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Sample records for human serum preliminary

  1. A preliminary screening study on the associated proteins in human psoriasis vulgaris by serum proteomics technologies

    Institute of Scientific and Technical Information of China (English)

    Zhankui Liu; Shengshun Tan; Chunshui Yu; Jinghua Fan; Zhuanli Bai; Junjie Li

    2007-01-01

    Objective:To investigate the optimum screening conditions of associated proteins in human psoriasis vulgaris by serum proteomics technique, and to screen the different expression proteins related with psoriasis vulgaris. Methods:Serum samples of peripheral blood were collected from newly diagnosed psoriasis vulgaris patients in the clinic, and 20 matched healthy persons.Serum albumin IgG was removed by filtering with ProteoExtract Albumin/IgG. After comparative proteomics analysis the different protein spots were identified using 2-DE and MS. Results :Electrophoresis figures with high resolution and reproducibility were obtained. Three different expression proteins were found only in the serum from psoriasis vulgaris patients,while nine other different proteins expressing from healthy volunteers. Conclusion:The protein expression was different in the serum between the psoriasis vulgaris patients and healthy volunteers. It was hoped that we could find the biomarkers related to psoriasis vulgaris by using proteomics.

  2. The human serum metabolome.

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    Nikolaos Psychogios

    Full Text Available 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.

  3. Preliminary investigation of human serum albumin-Vβ inhibition on toxic shock syndrome induced by staphylococcus enterotoxin B in vitro and in vivo.

    Science.gov (United States)

    Yuan, Qifeng; Li, Lin; Pian, Yaya; Hao, Huaijie; Zheng, Yuling; Zang, Yating; Jiang, Hua; Jiang, Yongqiang

    2016-04-01

    Staphylococcus enterotoxin B (SEB) is a superantigen that can induce massive activation of T cells with specific Vβ and inflammatory cytokine cascades, which mediate shock. To date, no SEB vaccine has been developed for preventing toxic shock syndrome (TSS). Here, we evaluated the therapeutic effect of a fusion protein human serum albumin-Vβ (HSA-Vβ) on TSS induced by SEB. Compared with Vβ, the preparation of HSA-Vβ was much easier to handle owing to its solubility. Affinity testing showed that HSA-Vβ had high affinity for SEB. In vitro results showed that HSA-Vβ could effectively inhibit interferon (IFN)-γ and tumor necrosis factor (TNF)-α secretion by human peripheral blood mononuclear cells. Moreover, in vivo, HSA-Vβ reduced IFN-γ and TNF-α levels in the serum and protected mice from SEB lethal challenge when administered simultaneously with SEB or 30 min after SEB. In summary, we simplified the preparation of Vβ by fusion with HSA, creating the HSA-Vβ protein, which effectively inhibited cytokine production and protected mice from lethal challenge with SEB. These data indicated that HSA-Vβ may represent a novel therapeutic strategy for the treatment of SEB-induced TSS.

  4. Novel hybrids of metronidazole and quinolones: synthesis, bioactive evaluation, cytotoxicity, preliminary antimicrobial mechanism and effect of metal ions on their transportation by human serum albumin.

    Science.gov (United States)

    Cui, Sheng-Feng; Peng, Li-Ping; Zhang, Hui-Zhen; Rasheed, Syed; Vijaya Kumar, Kannekanti; Zhou, Cheng-He

    2014-10-30

    A novel series of hybrids of metronidazole and quinolones as antimicrobial agents were designed and synthesized. Most prepared compounds exhibited good or even stronger antimicrobial activities in comparison with reference drugs. Furthermore, these highly active metronidazole-quinolone hybrids showed appropriate ranges of pKa, log P and aqueous solubility to pharmacokinetic behaviors and no obvious toxicity to A549 and human hepatocyte LO2 cells. Their competitive interactions with metal ions to HSA revealed that the participation of Mg(2+) ion in compound 7d-HSA association could result in a concentration increase of free compound 7d. Molecular modeling and experimental investigation of compound 7d with DNA suggested that possible antibacterial mechanism might be in relation with multiple binding sites between bioactive molecules and topo IV-DNA complex.

