buist, Marrije R.; Kenter, Gemma G.; Aerts, Johannes M. F. G.; Meijer, Danielle; Moerland, Perry D.

    2014-01-01

    Purpose To identify proteins and (molecular/biological) pathways associated with differences between benign and malignant epithelial ovarian tumors. Experimental Procedures Serum of six patients with a serous adenocarcinoma of the ovary was collected before treatment, with a control group consisting of six matched patients with a serous cystadenoma. In addition to the serum, homogeneous regions of cells exhibiting uniform histology were isolated from benign and cancerous tissue by laser microdissection. We subsequently employed label-free liquid chromatography tandem mass spectrometry (LC-MSe) to identify proteins in these serum and tissues samples. Analyses of differential expression between samples were performed using Bioconductor packages and in-house scripts in the statistical software package R. Hierarchical clustering and pathway enrichment analyses were performed, as well as network enrichment and interactome analysis using MetaCore. Results In total, we identified 20 and 71 proteins that were significantly differentially expressed between benign and malignant serum and tissue samples, respectively. The differentially expressed protein sets in serum and tissue largely differed with only 2 proteins in common. MetaCore network analysis, however inferred GCR-alpha and Sp1 as common transcriptional regulators. Interactome analysis highlighted 14-3-3 zeta/delta, 14-3-3 beta/alpha, Alpha-actinin 4, HSP60, and PCBP1 as critical proteins in the tumor proteome signature based on their relative overconnectivity. The data have been deposited to the ProteomeXchange with identifier PXD001084. Discussion Our analysis identified proteins with both novel and previously known associations to ovarian cancer biology. Despite the small overlap between differentially expressed protein sets in serum and tissue, APOA1 and Serotransferrin were significantly lower expressed in both serum and cancer tissue samples, suggesting a tissue-derived effect in serum. Pathway and subsequent

  20. 21 CFR 201.26 - Exceptions or alternatives to labeling requirements for human drug products held by the Strategic...

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 21 Food and Drugs 4 2014-04-01 2014-04-01 false Exceptions or alternatives to labeling requirements for human drug products held by the Strategic National Stockpile. 201.26 Section 201.26 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) DRUGS: GENERAL LABELING General Labeling Provisions §...

  1. Human serum activates CIDEB-mediated lipid droplet enlargement in hepatoma cells

    SciTech Connect

    Singaravelu, Ragunath; Lyn, Rodney K.; Srinivasan, Prashanth; Delcorde, Julie; Steenbergen, Rineke H.; Tyrrell, D. Lorne; Pezacki, John P.

    2013-11-15

    Highlights: •Human serum induced differentiation of hepatoma cells increases cellular lipid droplet (LD) size. •The observed increase in LD size correlates with increased PGC-1α and CIDEB expression. •Induction of CIDEB expression correlates with rescue of VLDL secretion and loss of ADRP. •siRNA knockdown of CIDEB impairs the human serum mediated increase in LD size. •This system represents a cost-efficient model to study CIDEB’s role in lipid biology. -- Abstract: Human hepatocytes constitutively express the lipid droplet (LD) associated protein cell death-inducing DFFA-like effector B (CIDEB). CIDEB mediates LD fusion, as well as very-low-density lipoprotein (VLDL) maturation. However, there are limited cell culture models readily available to study CIDEB’s role in these biological processes, as hepatoma cell lines express negligible levels of CIDEB. Recent work has highlighted the ability of human serum to differentiate hepatoma cells. Herein, we demonstrate that culturing Huh7.5 cells in media supplemented with human serum activates CIDEB expression. This activation occurs through the induced expression of PGC-1α, a positive transcriptional regulator of CIDEB. Coherent anti-Stokes Raman scattering (CARS) microscopy revealed a correlation between CIDEB levels and LD size in human serum treated Huh7.5 cells. Human serum treatment also resulted in a rapid decrease in the levels of adipose differentiation-related protein (ADRP). Furthermore, individual overexpression of CIDEB was sufficient to down-regulate ADRP protein levels. siRNA knockdown of CIDEB revealed that the human serum mediated increase in LD size was CIDEB-dependent. Overall, our work highlights CIDEB’s role in LD fusion, and presents a new model system to study the PGC-1α/CIDEB pathway’s role in LD dynamics and the VLDL pathway.

  2. Serum Phosphorus Levels in Premature Infants Receiving a Donor Human Milk Derived Fortifier

    PubMed Central

    Chetta, Katherine E.; Hair, Amy B.; Hawthorne, Keli M.; Abrams, Steven A.

    2015-01-01

    An elevated serum phosphorus (P) has been anecdotally described in premature infants receiving human milk fortified with donor human milk-derived fortifier (HMDF). No studies have prospectively investigated serum P in premature infants receiving this fortification strategy. In this single center prospective observational cohort study, extremely premature infants ≤1250 grams (g) birth weight (BW) were fed an exclusive human milk-based diet receiving HMDF and serum P levels were obtained. We evaluated 93 infants with a mean gestational age of 27.5 ± 2.0 weeks (Mean ± SD) and BW of 904 ± 178 g. Seventeen infants (18.3%) had at least one high serum P level with a mean serum P of 9.2 ± 1.1 mg/dL occurring at 19 ± 11 days of life. For all infants, the highest serum P was inversely correlated to the day of life of the infant (p < 0.001, R2 = 0.175) and positively correlated with energy density of HMDF (p = 0.035). Serum P was not significantly related to gender, BW, gestational age, or days to full feeds. We conclude that the incidence of hyperphosphatemia was mild and transient in this population. The risk decreased with infant age and was unrelated to gender, BW, or ethnicity. PMID:25912036

  3. Serum phosphorus levels in premature infants receiving a donor human milk derived fortifier.

    PubMed

    Chetta, Katherine E; Hair, Amy B; Hawthorne, Keli M; Abrams, Steven A

    2015-04-01

    An elevated serum phosphorus (P) has been anecdotally described in premature infants receiving human milk fortified with donor human milk-derived fortifier (HMDF). No studies have prospectively investigated serum P in premature infants receiving this fortification strategy. In this single center prospective observational cohort study, extremely premature infants ≤ 1250 grams (g) birth weight (BW) were fed an exclusive human milk-based diet receiving HMDF and serum P levels were obtained. We evaluated 93 infants with a mean gestational age of 27.5 ± 2.0 weeks (Mean ± SD) and BW of 904 ± 178 g. Seventeen infants (18.3%) had at least one high serum P level with a mean serum P of 9.2 ± 1.1 mg/dL occurring at 19 ± 11 days of life. For all infants, the highest serum P was inversely correlated to the day of life of the infant (p < 0.001, R2 = 0.175) and positively correlated with energy density of HMDF (p = 0.035). Serum P was not significantly related to gender, BW, gestational age, or days to full feeds. We conclude that the incidence of hyperphosphatemia was mild and transient in this population. The risk decreased with infant age and was unrelated to gender, BW, or ethnicity. PMID:25912036

  4. Iron absorption in humans: bovine serum albumin compared with beef muscle and egg white

    SciTech Connect

    Hurrell, R.F.; Lynch, S.R.; Trinidad, T.P.; Dassenko, S.A.; Cook, J.D.

    1988-01-01

    We studied the influence of bovine serum albumin and beef meat on nonheme iron absorption in humans and on dialyzable iron in vitro. The addition of serum albumin to a maize gruel had no significant effect on nonheme Fe absorption whereas the addition of beef meat caused a threefold increase. When added to a bread meal, serum albumin caused a modest 60% increase in nonheme Fe absorption and beef meat had no effect. When added to a protein-free meal, serum albumin reduced Fe absorption by 47% compared with a 72% reduction on addition of egg white. The bioavailability of nonheme Fe from meals containing serum albumin was consistently overestimated by the in vitro technique. We conclude that the facilitation of nonheme Fe absorption by meat is not a general property of all animal protein but is better explained by the action of one or more specific animal tissues.

  5. Human Serum-Specific Activation of Alternative Sigma Factors, the Stress Responders in Aggregatibacter actinomycetemcomitans.

    PubMed

    Tang-Siegel, Gaoyan; Bumgarner, Roger; Ruiz, Teresa; Kittichotirat, Weerayuth; Chen, Weizhen; Chen, Casey

    2016-01-01

    Aggregatibacter actinomycetemcomitans, a known pathogen causing periodontal disease and infective endocarditis, is a survivor in the periodontal pocket and blood stream; both environments contain serum as a nutrient source. To screen for unknown virulence factors associated with this microorganism, A. actinomycetemcomitans was grown in serum-based media to simulate its in vivo environment. Different strains of A. actinomycetemcomitans showed distinct growth phenotypes only in the presence of human serum, and they were grouped into high- and low-responder groups. High-responders comprised mainly serotype c strains, and showed an unusual growth phenomenon, featuring a second, rapid increase in turbidity after 9-h incubation that reached a final optical density 2- to 7-fold higher than low-responders. Upon further investigation, the second increase in turbidity was not caused by cell multiplication, but by cell death. Whole transcriptomic analysis via RNA-seq identified 35 genes that were up-regulated by human serum, but not horse serum, in high-responders but not in low-responders, including prominently an alternative sigma factor rpoE (σE). A lacZ reporter construct driven by the 132-bp rpoE promoter sequence of A. actinomycetemcomitans responded dramatically to human serum within 90 min of incubation only when the construct was carried by a high responder strain. The rpoE promoter is 100% identical among high- and low-responder strains. Proteomic investigation showed potential interactions between human serum protein, e.g. apolipoprotein A1 (ApoA1) and A. actinomycetemcomitans. The data clearly indicated a different activation process for rpoE in high- versus low-responder strains. This differential human serum-specific activation of rpoE, a putative extra-cytoplasmic stress responder and global regulator, suggests distinct in vivo adaptations among different strains of A. actinomycetemcomitans. PMID:27490177

  6. Human Serum-Specific Activation of Alternative Sigma Factors, the Stress Responders in Aggregatibacter actinomycetemcomitans

    PubMed Central

    Tang-Siegel, Gaoyan; Bumgarner, Roger; Ruiz, Teresa; Kittichotirat, Weerayuth; Chen, Weizhen; Chen, Casey

    2016-01-01

    Aggregatibacter actinomycetemcomitans, a known pathogen causing periodontal disease and infective endocarditis, is a survivor in the periodontal pocket and blood stream; both environments contain serum as a nutrient source. To screen for unknown virulence factors associated with this microorganism, A. actinomycetemcomitans was grown in serum-based media to simulate its in vivo environment. Different strains of A. actinomycetemcomitans showed distinct growth phenotypes only in the presence of human serum, and they were grouped into high- and low-responder groups. High-responders comprised mainly serotype c strains, and showed an unusual growth phenomenon, featuring a second, rapid increase in turbidity after 9-h incubation that reached a final optical density 2- to 7-fold higher than low-responders. Upon further investigation, the second increase in turbidity was not caused by cell multiplication, but by cell death. Whole transcriptomic analysis via RNA-seq identified 35 genes that were up-regulated by human serum, but not horse serum, in high-responders but not in low-responders, including prominently an alternative sigma factor rpoE (σE). A lacZ reporter construct driven by the 132-bp rpoE promoter sequence of A. actinomycetemcomitans responded dramatically to human serum within 90 min of incubation only when the construct was carried by a high responder strain. The rpoE promoter is 100% identical among high- and low-responder strains. Proteomic investigation showed potential interactions between human serum protein, e.g. apolipoprotein A1 (ApoA1) and A. actinomycetemcomitans. The data clearly indicated a different activation process for rpoE in high- versus low-responder strains. This differential human serum-specific activation of rpoE, a putative extra-cytoplasmic stress responder and global regulator, suggests distinct in vivo adaptations among different strains of A. actinomycetemcomitans. PMID:27490177