  5. Radioimmunoassay of secretin in human serum

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    Bonora, G.; Vezzadini, P.; Toni, R.; Labo, G. (Bologna Univ. (Italy))

    1984-06-27

    A sensitive radioimmunoassay for secretin has been developed. Antisera were raised against synthetic porcine secretin coupled to bovine serum albumin. N-..cap alpha..-desaminotyrosyl-..beta..-alanyl secretin was radioiodinated by a slight modification of the chloramine-T method. Pure synthetic porcine secretin was used as a standard. Free and bound hormone were separated by dextran-coated charcoal. No cross-reactivity was found with structurally and physiologically related peptides. The sensitivity of the assay was high enough to measure fasting secretin levels in human serum. Patients with acute or chronic pancreatitis had mean serum secretin concentration not significantly different from healthy subjects. In patients with pancreatic carcinoma the mean serum secretin concentration was significantly lower than in healthy subjects, although a wide overlap of the two groups was evident.

  6. Imidazole binding to human serum albumin.

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    Rodrigo, M C; Ceballos, A; Mariño, E; Cachaza, J M; Domínguez-Gil, A; Kuemmerle, H P

    1988-06-01

    Imidazole is a substance released by the organism when a new salicylate derivative, imidazole salicylate is administered. A study was made of the binding of imidazole to human serum albumin by an in vitro assay employing an ultrafiltration technique. For the concentration range that imidazole was found in plasma following administration of the drug to healthy volunteers, the mean binding percentages were: 12.1 +/- 1.8 and 19.7 +/- 3.1 at 37 degrees C and 25 degrees C, respectively. The results obtained in the study follow a model entailing three equal and independent binding sites of imidazole to serum albumin and the values of the corresponding constants were determined. Apparently, the presence in the plasma samples of sodium salicylate at a concentration of 100 micrograms/ml does not affect the binding of imidazole to human serum albumin.

  7. Purification of human serum hyaluronidase using chromatofocusing

    DEFF Research Database (Denmark)

    Fenger, M

    1982-01-01

    A commercial chromatofocusing system was applied to Cohn's fraction III of human serum to purify hyaluronidase (E.C.3.2.1.3.5). The protein that eluted in the pH range 4.7-5.3 was pooled and precipitated by adding ammonium sulphate to 50% saturation. This sequence of fractionation purified...

  8. Sexual hormone serum levels and temporomandibular disorders. A preliminary study.

    Science.gov (United States)

    Landi, Nicola; Lombardi, Ilaria; Manfredini, Daniele; Casarosa, Elena; Biondi, Katya; Gabbanini, Massimo; Bosco, Mario

    2005-02-01

    The aim of the present study was to investigate the role of sexual hormones in a young adult population affected by articular forms of temporomandibular disorders (TMD), measuring 17beta-estradiol and progesterone serum levels. In the study, we included 40 patients (20 males and 20 females) with a Research Diagnostic Criteria for Temporomandibular Disorders (RDC/TMD) axis I group II diagnosis of disk displacement and/or group III diagnosis of arthralgia, osteoarthritis or osteoarhrosis, and 32 healthy controls. In female patients, blood samples were collected in follicular and luteal phases of the same menstrual cycle, while only one blood sample was drawn in male patients. Serum levels of estradiol and progesterone were determined using a radioimmunoassay and the comparison between the two groups was performed using a t test. Regarding estradiol, our results showed significantly higher serum levels in patients affected by TMD than in healthy controls, both in males (p hormones, these data suggest that high serum estrogen levels might be implicated in the physiopathology of TMD.

  9. Interaction of Citrinin with Human Serum Albumin

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    Miklós Poór

    2015-12-01

    Full Text Available 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.

  10. Purification of human serum hyaluronidase using chromatofocusing

    DEFF Research Database (Denmark)

    Fenger, M

    1982-01-01

    A commercial chromatofocusing system was applied to Cohn's fraction III of human serum to purify hyaluronidase (E.C.3.2.1.3.5). The protein that eluted in the pH range 4.7-5.3 was pooled and precipitated by adding ammonium sulphate to 50% saturation. This sequence of fractionation purified...... hyaluronidase extensively by immunological criteria. It is shown that hyaluronidase is a population of enzymes displaying microheterogeneity. The commercial chromatofocusing system behaved as theoretically expected. The capacity of the gel is 3 mg per ml gel. Any overload will be trapped or precipitated...