  7. Oxidized Lipoprotein as a Major Vessel Cell Proliferator in Oxidized Human Serum

    PubMed Central

    Noguchi, Noriko

    2016-01-01

    Oxidative stress is correlated with the incidence of several diseases such as atherosclerosis and cancer, and oxidized biomolecules have been determined as biomarkers of oxidative stress; however, the detailed molecular relationship between generated oxidation products and the promotion of diseases has not been fully elucidated. In the present study, to clarify the role of serum oxidation products in vessel cell proliferation, which is related to the incidence of atherosclerosis and cancer, the major vessel cell proliferator in oxidized human serum was investigated. Oxidized human serum was prepared by free radical exposure, separated using gel chromatography, and then each fraction was added to several kinds of vessel cells including endothelial cells and smooth muscle cells. It was found that a high molecular weight fraction in oxidized human serum specifically induced vessel cell proliferation. Oxidized lipids were contained in this high molecular weight fraction, while cell proliferation activity was not observed in oxidized lipoprotein-deficient serum. Oxidized low-density lipoproteins induced vessel cell proliferation in a concentration-dependent manner. Taken together, these results indicate that oxidized lipoproteins containing lipid oxidation products function as a major vessel cell proliferator in oxidized human serum. These findings strongly indicate the relevance of determination of oxidized lipoproteins and lipid oxidation products in the diagnosis of vessel cell proliferation-related diseases such as atherosclerosis and cancer. PMID:27483438

  8. [sup 90]Y-labeled antibody uptake by human tumor xenografts and the effect of systemic administration of EDTA

    SciTech Connect

    Rowlinson-Busza, G.; Snook, D.; Epenetos, A.A. )

    1994-03-30

    A human tumor xenograft model was used to compare the tumor and normal tissue uptake of a tumor-associated monoclonal antibody radiolabeled with [sup 125]I or [sup 90]Y. Nude mice bearing SC xenografts of the human colon adenocarcinoma, HT29, were injected with a mixture of [sup 125]I- and [sup 90]Y-DTPA-labeled AUA1 monoclonal antibody, which recognizes an antigen expressed on the surface of the tumor cells. In addition, the effect of systemic ethylenediaminetetraacetic acid (EDTA) administration on [sup 90]Y-labeled antibody clearance, tumor uptake of antibody and bone accumulation of [sup 90]Y was studied in a nude mouse model of intraperitoneal cancer. Both the absolute amount (%id[center dot]g[sup -1]) and the tumor:normal tissue ratios were superior for the [sup 90]Y-labeled antibody, compared with the iodinated antibody, with the notable exception of bone. These results suggest that [sup 90]Y is a preferable isotope to iodine for radioimmunotherapy of solid masses, but that myelotoxicity due to bone uptake of released [sup 90]Y will limit the radiation dose which can be given when DTPA is used to chelate the [sup 90]Y. The [sup 90]Y-labeled antibody showed similar serum stability in vitro in the presence or absence of EDTA after incubation for up to 48 h. In vivo, urine excretion of [sup 90]Y was significantly enhanced in mice receiving daily injections of 20 mg EDTA for 3 days, commencing 2 h after intraperitoneal antibody administration, compared with control mice. There was no significant difference in the tumor uptake of [sup 90]Y-labeled antibody in EDTA-treated and control mice at any time-point up to 9 days postinjection. However, the bone levels of [sup 90]Y were significantly reduced in EDTA-treated mice at all times from 1 to 9 days. Based on these results, it should be possible to increase the amount of [sup 90]Y-labeled antibody administered, by chelating the released [sup 90]Y with systemic EDTA to facilitate its excretion. 50 refs., 5 figs.

  9. DTPA complexation of bismuth in human blood serum.

    PubMed

    Montavon, G; Le Du, A; Champion, J; Rabung, T; Morgenstern, A

    2012-07-28

    The in vivo(212)Pb/(212)Bi generator is promising for application in targeted alpha therapy (TAT) of cancer. One main limitation of its therapeutic application is due to potential release of (212)Bi from the radioconjugate upon radioactive decay of the mother nuclide (212)Pb, potentially leading to irradiation of healthy tissue. The objective of the present work is to assess whether the chelate CHX-A''-DTPA (N-(2-aminoethyl)-trans-1,2-diaminocyclohexane-N,N',N''-pentaacetic acid) bound to a biological carrier molecule may be able to re-complex released (212)Bi under in vivo conditions to limit its translocation from the target site. CHX-A''-DTPA was bound to bovine gamma globulin (BGG) to mimic a model conjugate and the stability of the Bi-CHX-A''-DTPA-BGG conjugate was studied in blood serum by ultrafiltration. TRLFS experiments using Cm(III) as a fluorescent probe demonstrated that linking CHX-A''-DTPA to BGG does not affect the coordination properties of the ligand. Furthermore, comparable stability constants were observed between Bi(III) and free CHX-A''-DTPA, BGG-bound CHX-A''-DTPA and DTPA. The complexation constants determined between Bi(III) and the chelate molecules are sufficiently high to allow ultra trace amounts of the ligand to efficiently compete with serum transferrin controlling Bi(III) speciation in blood plasma conditions. Nevertheless, CHX-A''-DTPA is not able to complex Bi(III) generated in blood serum because of the strong competition between Bi(III) and Fe(II) for the ligand. In other words, CHX-A''-DTPA is not "selective" enough to limit Bi(iii) release in the body when applying the (212)Pb/(212)Bi in vivo generator. PMID:22678751

  10. Investigation of serum proteome alterations in human glioblastoma multiforme.

    PubMed

    Gollapalli, Kishore; Ray, Sandipan; Srivastava, Rajneesh; Renu, Durairaj; Singh, Prateek; Dhali, Snigdha; Bajpai Dikshit, Jyoti; Srikanth, Rapole; Moiyadi, Aliasgar; Srivastava, Sanjeeva

    2012-08-01

    Glioblastoma multiforme (GBM) or grade IV astrocytoma is the most common and lethal adult malignant brain tumor. The present study was conducted to investigate the alterations in the serum proteome in GBM patients compared to healthy controls. Comparative proteomic analysis was performed employing classical 2DE and 2D-DIGE combined with MALDI TOF/TOF MS and results were further validated through Western blotting and immunoturbidimetric assay. Comparison of the serum proteome of GBM and healthy subjects revealed 55 differentially expressed and statistically significant (p <0.05) protein spots. Among the identified proteins, haptoglobin, plasminogen precursor, apolipoprotein A-1 and M, and transthyretin are very significant due to their functional consequences in glioma tumor growth and migration, and could further be studied as glioma biomarkers and grade-specific protein signatures. Analysis of the lipoprotein pattern indicated elevated serum levels of cholesterol, triacylglycerol, and low-density lipoproteins in GBM patients. Functional pathway analysis was performed using multiple software including ingenuity pathway analysis (IPA), protein analysis through evolutionary relationships (PANTHER), database for annotation, visualization and integrated discovery (DAVID), and GeneSpring to investigate the biological context of the identified proteins, which revealed the association of candidate proteins in a few essential physiological pathways such as intrinsic prothrombin activation pathway, plasminogen activating cascade, coagulation system, glioma invasiveness signaling, and PI3K signaling in B lymphocytes. A subset of the differentially expressed proteins was applied to build statistical sample class prediction models for discrimination of GBM patients and healthy controls employing partial least squares discriminant analysis (PLS-DA) and other machine learning methods such as support vector machine (SVM), Decision Tree and Naïve Bayes, and excellent

  11. DNA topology influences molecular machine lifetime in human serum.

    PubMed

    Goltry, Sara; Hallstrom, Natalya; Clark, Tyler; Kuang, Wan; Lee, Jeunghoon; Jorcyk, Cheryl; Knowlton, William B; Yurke, Bernard; Hughes, William L; Graugnard, Elton

    2015-06-21

    DNA nanotechnology holds the potential for enabling new tools for biomedical engineering, including diagnosis, prognosis, and therapeutics. However, applications for DNA devices are thought to be limited by rapid enzymatic degradation in serum and blood. Here, we demonstrate that a key aspect of DNA nanotechnology-programmable molecular shape-plays a substantial role in device lifetimes. These results establish the ability to operate synthetic DNA devices in the presence of endogenous enzymes and challenge the textbook view of near instantaneous degradation. PMID:25959862

  12. [Preparation of Recombinant Human Adenoviruses Labeled with miniSOG].

    PubMed

    Zou, Xiaohui; Xiao, Rong; Guo, Xiaojuan; Qu, Jianguo; Lu, Zhuozhuang; Hong, Tao

    2016-01-01

    We wished to study the intracellular transport of adenoviruses. We constructed a novel recombinant adenovirus in which the structural protein IX was labeled with a mini-singlet oxygen generator (miniSOG). The miniSOG gene was synthesized by overlapping extension polymerase chain reaction (PCR), cloned to the pcDNA3 vector, and expressed in 293 cells. Activation of miniSOG generated sufficient numbers of singlet oxygen molecules to catalyze polymerization of diaminobenzidine into an osmiophilic reaction product resolvable by transmission electron microscopy (TEM). To construct miniSOG-labelled recombinant adenoviruses, the miniSOG gene was subcloned downstream of the IX gene in a pShuttle plasmid. Adenoviral plasmid pAd5-IXSOG was generated by homologous recombination of the modified shuttle plasmid (pShuttle-IXSOG) with the backbone plasmid (pAdeasy-1) in the BJ5183 strain of Eschericia coli. Adenovirus HAdV-5-IXSOG was rescued by transfection of 293 cells with the linearized pAd5-IXSOG. After propagation, virions were purified using the CsC1 ultracentrifugation method. Finally, HAdV-5-IXSOG in 2.0 mL with a particle titer of 6 x 1011 vp/mL was obtained. Morphology of HAdV-5-IXSOG was verified by TEM. Fusion of IX with the miniSOG gene was confirmed by PCR. In conclusion, miniSOG-labeled recombinant adenoviruses were constructed, which could be valuable tools for virus tracking by TEM. PMID:27295881

  13. Experimental and theoretical investigation on the interaction between cyclovirobuxine D and human serum albumin

    NASA Astrophysics Data System (ADS)

    Yue, Yuanyuan; Liu, Ren; Liu, Jianming; Dong, Qiao; Fan, Jing

    2014-07-01

    Cyclovirobuxine D is an active compound extracted from the plant Buxux microphylla, and widely available as medications; however, its abuse may casts potential detrimental effects on human health. By using multispectroscopic techniques and molecular modeling, the interaction of cyclovirobuxine D with human serum albumin was investigated. The fluorescence results manifested that static type was the operative mechanism for the interaction with human serum albumin. The structural investigation of the complexed HSA through CD, three-dimensional, FT-IR and synchronous fluorescence shown the polypeptide chain of HSA partially destabilizing. Docking studies revealed the molecule to be bound in the subdomain IIA. Finally, we investigated the distance between the bound ligand and Trp-214 of human serum albumin.

  14. The importance of selecting a proper biological milieu for protein corona analysis in vitro: Human plasma versus human serum.

    PubMed

    Mirshafiee, Vahid; Kim, Raehyun; Mahmoudi, Morteza; Kraft, Mary L

    2016-06-01

    Nanoparticle (NP) exposure to biological fluids in the body results in protein binding to the NP surface, which forms a protein coating that is called the "protein corona". To simplify studies of protein-NP interactions and protein corona formation, NPs are incubated with biological solutions, such as human serum or human plasma, and the effects of this exposure are characterized in vitro. Yet, how NP exposure to these two different biological milieus affects protein corona composition and cell response has not been investigated. Here, we explore the differences between the protein coronas that form when NPs are incubated in human serum versus human plasma. NP characterization indicated that NPs that were exposed to human plasma had higher amounts of proteins bound to their surfaces, and were slightly larger in size than those exposed to human serum. In addition, significant differences in corona composition were also detected with gel electrophoresis and liquid chromatography-mass spectrometry/mass spectrometry, where a higher fraction of coagulation proteins and complement factors were found on the plasma-exposed NPs. Flow cytometry and confocal microscopy showed that the uptake of plasma-exposed NPs was higher than that of serum-exposed NPs by RAW 264.7 macrophage immune cells, but not by NIH 3T3 fibroblast cells. This difference is likely due to the elevated amounts of opsonins, such as fibrinogen, on the surfaces of the NPs exposed to plasma, but not serum, because these components trigger NP internalization by immune cells. As the human plasma better mimics the composition of the in vivo environment, namely blood, in vitro protein corona studies should employ human plasma, and not human serum, so the biological phenomena that is observed is more similar to that occurring in vivo. PMID:26643610

  15. Toward a Human Blood Serum Proteome: Analysis by Multidimensional Separation Coupled with Mass Spectrometry

    SciTech Connect

    Adkins, Joshua N.); Varnum, Susan M.); Auberry, Kenneth J.); Moore, Ronald J.); Angell, Nicolas; Smith, Richard D.); Springer, David L.); Pounds, Joel G.)

    2002-12-01

    Blood serum is a complex bodily fluid that contains proteins ranging in concentration over at least nine orders of magnitude. Using a combination of powerful mass spectrometry technologies with improvements in sample preparation, we have performed a proteomic analysis with sub-mL quantities of serum, and increased the measurable concentration range for proteins in blood serum beyond previous reports. We have detected 490 proteins in serum by online reversed-phase microcapillary liquid chromatography coupled with ion trap mass spectrometry. To perform this analysis, immunoglobulins were removed from serum using protein A/G, and the remaining proteins were digested with trypsin. Resulting peptides were separated by strong-cation exchange chromatography into distinct fractions prior to analysis. This separation resulted in an increase in the number of proteins detected in an individual serum sample by 3 to 5 fold. With this increase in the number of proteins identified we have detected some lower abundance serum proteins (ng/mL range) including human growth hormone, interleukin-12, and prostate-specific antigen. We also used SEQUEST to compare different protein databases with and without filtering. This comparison is plotted to allow for a quick visual assessment of different databases, as a subjective measure of quality. With this study, we have performed the most extensive analysis of serum proteins to date and laid the foundation for future refinements in the identification of novel protein biomarkers of disease.

  16. Increased Trypanosoma brucei cathepsin-L activity inhibits human serum-mediated trypanolysis

    PubMed Central

    Alsford, Sam

    2016-01-01

    Most African trypanosomes, including the veterinary species Trypanosoma brucei brucei and T. congolense are susceptible to lysis by human serum. A recent study by Alsford et al. [PLoS Pathogens (2014) 10, e1004130] has identified a T. b. brucei lysosomal cathepsin with an inhibitory effect on human serum’s trypanolytic action.

  17. DNA topology influences molecular machine lifetime in human serum

    NASA Astrophysics Data System (ADS)

    Goltry, Sara; Hallstrom, Natalya; Clark, Tyler; Kuang, Wan; Lee, Jeunghoon; Jorcyk, Cheryl; Knowlton, William B.; Yurke, Bernard; Hughes, William L.; Graugnard, Elton

    2015-06-01

    DNA nanotechnology holds the potential for enabling new tools for biomedical engineering, including diagnosis, prognosis, and therapeutics. However, applications for DNA devices are thought to be limited by rapid enzymatic degradation in serum and blood. Here, we demonstrate that a key aspect of DNA nanotechnology--programmable molecular shape--plays a substantial role in device lifetimes. These results establish the ability to operate synthetic DNA devices in the presence of endogenous enzymes and challenge the textbook view of near instantaneous degradation.DNA nanotechnology holds the potential for enabling new tools for biomedical engineering, including diagnosis, prognosis, and therapeutics. However, applications for DNA devices are thought to be limited by rapid enzymatic degradation in serum and blood. Here, we demonstrate that a key aspect of DNA nanotechnology--programmable molecular shape--plays a substantial role in device lifetimes. These results establish the ability to operate synthetic DNA devices in the presence of endogenous enzymes and challenge the textbook view of near instantaneous degradation. Electronic supplementary information (ESI) available: DNA sequences, fluorophore and quencher properties, equipment design, and degradation studies. See DOI: 10.1039/c5nr02283e

  18. The TgsGP gene is essential for resistance to human serum in Trypanosoma brucei gambiense.

    PubMed

    Capewell, Paul; Clucas, Caroline; DeJesus, Eric; Kieft, Rudo; Hajduk, Stephen; Veitch, Nicola; Steketee, Pieter C; Cooper, Anneli; Weir, William; MacLeod, Annette

    2013-01-01

    Trypanosoma brucei gambiense causes 97% of all cases of African sleeping sickness, a fatal disease of sub-Saharan Africa. Most species of trypanosome, such as T. b. brucei, are unable to infect humans due to the trypanolytic serum protein apolipoprotein-L1 (APOL1) delivered via two trypanosome lytic factors (TLF-1 and TLF-2). Understanding how T. b. gambiense overcomes these factors and infects humans is of major importance in the fight against this disease. Previous work indicated that a failure to take up TLF-1 in T. b. gambiense contributes to resistance to TLF-1, although another mechanism is required to overcome TLF-2. Here, we have examined a T. b. gambiense specific gene, TgsGP, which had previously been suggested, but not shown, to be involved in serum resistance. We show that TgsGP is essential for resistance to lysis as deletion of TgsGP in T. b. gambiense renders the parasites sensitive to human serum and recombinant APOL1. Deletion of TgsGP in T. b. gambiense modified to uptake TLF-1 showed sensitivity to TLF-1, APOL1 and human serum. Reintroducing TgsGP into knockout parasite lines restored resistance. We conclude that TgsGP is essential for human serum resistance in T. b. gambiense. PMID:24098129

  19. The TgsGP Gene Is Essential for Resistance to Human Serum in Trypanosoma brucei gambiense

    PubMed Central

    DeJesus, Eric; Kieft, Rudo; Hajduk, Stephen; Veitch, Nicola; Steketee, Pieter C.; Cooper, Anneli; Weir, William; MacLeod, Annette

    2013-01-01

    Trypanosoma brucei gambiense causes 97% of all cases of African sleeping sickness, a fatal disease of sub-Saharan Africa. Most species of trypanosome, such as T. b. brucei, are unable to infect humans due to the trypanolytic serum protein apolipoprotein-L1 (APOL1) delivered via two trypanosome lytic factors (TLF-1 and TLF-2). Understanding how T. b. gambiense overcomes these factors and infects humans is of major importance in the fight against this disease. Previous work indicated that a failure to take up TLF-1 in T. b. gambiense contributes to resistance to TLF-1, although another mechanism is required to overcome TLF-2. Here, we have examined a T. b. gambiense specific gene, TgsGP, which had previously been suggested, but not shown, to be involved in serum resistance. We show that TgsGP is essential for resistance to lysis as deletion of TgsGP in T. b. gambiense renders the parasites sensitive to human serum and recombinant APOL1. Deletion of TgsGP in T. b. gambiense modified to uptake TLF-1 showed sensitivity to TLF-1, APOL1 and human serum. Reintroducing TgsGP into knockout parasite lines restored resistance. We conclude that TgsGP is essential for human serum resistance in T. b. gambiense. PMID:24098129

  20. Calibration-free concentration analysis of protein biomarkers in human serum using surface plasmon resonance.

    PubMed

    Grover Shah, Veenita; Ray, Sandipan; Karlsson, Robert; Srivastava, Sanjeeva

    2015-11-01

    In complex biological samples such as serum, determination of specific and active concentration of target proteins, independent of a calibration curve, will be valuable in many applications. Calibration-free concentration analysis (CFCA) is a surface plasmon resonance (SPR)-based label-free approach, which calculates active concentration of proteins using their known diffusion coefficient and observed changes in binding rates at different flow rates under diffusion-limited conditions. Here, for the first time we demonstrate the application of CFCA for determining protein biomarker abundance, specifically serum amyloid A (SAA), directly in the serum samples of patients suffering from different infectious and non-infectious diseases. The assay involves preparation of appropriate reaction surfaces by immobilizing antibodies on CM5 chips via amine coupling followed by serum sample preparation and injection over activated and reference surfaces at flow-rates of 5 and 100 μL/min. The system was validated in healthy and diseased (infectious and non-infectious) serum samples by quantifying two different proteins: β2-microglobulin (β2M) and SAA. All concentration assays were performed for nearly 100 serum samples, which showed reliable quantification in unattended runs with high accuracy and sensitivity. The method could detect the serum β2M to as low as 13 ng/mL in 1000-fold serum dilution, indicating the possible utility of this approach to detect low abundance protein biomarkers in body fluids. Applying the CFCA approach, significant difference in serum abundance of SAA was identified in diseased subjects as compared to the healthy controls, which correlated well with our previous proteomic investigations. Estimation of SAA concentration for a subset of healthy and diseased sera was also performed using ELISA, and the trend was observed to be similar in both SPR assay and ELISA. The reproducibility of CFCA in various serum samples made the interpretation of assay

  1. Serum Autotaxin/ENPP2 Correlates with Insulin Resistance in Older Humans with Obesity

    PubMed Central

    Reeves, Valerie L.; Trybula, Joy S.; Wills, Rachel C.; Goodpaster, Bret H.; Dubé, John J.; Kienesberger, Petra C.; Kershaw, Erin E.

    2015-01-01

    Objective Autotaxin (ATX) is an adipocyte-derived lysophospholipase D that generates the lipid signaling molecule lysophosphatidic acid (LPA). The ATX/LPA pathway in adipose tissue has recently been implicated in obesity and insulin resistance in animal models, but the role of circulating ATX in humans remains unclear. The aim of the present study was to determine the relationship between serum ATX and insulin resistance. Methods In this retrospective study, older (60–75 years), non-diabetic human participants with overweight or obesity (BMI 25–37 kg/m2), were characterized for metabolic phenotype including measures of energy, glucose, and lipid homeostasis. The relationship between serum ATX and metabolic parameters was then determined using correlative and predictive statistics. Results Serum ATX was higher in females than in males. After controlling for sex, serum ATX correlated with multiple measures of adiposity and glucose homeostasis/insulin action. Serum ATX and BMI also independently predicted glucose infusion rate during a hyperinsulinemic euglycemic clamp and homeostatic model assessment of insulin resistance after controlling for sex and medication use. Conclusion Serum ATX correlates with and predicts measures of glucose homeostasis and insulin sensitivity in older humans, suggesting that it may be a potential pathogenic factor and/or diagnostic/therapeutic target for insulin resistance in this population. PMID:26727116

  2. ASTHMATIC HUMAN SERUM IGE-REACTIVITY WITH MOLD EXTRACTS

    EPA Science Inventory

    Although molds have demonstrated the ability to induce allergic asthma-like responses in mouse models, their role in human disease is unclear. This study was undertaken to provide insight into the prevalence of human IgE-reactivity and identify the target mold protein(s). The st...

  3. A biosensor of high-density lipoprotein of human serum on a liquid crystal and polymer composite film

    NASA Astrophysics Data System (ADS)

    Lin, Yi-Hsin; Chang, Kai-Han; Chu, Wei-Lin; Tsou, Yu-Shih; Wu, Li-Ching; Li, Chien-Feng

    2013-10-01

    A biosensor for the concentration of high-density lipoprotein (HDL) in human serum on a liquid crystal and polymer composite film (LCPCF) is demonstrated. The sensing mechanism is based on a polar-polar interaction between orientation of LC directors and HDL in human serum. The concentration of polar HDL in human serum affects the orientations of LC directors at the interface between LCPCF and the human serum. In addition, the surface free energy of LCPCF changes with the applied voltage due to the electrically tunable orientations of LC directors anchored among the polymer grains of LCPCF. As a result, the droplet motion of human serum on LCPCF under applied voltages can sense the concentration of HDL in human serum.

  4. Photoactivable analogs for labeling 25-hydroxyvitamin D3 serum binding protein and for 1,25-dihydroxyvitamin D3 intestinal receptor protein

    NASA Technical Reports Server (NTRS)

    Kutner, A.; Link, R. P.; Schnoes, H. K.; DeLuca, H. F.

    1986-01-01

    3-Azidobenzoates and 3-azidonitrobenzoates of 25-hydroxyvitamin D3 as well as 3-deoxy-3-azido-25-hydroxyvitamin D3 and 3-deoxy-3-azido-1,25-dihydroxyvitamin D3 were prepared as photoaffinity labels for vitamin D serum binding protein and 1,25-dihydroxyvitamin D3 intestinal receptor protein. The compounds prepared were easily activated by short- or long-wavelength uv light, as monitored by uv and ir spectrometry. The efficacy of the compounds to compete with 25-hydroxyvitamin D3 or 1,25-dihydroxyvitamin D3 for the binding site of serum binding protein and receptor, respectively, was studied to evaluate the vitamin D label with the highest affinity for the protein. The presence of an azidobenzoate or azidonitrobenzoate substituent at the C-3 position of 25-OH-D3 significantly decreased (10(4)- to 10(6)-fold) the binding activity. However, the labels containing the azido substituent attached directly to the vitamin D skeleton at the C-3 position showed a high affinity, only 20- to 150-fold lower than that of the parent compounds with their respective proteins. Therefore, 3-deoxy-3-azidovitamins present potential ligands for photolabeling of vitamin D proteins and for studying the structures of the protein active sites.

  5. In vitro studies on the impact of human serum on the antibacterial effect of faropenem.

    PubMed

    MacGowan, A; Bowker, K

    2004-02-01

    The interaction between faropenem and serum in determining antibacterial effect was studied using three target pathogens and two different in vitro methodologies. Strains of Staphylococcus aureus, Streptococcus pneumoniae, capsulate and non capsulate Haemophilus influenzae, all faropenem MIC 0.12 mg/L, were tested in a range of pharmacologically realistic faropenem concentrations with various proportions of serum up to 75%. Using simulated serum bactericidal titres the presence of human serum reduced the activity of faropenem against S. pneumoniae as did heat-treated serum for non-capsulate H. influenzae. Serum on its own was bactericidal against H. influenzae probably due to the presence of complement. The antibacterial effects of combinations of faropenem and serum was assessed in time-kill curves by calculation of the area-under-the bacterial-kill-curve (AUBKC). This was then related to faropenem concentration and the proportion of serum using three-dimensional plots. Serum on its own was mildly inhibitory of the growth of S. aureus, supported improved growth of S. pneumoniae at some proportions and was rapidly bactericidal to H. influenzae, especially the non-capsulate strains. Faropenem had a marked antibacterial effect against all three species in the range 0-2.5 mg/L. Increasing the faropenem concentration from 2.5-10 mg/L produced little or no additional effect. The combination of serum and faropenem had little impact on the antibacterial effect against S. pneumoniae and S. aureus but free drug concentrations were likely to be greater than the MIC in all the combinations used. Against capsulate H. influenzae the effect of serum and faropenem was broadly equivalent while against non-capsulate strains the activity of serum was so great it is difficult to assess the impact of faropenem alone. The interaction between serum and antibiotic in determining antibacterial effect is complex and critically dependent on the proportions of serum and drug concentrations

  6. The influence of fatty acids on theophylline binding to human serum albumin. Comparative fluorescence study

    NASA Astrophysics Data System (ADS)

    Maciążek-Jurczyk, M.; Sułkowska, A.; Bojko, B.; Równicka-Zubik, J.; Szkudlarek-Haśnik, A.; Zubik-Skupień, I.; Góra, A.; Dubas, M.; Korzonek-Szlacheta, I.; Wielkoszyński, T.; Żurawiński, W.; Sosada, K.

    2012-04-01

    Theophylline, popular diuretic, is used to treat asthma and bronchospasm. In blood it forms complexes with albumin, which is also the main transporter of fatty acids. The aim of the present study was to describe the influence of fatty acids (FA) on binding of theophylline (Th) to human serum albumin (HSA) in the high affinity binding sites. Binding parameters have been obtained on the basis of the fluorescence analysis. The data obtained for the complex of Th and natural human serum albumin (nHSA) obtained from blood of obese patients qualified for surgical removal of stomach was compared with our previous studies on the influence of FA on the complex of Th and commercially available defatted human serum albumin (dHSA).

  7. Determination of xanthine oxidase in human serum by a competitive enzyme-linked immunosorbent assay (ELISA).

    PubMed

    Battelli, M G; Abbondanza, A; Musiani, S; Buonamici, L; Strocchi, P; Tazzari, P L; Gramantieri, L; Stirpe, F

    1999-03-01

    Xanthine oxidase was purified from human milk and used to immunise rabbits. A competitive immunoenzymatic assay with purified enzyme and rabbit antiserum was optimised to measure xanthine oxidase in human serum, the lowest detectable amount being 0.03 pmol of enzymatic protein. Thus, the test (i) is sensitive enough to determine xanthine oxidase in human serum, being more sensitive than the spectrophotometric method, (ii) it is more convenient for clinical laboratories than other sensitive tests and (iii) it has the advantage over the enzyme activity-based assays of also detecting inactive enzyme molecules. A competitive enzyme-linked immunosorbent assay (ELISA) was used to measure the serum xanthine oxidase level in healthy donors and in patients with liver diseases, and it was found that any concentration below 1 mg/L is in the normal range. PMID:10217635

  8. 21 CFR 809.11 - Exceptions or alternatives to labeling requirements for in vitro diagnostic products for human...

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... requirements for in vitro diagnostic products for human use held by the Strategic National Stockpile. 809.11... (CONTINUED) MEDICAL DEVICES IN VITRO DIAGNOSTIC PRODUCTS FOR HUMAN USE Labeling § 809.11 Exceptions or alternatives to labeling requirements for in vitro diagnostic products for human use held by the...

  9. 21 CFR 809.11 - Exceptions or alternatives to labeling requirements for in vitro diagnostic products for human...

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... requirements for in vitro diagnostic products for human use held by the Strategic National Stockpile. 809.11... (CONTINUED) MEDICAL DEVICES IN VITRO DIAGNOSTIC PRODUCTS FOR HUMAN USE Labeling § 809.11 Exceptions or alternatives to labeling requirements for in vitro diagnostic products for human use held by the...

  10. 21 CFR 809.11 - Exceptions or alternatives to labeling requirements for in vitro diagnostic products for human...

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... requirements for in vitro diagnostic products for human use held by the Strategic National Stockpile. 809.11... (CONTINUED) MEDICAL DEVICES IN VITRO DIAGNOSTIC PRODUCTS FOR HUMAN USE Labeling § 809.11 Exceptions or alternatives to labeling requirements for in vitro diagnostic products for human use held by the...

  11. 21 CFR 809.11 - Exceptions or alternatives to labeling requirements for in vitro diagnostic products for human...

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... requirements for in vitro diagnostic products for human use held by the Strategic National Stockpile. 809.11... (CONTINUED) MEDICAL DEVICES IN VITRO DIAGNOSTIC PRODUCTS FOR HUMAN USE Labeling § 809.11 Exceptions or alternatives to labeling requirements for in vitro diagnostic products for human use held by the...

  12. Labeling of human mesenchymal stem cell: Comparison between paramagnetic and superparamagnetic agents

    NASA Astrophysics Data System (ADS)

    Yang, Chung-Yi; Tai, Ming-Fong; Chen, Shin-Tai; Wang, Yi-Ting; Chen, Ya-Fang; Hsiao, Jong-Kai; Wang, Jaw-Lin; Liu, Hon-Man

    2009-04-01

    Paramagnetic and superparamagnetic substances are used to trace stem cell in living organisms under magnetic resonance imaging (MRI). We compared paramagnetic and superparamagnetic substance for their labeling efficiency by using clinically widely used gadolinium chelates and iron oxide nanoparticles. Without the aid of transfection agent, human mesenchymal stem cells were labeled with each agent separately in different concentration and the optimized concentration was determined by maintaining same cell viability as unlabeled cells. Iron oxide nanoparticle labeling has a detecting threshold of 12 500 cells in vitro, while gadolinium chelates labeling could be detected for at least 50 000 cells. In life animal study, we found there is an eightfold sensitivity in cells labeled with iron oxide superparamagnetic nanoparticles; however, the magnetic susceptibility artifact would obscure the detail of adjacent anatomical structures. We conclude that labeling stem cells with superparamagnetic substance is more efficacious. However, the cells labeled by superparamagnetic nanoparticles might interfere with the interpretation of anatomical structure. These findings would be beneficial to applications of magnetic substances toward stem cell biology and tissue engineering.

  13. Visualization of antigen-specific human cytotoxic T lymphocytes labeled with superparamagnetic iron-oxide particles.

    PubMed

    Beer, Ambros J; Holzapfel, Konstantin; Neudorfer, Juliana; Piontek, Guido; Settles, Marcus; Krönig, Holger; Peschel, Christian; Schlegel, Jürgen; Rummeny, Ernst J; Bernhard, Helga

    2008-06-01

    New technologies are needed to characterize the migration and survival of antigen-specific T cells in vivo. In this study, we developed a novel technique for the labeling of human cytotoxic T lymphocytes with superparamagnetic iron-oxide particles and the subsequent depiction with a conventional 1.5-T magnetic resonance scanner. Antigen-specific CD8(+) T lymphocytes were labeled with ferucarbotran by lipofection. The uptake of ferucarbotran was confirmed by immunofluorescence microscopy using a dextran-specific antibody, and the intracellular enrichment of iron was measured by atomic absorption spectrometry. The imaging of T cells was performed by magnetic resonance on day 0, 2, 7 and 14 after the labeling procedure. On day 0 and 2 post labeling, a pronounced shortening of T2*-relaxation times was observed, which diminished after 7 days and was not detectable anymore after 14 days, probably due to the retained mitotic activity of the labeled T cells. Of importance, the antigen-specific cytolytic activity of the T cells was preserved following ferucarbotran labeling. Efficient ferucarbotran labeling of functionally active T lymphocytes and their detection by magnetic resonance imaging allows the in vivo monitoring of T cells and, subsequently, will impact the further development of T cell-based therapies. PMID:18286290

  14. Serum-Induced Keratinization Processes in an Immortalized Human Meibomian Gland Epithelial Cell Line

    PubMed Central

    Hampel, Ulrike; Schröder, Antje; Mitchell, Todd; Brown, Simon; Snikeris, Peta; Garreis, Fabian; Kunnen, Carolina; Willcox, Mark; Paulsen, Friedrich

    2015-01-01

    Purpose The aim of this study was to evaluate a human meibomian gland epithelial cell line (HMGEC) as a model for meibomian gland (patho)physiology in vitro. Methods HMGEC were cultured in the absence or presence of serum. Sudan III lipid staining, ultrastructural analysis and lipidomic analyses were performed. Impedance sensing, desmoplakin 1/2 mRNA and cytokeratin (CK) 1, 5, 6, 14 levels were evaluated. Serum containing medium supplemented with higher serum, glucose, an omega-3 lipid cocktail, eicosapentaenoic acid or sebomed medium were investigated for lipid accumulation and ultrastructural morphology. Results Lipid droplet accumulation in HMGEC was induced by serum containing media after 1 day, but decreased over time. Cultivation in serum induced desmosome and cytokeratin filament formation. Desmoplakin 1/2 gene levels were significantly upregulated after 1d of serum treatment. Furthermore, the normalized impedance increased significantly. Lipidome analysis revealed high levels of phospholipids (over 50%), but very low levels of wax ester and cholesteryl esters (under 1%). Stimulation with eicosapentaenoic acid increased lipid accumulation after one day. Conclusion Serum treatment of HMGEC caused lipid droplet formation to some extent but also induced keratinization. The cells did not produce typical meibum lipids under these growth conditions. HMGEC are well suited to study (hyper)keratinization processes of meibomian gland epithelial cells in vitro. PMID:26042605

  15. Alcohol-induced alterations in serum immunoglobulin e (IgE) levels in human subjects.

    PubMed

    González-Quintela, Arturo; Vidal, Carmen; Gude, Francisco

    2002-05-01

    The association of alcohol intake with total serum IgE concentrations in humans is discussed in the present review. The possible relationship of regular alcohol intake with both the risk of allergic sensitization and serum allergen-specific IgE values is also reviewed. Several studies consistently show that total serum IgE concentrations are increased in alcoholics when compared with healthy controls. Total serum IgE levels decrease after ethanol abstinence in alcoholics. Total serum IgE is increased in moderate alcohol consumers with respect to abstainers. Alcohol consumption in mothers may be associated with increased cord blood IgE levels in their offspring. IgE elevation in alcohol consumers is independent of potential confounders such as age, sex, liver disease, cigarette smoking or atopic status. Experimental studies in animals further support that ethanol administration is followed by an increase in serum IgE concentrations. In atopic patients, regular alcohol consumption is associated with increased serum specific IgE levels against some aeroallergens. Preliminary reports suggest that alcohol intake is associated to variable risk of sensitization to some aeroallergens. The possible mechanisms of alcohol-induced alterations in IgE levels and IgE-mediated diseases are discussed. PMID:11991851

  16. Visual Assay of Total Iron in Human Serum with Bathophenanthrolin Disulfonate-accommodated MCM-41.

    PubMed

    Sakamoto, Misato; Hizawa, Keita; Hosaka, Manabu; Sugawara, Masao

    2016-01-01

    A simple visual method for determining the total iron in human serum is proposed based on color development in the nanospace of mesoporous silica MCM-41 and a chromogenic ligand bathophenathroline disulfonate (BPS). Observing the color intensity of a complex between iron(II) and BPS devloped on the MCM-41 material by the naked eye enabled us to quntify iron(II) with a detection limit of 0.5 μM. The BPS-loaded MCM-41 was successfully applied for quantifying the total iron in human serum. PMID:26860573

  17. Optical diagnosis of dengue virus infection in human blood serum using Raman spectroscopy

    NASA Astrophysics Data System (ADS)

    Saleem, M.; Bilal, M.; Anwar, S.; Rehman, A.; Ahmed, M.

    2013-03-01

    We present the optical diagnosis of dengue virus infection in human blood serum using Raman spectroscopy. Raman spectra were acquired from 18 blood serum samples using a laser at 532 nm as the excitation source. A multivariate regression model based on partial least-squares regression is developed that uses Raman spectra to predict dengue infection with leave-one-sample-out cross validation. The prediction of dengue infection by our model yields correlation coefficient r2 values of 0.9998 between the predicted and reference clinical results. The model was tested for six unknown human blood sera and found to be 100% accurate in accordance with the clinical results.

  18. Induced Long-Range Attractive Potentials of Human Serum Albumin by Ligand Binding

    SciTech Connect

    Sato, Takaaki; Komatsu, Teruyuki; Nakagawa, Akito; Tsuchida, Eishun

    2007-05-18

    Small-angle x-ray scattering and dielectric spectroscopy investigation on the solutions of recombinant human serum albumin and its heme hybrid revealed that heme incorporation induces a specific long-range attractive potential between protein molecules. This is evidenced by the enhanced forward intensity upon heme binding, despite no hindrance to rotatory Brownian motion, unbiased colloid osmotic pressure, and discontiguous nearest-neighbor distance, confirming monodispersity of the proteins. The heme-induced potential may play a trigger role in recognition of the ligand-filled human serum albumins in the circulatory system.

  19. Determination of glyphosate, glyphosate metabolites, and glufosinate in human serum by gas chromatography-mass spectrometry.

    PubMed

    Motojyuku, Megumi; Saito, Takeshi; Akieda, Kazuki; Otsuka, Hiroyuki; Yamamoto, Isotoshi; Inokuchi, Sadaki

    2008-11-15

    This paper describes an assay for the determination of glyphosate (GLYP), glyphosate metabolites [(aminomethyl) phosphonic acid] (AMPA), and glufosinate (GLUF) in human serum. After protein precipitation using acetonitrile and solid-phase extraction, serum samples were derivatized and analyzed by gas chromatography-mass spectrometry (GC-MS). The assay was linear over a concentration range of 3-100.0 microg/ml for GLYP, AMPA, and GLUF. The overall recoveries for the three compounds were >73%. The intra- and inter-day variations were <15%. Precision and accuracy were 6.4-10.6% and 88.2-103.7%, respectively. The validated method was applied to quantify the GLYP and AMPA content in the serum of a GLYP-poisoned patient. In conclusion, the method was successfully applied for the determination of GLYP and its metabolite AMPA in serum obtained from patient of GLYP-poisoning. PMID:18945648

  20. Binding of radioiodinated human. beta. -endorphin to serum proteins from rats and humans, determined by several methods

    SciTech Connect

    Sato, H.; Sugiyama, Y.; Sawada, Y.; Iga, T.; Hanano, M.

    1985-10-07

    Binding of immunoreactive radioiodinated human ..beta..-endorphin (/sup 125/I-..beta..-EP) to rat serum was demonstrated by gel filtration of /sup 125/I-..beta..-EP in pooled rat serum on Sephadex G-200. Two radioactive peaks associated with proteins eluted from the column. The first peak eluted at the void volume containing lipoproteins, ..cap alpha../sub 2/- and ..beta../sub 2/-macroglobulins, and the second peak at the fraction of albumin. Binding of /sup 125/I-..beta..-EP to albumin was directly proved by gel filtration of /sup 125/I-..beta..-EP in buffer containing 4% human serum albumin on Sephadex G-200. Equilibrium dialysis was not applicable to investigating the interaction of /sup 125/I-..beta..-EP with serum proteins, because of the intense nonspecific adsorption to the semi-permeable membrane and the degradation of the peptide during dialysis. Therefore, in order to quantitatively evaluate the binding of /sup 125/I-..beta..-EP in sera from rats and humans, the authors utilized four other methods (ultrafiltration, charcoal adsorption, polyethylene glycol precipitation and equilibrium gel filtration). These methods corresponded well with each other and indicated 35-44% binding of /sup 125/I-..beta..-EP in rat serum. Binding of /sup 125/I-..beta..-EP in normal human serum was 36%, determined by ultrafiltration. Serum protein binding of /sup 125/I-..beta..-EP was concentration independent over the concentration range studied (1-1000 nM). 23 references, 4 figures, 1 table.

  1. Gastroenteric cancers detection by Raman spectroscopy of human serum

    NASA Astrophysics Data System (ADS)

    Li, Xiaozhou; Lin, Junxiu

    2003-06-01

    To investigate the spectral specialities of stomach cancer serum for diagnosis, fluorescence and Raman spectra of normal, stomach cancer, esophagus cancer and atophic gastritis sera were measured in the visible region in this study. All spectra except esophagus cancer were characterized by three sharp peaks. The intensity of each peak was different in different spectrum. After sampels were radiated by laser, fluorescence weakened along wiht red shift of its band center, and spectral changes of normal and stomach cancer cases were different from other samples. It was also observed that spectral changes of atophic gastritis were very similar with stomach cancer after radiated by laser, however, there are still some distinctions that can be used to differentiate them from each other. A notable difference is that the relative intensity of peak C excited by 488.0nm is higher than excited by 514.5nm in spectrum of stomach cancer, whereas lower in other cases. We utilized it as a criterion and got an accuracy of 80.77% in stomach cancer detection.

  2. Oral iron acutely elevates bacterial growth in human serum

    PubMed Central

    Cross, James H.; Bradbury, Richard S.; Fulford, Anthony J.; Jallow, Amadou T.; Wegmüller, Rita; Prentice, Andrew M.; Cerami, Carla

    2015-01-01

    Iron deficiency is the most common nutrient deficiency worldwide and routine supplementation is standard policy for pregnant mothers and children in most low-income countries. However, iron lies at the center of host-pathogen competition for nutritional resources and recent trials of iron administration in African and Asian children have resulted in significant excesses of serious adverse events including hospitalizations and deaths. Increased rates of malaria, respiratory infections, severe diarrhea and febrile illnesses of unknown origin have all been reported, but the mechanisms are unclear. We here investigated the ex vivo growth characteristics of exemplar sentinel bacteria in adult sera collected before and 4 h after oral supplementation with 2 mg/kg iron as ferrous sulfate. Escherichia coli, Yersinia enterocolitica and Salmonella enterica serovar Typhimurium (all gram-negative bacteria) and Staphylococcus epidermidis (gram-positive) showed markedly elevated growth in serum collected after iron supplementation. Growth rates were very strongly correlated with transferrin saturation (p < 0.0001 in all cases). Growth of Staphylococcus aureus, which preferentially scavenges heme iron, was unaffected. These data suggest that even modest oral supplements with highly soluble (non-physiological) iron, as typically used in low-income settings, could promote bacteremia by accelerating early phase bacterial growth prior to the induction of immune defenses. PMID:26593732

  3. Chemotyping the distribution of vitamin D metabolites in human serum

    NASA Astrophysics Data System (ADS)

    Müller, Miriam J.; Stokes, Caroline S.; Lammert, Frank; Volmer, Dietrich A.

    2016-02-01

    Most studies examining the relationships between vitamin D and disease or health focus on the main 25-hydroxyvitamin D3 (25(OH)D3) metabolite, thus potentially overlooking contributions and dynamic effects of other vitamin D metabolites, the crucial roles of several of which have been previously demonstrated. The ideal assay would determine all relevant high and low-abundant vitamin D species simultaneously. We describe a sensitive quantitative assay for determining the chemotypes of vitamin D metabolites from serum after derivatisation and ultra-high performance liquid chromatography-electrospray ionisation-tandem mass spectrometry (UHPLC-ESI-MS/MS). We performed a validation according to the ‘FDA Guidance for Industry Bioanalytical Method Validation’. The proof-of-concept of the method was then demonstrated by following the metabolite concentrations in patients with chronic liver diseases (CLD) during the course of a vitamin D supplementation study. The new quantitative profiling assay provided highly sensitive, precise and accurate chemotypes of the vitamin D metabolic process rather than the usually determined 25(OH)D3 concentrations.

  4. Quantification of nerve agent biomarkers in human serum and urine.

    PubMed

    Røen, Bent Tore; Sellevåg, Stig Rune; Lundanes, Elsa

    2014-12-01

    A novel method for rapid and sensitive quantification of the nerve agent metabolites ethyl, isopropyl, isobutyl, cyclohexyl, and pinacolyl methylphosphonic acid has been established by combining salting-out assisted liquid-liquid extraction (SALLE) and online solid phase extraction-liquid chromatography-tandem mass spectrometry (SPE-LC-MS/MS). The procedure allows confirmation of nerve agent exposure within 30 min from receiving a sample, with very low detection limits for the biomarkers of 0.04-0.12 ng/mL. Sample preparation by SALLE was performed in less than 10 min, with a common procedure for both serum and urine. Analyte recoveries of 70-100% were obtained using tetrahydrofuran as extraction solvent and Na2SO4 to achieve phase separation. After SALLE, selective analyte retention was obtained on a ZrO2 column by Lewis acid-base and hydrophilic interactions with acetonitrile/1% CH3COOH (82/18) as the loading mobile phase. The phosphonic acids were backflush-desorbed onto a polymeric zwitterionic column at pH 9.8 and separated by hydrophilic interaction liquid chromatography. The method was linear (R(2) ≥ 0.995) from the limits of quantification to 50 ng/mL, and the within- and between-assay repeatability at 20 ng/mL were below 5% and 10% relative standard deviation, respectively. PMID:25371246

  5. Loss-of-function variants influence the human serum metabolome.

    PubMed

    Yu, Bing; Li, Alexander H; Metcalf, Ginger A; Muzny, Donna M; Morrison, Alanna C; White, Simon; Mosley, Thomas H; Gibbs, Richard A; Boerwinkle, Eric

    2016-08-01

    The metabolome is a collection of small molecules resulting from multiple cellular and biological processes that can act as biomarkers of disease, and African-Americans exhibit high levels of genetic diversity. Exome sequencing of a sample of deeply phenotyped African-Americans allowed us to analyze the effects of annotated loss-of-function (LoF) mutations on 308 serum metabolites measured by untargeted liquid and gas chromatography coupled with mass spectrometry. In an independent sample, we identified and replicated four genes harboring six LoF mutations that significantly affected five metabolites. These sites were related to a 19 to 45% difference in geometric mean metabolite levels, with an average effect size of 25%. We show that some of the affected metabolites are risk predictors or diagnostic biomarkers of disease and, using the principle of Mendelian randomization, are in the causal pathway of disease. For example, LoF mutations in SLCO1B1 elevate the levels of hexadecanedioate, a fatty acid significantly associated with increased blood pressure levels and risk of incident heart failure in both African-Americans and an independent sample of European-Americans. We show that SLCO1B1 LoF mutations significantly increase the risk of incident heart failure, thus implicating the metabolite in the causal pathway of disease. These results reveal new avenues into gene function and the understanding of disease etiology by integrating -omic technologies into a deeply phenotyped population study. PMID:27602404

  6. Loss-of-function variants influence the human serum metabolome

    PubMed Central

    Yu, Bing; Li, Alexander H.; Metcalf, Ginger A.; Muzny, Donna M.; Morrison, Alanna C.; White, Simon; Mosley, Thomas H.; Gibbs, Richard A.; Boerwinkle, Eric

    2016-01-01

    The metabolome is a collection of small molecules resulting from multiple cellular and biological processes that can act as biomarkers of disease, and African-Americans exhibit high levels of genetic diversity. Exome sequencing of a sample of deeply phenotyped African-Americans allowed us to analyze the effects of annotated loss-of-function (LoF) mutations on 308 serum metabolites measured by untargeted liquid and gas chromatography coupled with mass spectrometry. In an independent sample, we identified and replicated four genes harboring six LoF mutations that significantly affected five metabolites. These sites were related to a 19 to 45% difference in geometric mean metabolite levels, with an average effect size of 25%. We show that some of the affected metabolites are risk predictors or diagnostic biomarkers of disease and, using the principle of Mendelian randomization, are in the causal pathway of disease. For example, LoF mutations in SLCO1B1 elevate the levels of hexadecanedioate, a fatty acid significantly associated with increased blood pressure levels and risk of incident heart failure in both African-Americans and an independent sample of European-Americans. We show that SLCO1B1 LoF mutations significantly increase the risk of incident heart failure, thus implicating the metabolite in the causal pathway of disease. These results reveal new avenues into gene function and the understanding of disease etiology by integrating -omic technologies into a deeply phenotyped population study. PMID:27602404

  7. Nonlocal atlas-guided multi-channel forest learning for human brain labeling

    PubMed Central

    Ma, Guangkai; Gao, Yaozong; Wu, Guorong; Wu, Ligang; Shen, Dinggang

    2016-01-01

    Purpose: It is important for many quantitative brain studies to label meaningful anatomical regions in MR brain images. However, due to high complexity of brain structures and ambiguous boundaries between different anatomical regions, the anatomical labeling of MR brain images is still quite a challenging task. In many existing label fusion methods, appearance information is widely used. However, since local anatomy in the human brain is often complex, the appearance information alone is limited in characterizing each image point, especially for identifying the same anatomical structure across different subjects. Recent progress in computer vision suggests that the context features can be very useful in identifying an object from a complex scene. In light of this, the authors propose a novel learning-based label fusion method by using both low-level appearance features (computed from the target image) and high-level context features (computed from warped atlases or tentative labeling maps of the target image). Methods: In particular, the authors employ a multi-channel random forest to learn the nonlinear relationship between these hybrid features and target labels (i.e., corresponding to certain anatomical structures). Specifically, at each of the iterations, the random forest will output tentative labeling maps of the target image, from which the authors compute spatial label context features and then use in combination with original appearance features of the target image to refine the labeling. Moreover, to accommodate the high inter-subject variations, the authors further extend their learning-based label fusion to a multi-atlas scenario, i.e., they train a random forest for each atlas and then obtain the final labeling result according to the consensus of results from all atlases. Results: The authors have comprehensively evaluated their method on both public LONI_LBPA40 and IXI datasets. To quantitatively evaluate the labeling accuracy, the authors use the

  8. (/sup 3/H)cholesteryl ester labeling and transfer among human and honhuman primate plasma lipoproteins

    SciTech Connect

    Thomas, M.S.; Rudel, L.L.

    1983-04-01

    Aliquots of human and nonhuman primate plasma containing 5,5'-dithiobis (2-nitrobenzoic acid) were incubated at 37/sup 0/C in tubes previously coated with trace amounts of tritium-labeled cholesteryl oleate ((/sup 3/H)CO). Initially, cholesteryl esters were transferred at a rapid rate into plasma after which the rate slowed. During 24 h of incubation, an average of 55% of the (/sup 3/H)CO transferred from the side of the tube into African green monkey plasma, 44% into human plasma and 21% into rat plasma. Greater than 98% of the radioactive ester transferred into plasma was found to be associated with plasma lipoproteins that were then rapidly separated using vertical rotor density gradient ultracentrifugation. In very low density lipoprotein (VLDL)-poor plasma after 30 min incubations, high density lipoproteins (HDL) contained most of the (/sup 3/H)CO while 5- to 24-h incubations resulted in increased labeling of low density proteins (LDL). In VLDL-rich plasma, it was found that in addition to the labeling of HDL, VLDL contained about 25% of the labeled cholesteryl esters after 30-min incubations and, as above, the proportion in LDL subsequently increased. Compositional analyses showed that intermediate-sized LDL (ILDL) were accumulating cholesteryl ester mass while transfer occurred. LDL labeled using this method were injected intravenously into monkeys and their removal from plasma was found to be similar to that found for LDL labeled in vivo. It was concluded that this method of plasma lipoprotein cholesteryl ester labeling, presumably a result of cholesteryl ester transfer protein activity, was efficient, resulted in lipoproteins labeled only in the cholesteryl ester moiety, and induced minimal modification of lipoprotein particles that did not alter their biological activity.

  9. Biodegradable human serum albumin nanoparticles as contrast agents for the detection of hepatocellular carcinoma by magnetic resonance imaging.

    PubMed

    Watcharin, Waralee; Schmithals, Christian; Pleli, Thomas; Köberle, Verena; Korkusuz, Hüdayi; Huebner, Frank; Zeuzem, Stefan; Korf, Hans W; Vogl, Thomas J; Rittmeyer, Claudia; Terfort, Andreas; Piiper, Albrecht; Gelperina, Svetlana; Kreuter, Jörg

    2014-05-01

    Tumor visualization by magnetic resonance imaging (MRI) and nanoparticle-based contrast agents may improve the imaging of solid tumors such as hepatocellular carcinoma (HCC). In particular, human serum albumin (HSA) nanoparticles appear to be a suitable carrier due to their safety and feasibility of functionalization. In the present study HSA nanoparticles were conjugated with gadolinium diethylenetriaminepentaacetic acid (Gd-DTPA) using carbodiimide chemistry. The nanoparticles had a uniform spherical shape and a diameter of 235±19nm. For better optical visualization in vitro and in vivo, the HSA-Gd nanoparticles were additionally labeled with rhodamine 123. As shown by confocal microscopy and flow cytometry analysis, the fluorescent nanoparticles were readily taken up by Huh-7 hepatocellular carcinoma cells. After 24h incubation in blood serum, less than 5% of the Gd(III) was released from the particles, which suggests that this nanoparticulate system may be stable in vivo and, therefore, may serve as potentially safe T1 MRI contrast agent for MRI of hepatocellular carcinoma. PMID:24365328

  10. A magnetic nanoparticle-based time-resolved fluoroimmunoassay for determination of the cytokeratin 19 fragment in human serum.

    PubMed

    Lin, Guanfeng; Liu, Tiancai; Hou, Jingyuan; Ren, Zhiqi; Zhou, Jianwei; Liang, Qianni; Chen, Zhenhua; Dong, Wenqi; Wu, Yingsong

    2015-03-01

    A sensitive, rapid and novel measurement method for cytokeratin 19 fragment (CYFRA 21-1) in human serum by magnetic particle-based time-resolved fluoroimmunoassay (TRFIA) is described. Built on a sandwich-type immunoassay format, analytes in samples were captured by one monoclonal antibody coating onto the surface of magnetic beads and "sandwiched" by another monoclonal antibody labeled with europium chelates. The coefficient variations of the method were lower than 7%, and the recoveries were in the range of 90-110% for serum samples. The lower limit of quantitation of the present method for CYFRA 21-1 was 0.78 ng/ml. The correlation coefficient of CYFRA 21-1 values obtained by our novel TRFIA and CLIA was 0.980. The present novel TRFIA demonstrated high sensitivity, wider effective detection range and excellent reproducibility for determination of CYFRA 21-1 can be useful for early screening and prognosis evaluation of patients with non-small cell lung cancer. PMID:25666714

  11. Human Serum Promotes Candida albicans Biofilm Growth and Virulence Gene Expression on Silicone Biomaterial

    PubMed Central

    Samaranayake, Yuthika Hemamala; Cheung, Becky P. K.; Yau, Joyce Y. Y.; Yeung, Shadow K. W.; Samaranayake, Lakshman P.

    2013-01-01

    Objectives Systemic candidal infections are a common problem in hospitalized patients due to central venous catheters fabricated using silicone biomaterial (SB). We therefore evaluated the effect of human serum on C. albicans biofilm morphology, growth, and the expression of virulence-related genes on SB in vitro. Methods We cultivated C. albicans SC5314 (wild-type strain, WT) and its derivative HLC54 (hyphal mutant, HM) for 48 h in various conditions, including the presence or absence of SB discs, and human serum. The growth of planktonic and biofilm cells of both strains was monitored at three time points by a tetrazolium salt reduction assay and by scanning electron microscopy. We also analyzed by RT-PCR its expression of the virulence-related genes ALS3, HWP1, EAP1, ECE1, SAP1 - SAP10, PLB1, PLB2, PLC and PLD. Results At each time point, planktonic cells of WT strain cultured in yeast nitrogen base displayed a much higher expression of EAP1 and HWP1, and a moderately higher ALS3 expression, than HM cells. In planktonic cells, expression of the ten SAP genes was higher in the WT strain initially, but were highly expressed in the HM strain by 48 h. Biofilm growth of both strains on SB was promoted in the presence of human serum than in its absence. Significant upregulation of ALS3, HWP1, EAP1, ECE1, SAP1, SAP4, SAP6 - SAP10, PLB1, PLB2 and PLC was observed for WT biofilms grown on serum-treated SB discs for at least one time point, compared with biofilms on serum-free SB discs. Conclusions Human serum stimulates C. albicans biofilm growth on SB discs and upregulates the expression of virulence genes, particularly adhesion genes ALS3 and HWP1, and hydrolase-encoding genes SAP, PLB1 and PLB2. This response is likely to promote the colonization of this versatile pathogen within the human host. PMID:23704884

  12. Measurment of rhesus monkey (Macaca mulatta) apolipoprotein B in serum by radioimmunoassay: comparison of immunoreactivities of rhesus and human low density lipoproteins.

    PubMed

    Karlin, J B; Juhn, D J; Fless, G; Scanu, A M; Rubenstein, A H

    1978-02-01

    A sensitive and specific double antibody radio-immunoassay for the major apolipoprotein (apoB) of rhesus (Macaca mulatta) serum very low density lipoprotein (VLDL) and low density lipoprotein (LDL) is described. The anti-serum was raised to LDL (d 1.030-1.040 g/ml) and the LDL(2) (d 1.020-1.050 g/ml) was labeled with (125)I by the chloramine-T or iodine monochloride method. The assay, which was sensitive to 0.02-0.5 micro g of LDL(2), had an inter-assay coefficient of variation of 4.5%. This assay was successfully used to measure apoB in the whole serum and low density lipoproteins of control monkeys maintained on a standard Purina monkey chow (PMC) diet and of three groups of monkeys fed atherogenic diets: an "average American diet," a 25% peanut oil and 2% cholesterol-supplemented PMC diet, and a 25% coconut oil and 2% cholesterol-supplemented PMC diet. The control monkeys (n = 13) had a serum cholesterol of 146 +/- 28 mg/dl and an apoB of 50 +/- 18 mg/dl. In the monkeys maintained on the atherogenic diets the serum apoB was elevated: 103 +/- 28 mg/dl (American), 102 +/- 35 mg/dl (peanut oil), and 312 +/- 88 mg/dl (coconut oil). The values for serum total cholesterol were 333 +/- 65 mg/dl (American), 606 +/- 212 mg/dl (peanut oil), and 864 +/- 233 mg/dl (coconut oil) and were elevated relative to controls (P < 0.001). For each of the diets, total serum cholesterol correlated with serum apoB (P < 0.001). The slopes of the regression lines of serum apoB vs. cholesterol for the monkeys on the PMC, American, and coconut oil diets were similar (m = 0.531, 0.401, and 0.359, respectively), but differed from that of monkeys on the peanut oil diet (m = 0.121). The immunoreactivities of rhesus and human LDL were compared using specific antisera raised against these antigens. In homologous assay systems, monkey and human LDL exhibited unique immunological determinants. The same results were obtained with the delipidated preparations of the two LDLs using antisera raised

  13. Human Serum Eye Drops in Eye Alterations: An Insight and a Critical Analysis

    PubMed Central

    De Pascale, Maria Rosaria; Lanza, Michele; Sommese, Linda; Napoli, Claudio

    2015-01-01

    Human serum contains a physiological plethora of bioactive elements naturally released by activated platelets which might have a significant effect on the regeneration of corneal layers by stimulating the cell growth. This mechanism supported the use of human serum eye drops in some ocular diseases associated with dystrophic changes and alterations of the tear film, such as persistent corneal epithelial defects and dry eye syndrome. We focused our effort on potential benefits and limitations of the use of human serum eye drops when conventional therapies failed. We reviewed the recent literature by reporting published studies from 2010 to 2014. Despite the limited evaluated study populations, most of the clinical studies have confirmed that serum eye drop therapy is effective in corneal healing by reducing ocular symptom, particularly during the short-term follow-up. In addition, three recent published studies have shown the efficacy of the serum eye drop therapy in comparison to traditional ones in intractable patients. Besides, reported ongoing clinical studies confirmed the open debate regarding the use of biologic tools for cornea regeneration. Results from these studies might open novel challenges and perspectives in the therapy of such refractory patients. PMID:26504592

  14. Serum Supplementation Modulates the Effects of Dibutyltin on Human Natural Killer Cell Function

    PubMed Central

    DeWitt, Jamie C.; Luebke, Robert W.

    2008-01-01

    Natural killer (NK) cells are a subset of lymphocytes capable of killing tumor cells, virally infected cells and antibody-coated cells. Dibutyltin (DBT) dichloride is an organotin used as a stabilizer in polyvinylchloride (PVC) plastics and as a deworming product in poultry. DBT may leach from PVC water supply pipes and therefore poses a potential risk to human health. We previously reported diminished NK cells lysis of tumor cells following exposure to DBT in serum-free cell culture medium. However, under in vivo conditions, circulating cells will be exposed to DBT in the presence of 100% plasma; thus we investigated whether serum supplementation and incubation time modulates DBT effects on NK cell killing and the accumulation of DBT in freshly isolated NK cells, to determine whether a serum-free model accurately predicts possible effects of DBT on human NK cells under in vivo conditions. Lytic function was decreased by approximately 35% at an intracellular DBT (DBTi) concentration of 200μM and nearly complete loss of lytic function was observed at DBTi above 300μM for one h. However, an intracellular concentration of 50μM DBT, achieved over 24 h of exposure in 50% serum, reduced lytic function by 50%. Thus, conditions that reflect prolonged contact with circulating DBT, in the presence of serum, suggest that NK cell activity is decreased at lower DBTi. These data indicate that the model is useful in predicting potential human effects of relatively low DBTi concentrations. PMID:18441343

  15. Quantification of Sorafenib in Human Serum by Competitive Enzyme-Linked Immunosorbent Assay.

    PubMed

    Saita, Tetsuya; Yamamoto, Yuta; Noda, Satoshi; Shioya, Makoto; Hira, Daiki; Andoh, Akira; Morita, Shin-Ya; Terada, Tomohiro; Shin, Masashi

    2015-01-01

    The multikinase inhibitor sorafenib has been used in the treatment of hepatocellular carcinoma, renal cell carcinoma, and differentiated thyroid carcinoma. Here we have demonstrated the production of the first specific antibody against sorafenib. Anti-sorafenib serum was obtained by immunizing mice with an antigen conjugated with bovine serum albumin and carboxylic modified 4-(4-aminophenoxy)-N-methyl-2-pyridinecarboxamide (AMPC) using the N-succinimidyl ester method. Enzyme labeling of sorafenib with horseradish peroxidase was similarly performed using carboxylic modified AMPC. A simple competitive enzyme-linked immunosorbent assay (ELISA) for sorafenib was developed using the principle of direct competition between sorafenib and the enzyme marker for anti-sorafenib antibody, which had been adsorbed by the plastic surface of a microtiter plate. Serum sorafenib concentrations lower than 0.04 µg/mL were reproducibly measurable using the ELISA. This ELISA was specific to sorafenib and showed very slight cross-reactivity (2.5%) with a major metabolite, sorafenib N-oxide. The values of serum sorafenib levels from 32 patients measured by this ELISA were comparable with those measured by HPLC, and there was a strong correlation between the values determined by the two methods (Y=1.016X-0.137, r=0.979). The specificity and sensitivity of the ELISA for sorafenib should provide a valuable new tool for use in therapeutic drug monitoring and pharmacokinetic studies of sorafenib. PMID:26521829

  16. Use of In Vitro Assays To Determine Effects of Human Serum on Biological Characteristics of Acanthamoeba castellanii

    PubMed Central

    Sissons, James; Alsam, Selwa; Stins, Monique; Rivas, Antonio Ortega; Morales, Jacob Lorenzo; Faull, Jane; Khan, Naveed Ahmed

    2006-01-01

    Normal human serum inhibits Acanthamoeba (encephalitis isolate) binding to and cytotoxicity of human brain microvascular endothelial cells, which constitute the blood-brain barrier. Zymographic assays revealed that serum inhibits extracellular protease activities of acanthamoebae. But it is most likely that inhibition of specific properties of acanthamoebae is a consequence of the initial amoebicidal-amoebistatic effects induced by serum. For example, serum exhibited amoebicidal effects; i.e., up to 50% of the exposed trophozoites were killed. The residual subpopulation, although viable, remained static over longer incubations. Interestingly, serum enhanced the phagocytic ability of acanthamoebae, as measured by bacterial uptake. Overall, our results demonstrate that human serum has inhibitory effects on Acanthamoeba growth and viability, protease secretions, and binding to and subsequent cytotoxicity for brain microvascular endothelial cells. Conversely, Acanthamoeba phagocytosis was stimulated by serum. PMID:16825391

  17. Label-free electrochemical aptasensing of the human prostate-specific antigen using gold nanospears.

    PubMed

    Rahi, A; Sattarahmady, N; Heli, H

    2016-08-15

    Gold nanospears were electrodeposited with the assistance of arginine as a soft template and precise selection of experimental parameters. The nanospears were then employed as a transducer to immobilize an aptamer of prostate-specific antigen (PSA) and fabrication of a label-free electrochemical aptasensor. The aptasensor was employed for the detection of PSA with a linear concentration range of 0.125-200ngmL(-1) and a limit of detection of 50pgmL(-1). The aptasensor was successfully applied to detect PSA in blood serum samples of healthy and patient persons. PMID:27260456

  18. A flow injection sampling resonance light scattering system for total protein determination in human serum

    NASA Astrophysics Data System (ADS)

    Dong, Lijun; Li, Ying; Zhang, Yaheng; Chen, Xingguo; Hu, Zhide

    2007-04-01

    A novel flow injection method with resonance light scattering detection was developed for the determination of total protein concentrations. This method is based on the enhancement of RLS signals from Methyl Blue (MB) by protein. The enhanced RLS intensities at 333 nm, in a pH 4.1 acidic aqueous solution, were proportional to the protein concentration over the range 2.0-37.3 and 1.0-36.0 μg ml -1 for human serum albumin (HSA) and bovine serum albumin (BSA), respectively. The corresponding limits of detection (3 σ) of 45 ng ml -1 for HSA and 80 ng ml -1 for BSA were attained. The method was successfully applied to the quantification of total proteins in human serum samples, the maximum relative error is less than 1% and the recovery is between 98% and 102%. The sample throughput was 60 h -1.

  19. Detection of α-fetoprotein in human serum using carbon nanotube transistor

    NASA Astrophysics Data System (ADS)

    So, Hye-Mi; Park, Dong-Won; Lee, Seong-Kyu; Kim, Beom Soo; Chang, Hyunju; Lee, Jeong-O.

    2009-03-01

    We have fabricated antibody-coated carbon nanotube field effect transistor (CNT-FET) sensor for the detection of α-fetoprotein (AFP), single chain glycoprotein of 70 kDa that is normally expressed in the fetal liver, in human serum. The AFP-specific antibodies were immobilized on CNT with linker molecule such as pyrenebutyric acid N-hydroxysuccinimide ester. To prevent nonspecific adsorption of antigen, we performed blocking procedure using bovine serum albumin (BSA). Antibody-antigen binding was determined by measuring electrical conductance change of FET and took an average of thereshold voltage change before and after binding. Also we checked concentration-dependent conductance change in human serum using both p-type SWNT-FETs and n-type SWNT-FETs.

  20. Transport of circulating serum cholesterol by human renal cell carcinoma

    SciTech Connect

    Clayman, R.V.; Figenshau, R.S.; Prigge, W.F.; Forstrom, L.; Gebhard, R.L.

    1987-06-01

    Clear cell renal cancer contains a large quantity of cholesterol ester (300-mg./gm. protein). To determine whether abnormalities in cholesterol transport could account for this sterol accumulation, the uptake, release, and imaging capabilities of intravenously injected /sup 131/I-6-iodomethyl-29-norcholesterol, a cholesterol analogue, were studied preoperatively in five patients with clear cell renal cancer. At surgery, samples of the liver, tumor, adrenal, and non-tumor kidney were obtained for analysis. /sup 131/I-sterol uptake by the tumor, when normalized for cholesterol content, was less than for adrenal, liver or kidney. In contrast, release of preloaded /sup 131/I-sterol from the human tumors was consistently slower than for normal kidney. The reduced release of free cholesterol from renal cancer cells may, in part, be responsible for the accumulation of cholesterol in human renal cancer.

  1. Computer-Modelling of Metal Speciation in Human Blood Serum

    NASA Astrophysics Data System (ADS)

    Letkeman, Peter

    1996-02-01

    This paper briefly describes two computer programs, BEST and ECCLES, both available on disc, that can generate distribution diagrams for various metal-complexes in human blood plasma. Two heavy metals, lead and mercury, are used as examples. The species distribution vs pH diagrams are based on formation constants obtained from our own potentiometric study of the mercury-glutathione system. The efficacy of a chelating agent for mobilizing a metal ion from the liable metal-protein complex portion of blood, PMI index, is discussed as well. The paper points out that computer modelling of metal speciation in human blood plasma has led to the design of important new therapeutic chelating agents.

  2. Label-Free Characterization of Emerging Human Neuronal Networks

    NASA Astrophysics Data System (ADS)

    Mir, Mustafa; Kim, Taewoo; Majumder, Anirban; Xiang, Mike; Wang, Ru; Liu, S. Chris; Gillette, Martha U.; Stice, Steven; Popescu, Gabriel

    2014-03-01

    The emergent self-organization of a neuronal network in a developing nervous system is the result of a remarkably orchestrated process involving a multitude of chemical, mechanical and electrical signals. Little is known about the dynamic behavior of a developing network (especially in a human model) primarily due to a lack of practical and non-invasive methods to measure and quantify the process. Here we demonstrate that by using a novel optical interferometric technique, we can non-invasively measure several fundamental properties of neural networks from the sub-cellular to the cell population level. We applied this method to quantify network formation in human stem cell derived neurons and show for the first time, correlations between trends in the growth, transport, and spatial organization of such a system. Quantifying the fundamental behavior of such cell lines without compromising their viability may provide an important new tool in future longitudinal studies.

  3. Candida albicans Shaving to Profile Human Serum Proteins on Hyphal Surface

    PubMed Central

    Marín, Elvira; Parra-Giraldo, Claudia M.; Hernández-Haro, Carolina; Hernáez, María L.; Nombela, César; Monteoliva, Lucía; Gil, Concha

    2015-01-01

    Candida albicans is a human opportunistic fungus and it is responsible for a wide variety of infections, either superficial or systemic. C. albicans is a polymorphic fungus and its ability to switch between yeast and hyphae is essential for its virulence. Once C. albicans obtains access to the human body, the host serum constitutes a complex environment of interaction with C. albicans cell surface in bloodstream. To draw a comprehensive picture of this relevant step in host-pathogen interaction during invasive candidiasis, we have optimized a gel-free shaving proteomic strategy to identify both, human serum proteins coating C. albicans cells and fungi surface proteins simultaneously. This approach was carried out with normal serum (NS) and heat inactivated serum (HIS). We identified 214 human and 372 C. albicans unique proteins. Proteins identified in C. albicans included 147 which were described as located at the cell surface and 52 that were described as immunogenic. Interestingly, among these C. albicans proteins, we identified 23 GPI-anchored proteins, Gpd2 and Pra1, which are involved in complement system evasion and 7 other proteins that are able to attach plasminogen to C. albicans surface (Adh1, Eno1, Fba1, Pgk1, Tdh3, Tef1, and Tsa1). Furthermore, 12 proteins identified at the C. albicans hyphae surface induced with 10% human serum were not detected in other hypha-induced conditions. The most abundant human proteins identified are involved in complement and coagulation pathways. Remarkably, with this strategy, all main proteins belonging to complement cascades were identified on the C. albicans surface. Moreover, we identified immunoglobulins, cytoskeletal proteins, metabolic proteins such as apolipoproteins and others. Additionally, we identified more inhibitors of complement and coagulation pathways, some of them serpin proteins (serine protease inhibitors), in HIS vs. NS. On the other hand, we detected a higher amount of C3 at the C. albicans surface in

  4. Human Ozone (O3) Exposure Alters Serum Profile of Lipid Metabolites

    EPA Science Inventory

    HUMAN OZONE (O3) EXPOSURE ALTERS SERUM PROFILE OF LIPID METABOLITES Miller, D B.1; Kodavanti, U P.2 Karoly, E D.3; Cascio W.E2, Ghio, A J. 21. UNC-Chapel Hill, Chapel Hill, N.C., United States. 2. NHEERL, U.S. EPA, RTP, N.C., United States. 3. METABOLON INC., Durham, N.C., United...

  5. Synergistic action of photosensitizers and normal human serum in a bactericidal process. I. Effect of chlorophylls.

    PubMed

    Jankowski, Andrzej; Jankowski, Stanisław; Mirończyk, Agnieszka

    2003-01-01

    Susceptibility of some Gram-negative strains against the bactericidal action of normal human serum (NHS) and of chlorophyll, which induces production of reactive oxygen species by light, was studied. A synergistic bactericidal activity of NHS and chlorophyll against E. coli K1 and Shigella flexneri strains was observed. PMID:15095924

  6. Determination of element levels in human serum: Total reflection X-ray fluorescence applications

    NASA Astrophysics Data System (ADS)

    Majewska, U.; Łyżwa, P.; Łyżwa, K.; Banaś, D.; Kubala-Kukuś, A.; Wudarczyk-Moćko, J.; Stabrawa, I.; Braziewicz, J.; Pajek, M.; Antczak, G.; Borkowska, B.; Góźdź, S.

    2016-08-01

    Deficiency or excess of elements could disrupt proper functioning of the human body and could lead to several disorders. Determination of their concentrations in different biological human fluids and tissues should become a routine practice in medical treatment. Therefore the knowledge about appropriate element concentrations in human organism is required. The purpose of this study was to determine the concentration of several elements (P, S, Cl, K, Ca, Cr, Fe, Cu, Zn, Se, Br, Rb, Pb) in human serum and to define the reference values of element concentration. Samples of serum were obtained from 105 normal presumably healthy volunteers (66 women aged between 15 and 78 years old; 39 men aged between 15 and 77 years old). Analysis has been done for the whole studied population and for subgroups by sex and age. It is probably first so a wide study of elemental composition of serum performed in the case of Świętokrzyskie region. Total reflection X-ray fluorescence (TXRF) method was used to perform the elemental analysis. Spectrometer S2 Picofox (Bruker AXS Microanalysis GmbH) was used to identify and measure elemental composition of serum samples. Finally, 1st and 3rd quartiles were accepted as minimum and maximum values of concentration reference range.

  7. The distribution of iron between the metal-binding sites of transferrin human serum.

    PubMed

    Williams, J; Moreton, K

    1980-02-01

    The Makey & Seal [(1976) Biochim. Biophys. Acta 453, 250--256] method of polyacrylamide-gel electrophoresis in buffer containing 6 M-urea was used to determine the distribution of iron between the N-terminal and C-terminal iron-binding sites of transferrin in human serum. In fresh serum the two sites are unequally occupied; there is preferential occupation of the N-terminal site. On incubation of the serum at 37 degrees C the preference of iron for the N-terminal site becomes more marked. On storage of serum at -15 degrees C the iron distribution changes so that there is a marked preference for the C-terminal site. Dialysis of serum against buffer at pH 7.4 also causes iron to be bound much more strongly by the C-terminal than by the N-terminal site. The original preference for the N-terminal site can be resroted to the dialysed serum by addition of the diffusible fraction. PMID:7396826

  8. The distribution of iron between the metal-binding sites of transferrin human serum.

    PubMed Central

    Williams, J; Moreton, K

    1980-01-01

    The Makey & Seal [(1976) Biochim. Biophys. Acta 453, 250--256] method of polyacrylamide-gel electrophoresis in buffer containing 6 M-urea was used to determine the distribution of iron between the N-terminal and C-terminal iron-binding sites of transferrin in human serum. In fresh serum the two sites are unequally occupied; there is preferential occupation of the N-terminal site. On incubation of the serum at 37 degrees C the preference of iron for the N-terminal site becomes more marked. On storage of serum at -15 degrees C the iron distribution changes so that there is a marked preference for the C-terminal site. Dialysis of serum against buffer at pH 7.4 also causes iron to be bound much more strongly by the C-terminal than by the N-terminal site. The original preference for the N-terminal site can be resroted to the dialysed serum by addition of the diffusible fraction. Images Fig. 1. PMID:7396826

  9. Identification of Serum Biomarkers for Gastric Cancer Diagnosis Using a Human Proteome Microarray.

    PubMed

    Yang, Lina; Wang, Jingfang; Li, Jianfang; Zhang, Hainan; Guo, Shujuan; Yan, Min; Zhu, Zhenggang; Lan, Bin; Ding, Youcheng; Xu, Ming; Li, Wei; Gu, Xiaonian; Qi, Chong; Zhu, Heng; Shao, Zhifeng; Liu, Bingya; Tao, Sheng-Ce

    2016-02-01

    We aimed to globally discover serum biomarkers for diagnosis of gastric cancer (GC). GC serum autoantibodies were discovered and validated using serum samples from independent patient cohorts encompassing 1,401 participants divided into three groups, i.e. healthy, GC patients, and GC-related disease group. To discover biomarkers for GC, the human proteome microarray was first applied to screen specific autoantibodies in a total of 87 serum samples from GC patients and healthy controls. Potential biomarkers were identified via a statistical analysis protocol. Targeted protein microarrays with only the potential biomarkers were constructed and used to validate the candidate biomarkers using 914 samples. To provide further validation, the abundance of autoantibodies specific to the biomarker candidates was analyzed using enzyme-linked immunosorbent assays. Receiver operating characteristic curves were generated to evaluate the diagnostic accuracy of the serum biomarkers. Finally, the efficacy of prognosis efficacy of the final four biomarkers was evaluated by analyzing the clinical records. The final panel of biomarkers consisting of COPS2, CTSF, NT5E, and TERF1 provides high diagnostic power, with 95% sensitivity and 92% specificity to differentiate GC patients from healthy individuals. Prognosis analysis showed that the panel could also serve as independent predictors of the overall GC patient survival. The panel of four serum biomarkers (COPS2, CTSF, NT5E, and TERF1) could serve as a noninvasive diagnostic index for GC, and the combination of them could potentially be used as a predictor of the overall GC survival rate. PMID:26598640

  10. Novel Transgenic Mouse Model for Studying Human Serum Albumin as a Biomarker of Carcinogenic Exposure.

    PubMed

    Sheng, Jonathan; Wang, Yi; Turesky, Robert J; Kluetzman, Kerri; Zhang, Qing-Yu; Ding, Xinxin

    2016-05-16

    Albumin is a commonly used serum protein for studying human exposure to xenobiotic compounds, including therapeutics and environmental pollutants. Often, the reactivity of albumin with xenobiotic compounds is studied ex vivo with human albumin or plasma/serum samples. Some studies have characterized the reactivity of albumin with chemicals in rodent models; however, differences between the orthologous peptide sequences of human and rodent albumins can result in the formation of different types of chemical-protein adducts with different interaction sites or peptide sequences. Our goal is to generate a human albumin transgenic mouse model that can be used to establish human protein biomarkers of exposure to hazardous xenobiotics for human risk assessment via animal studies. We have developed a human albumin transgenic mouse model and characterized the genotype and phenotype of the transgenic mice. The presence of the human albumin gene in the genome of the model mouse was confirmed by genomic PCR analysis, whereas liver-specific expression of the transgenic human albumin mRNA was validated by RT-PCR analysis. Further immunoblot and mass spectrometry analyses indicated that the transgenic human albumin protein is a full-length, mature protein, which is less abundant than the endogenous mouse albumin that coexists in the serum of the transgenic mouse. The transgenic protein was able to form ex vivo adducts with a genotoxic metabolite of 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine, a procarcinogenic heterocyclic aromatic amine formed in cooked meat. This novel human albumin transgenic mouse model will facilitate the development and validation of albumin-carcinogen adducts as biomarkers of xenobiotic exposure and/or toxicity in humans. PMID:27028147