  11. Induction and properties of guinea pig serum interferon. Preliminary report.

    Science.gov (United States)

    Nolewajka, E; Mikolajski, K; Kapp-Burzyńska, Z; Trzeciak, J; Wrona, M

    1977-01-01

    Guinea pigs, 250-350 g body weight, both sexes, were injected with 5X10(8.5) EID50 NDV (Radom strain) intracardially and intraperitoneally simultaneously. The animals were bled by cardiac puncture 0, 3, 6, 12, 24 and 48 hours after injection. After virus inactivation, serum interferon titration was performed in cultures of guinea pig embryo kidney cells with 50 percent plaque inhibition test using VSV. The highest interferon titer (64 u./ml) was found after 6 hours of inductor injection. Interferon titer decreased quickly and after 12 hours it was lower than 16 u./ml. Guinea pig serum interferon induced by NDV was resistant to pH 2 and 56 degrees C during 1 hour. Interferon was inactivated by trypsin. The decribed interferon did not protect heterologous species cells (swine) against Teschen Disease Virus infection. Other properties of this interferon are being studied.

  12. Serum thyroid stimulating hormone (TSH) in malnutrition: preliminary results.

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    Osman, A; Khalid, B A; Tan, T T; Wan Nazaimoon, W M; Wu, L L; Ng, M L

    1993-06-01

    This is a report of a cross sectional study involving 3 groups of children, moderately malnourished (BMI BMI 15-18) and well nourished (BMI > 18) to determine the differences in hormonal and biochemical parameters between the groups. The children were of age range from 7-17 years old. The children were from the same area with exposure to the same food, drinking water and environment. There were significant differences in the nutritional indices between the three groups. No differences were observed in levels of triiodothyronine (T3), thyroxine (T4) and T3:T4 ratio. Significant difference however was found in the TSH levels using highly sensitive IRMA TSH assays. Moderately malnourished children had higher TSH levels (p < 0.05) compared to mildly malnourished and well-nourished children. No difference was found between the mildly malnourished and well-nourished groups. There were no significant differences in serum cortisols done at similar times, fasting growth hormone and calcium. Serum alanine transminase (ALT) however was higher in moderately malnourished than in well-nourished children. Thus using highly sensitive IRMA TSH assays, we were able to detect differences in TSH levels even though T3, T4 and T3:T4 ratio, cortisol, growth hormone and calcium were normal, implying in moderately malnourished children, a higher TSH drive to maintain euthyroid state.

  13. Proteomic analysis for detecting serum biomarkers related to smoking in humans

    Institute of Scientific and Technical Information of China (English)

    XIAO Dan; ZHAO Li-juan; CHU Shui-lian; JING Hang; WANG Chen

    2012-01-01

    Background Smoking is the leading cause of death in the world.This study focused on the difference of the serum proteomic profiling between healthy smokers and nonsmokers in order to find smoking-specific serum biomarkers.Methods Pattern-based proteomic profiling of 100 serum samples (from 50 Chinese male smokers and 50 matched nonsmokers) was performed through magnetic bead fractionation coupled with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry analysis (MALDI-TOF-MS) and resulting data were statistically analyzed by Ciphergen ProteinChip software 3.0.2.Results We found 72 serum peaks were significantly different between smokers and nonsmokers (P <0.05).Marker peaks of mass-to-charge ratio (m/z) 3159.13,7561.03 and 9407.32 were smoking-specific.Conclusion The preliminary data suggested that smoking-specific serum biomarkers could be detected in humans.

  14. Measuring hepatic functional reserve using low temporal resolution Gd-EOB-DTPA dynamic contrast-enhanced MRI: a preliminary study comparing galactosyl human serum albumin scintigraphy with indocyanine green retention

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    Saito, Kazuhiro; Hashimoto, Tsuyoshi; Araki, Yoichi; Akata, Soichi; Tokuuye, Koichi [Tokyo Medical University, Department of Radiology, Tokyo (Japan); Ledsam, Joseph; Sourbron, Steven [University of Leeds, Division of Medical Physics, Leeds (United Kingdom)

    2014-01-15

    To investigate if tracer kinetic modelling of low temporal resolution dynamic contrast-enhanced (DCE) MRI with Gd-EOB-DTPA could replace technetium-99 m galactosyl human serum albumin (GSA) single positron emission computed tomography (SPECT) and indocyanine green (ICG) retention for the measurement of liver functional reserve. Twenty eight patients awaiting liver resection for various cancers were included in this retrospective study that was approved by the institutional review board. The Gd-EOB-DTPA MRI sequence acquired five images: unenhanced, double arterial phase, portal phase, and 4 min after injection. Intracellular contrast uptake rate (UR) and extracellular volume (Ve) were calculated from DCE-MRI, along with the ratio of GSA radioactivity of liver to heart-plus-liver and per cent of cumulative uptake from 15-16 min (LHL15 and LU15, respectively) from GSA-scintigraphy. ICG retention at 15 min, Child-Pugh cirrhosis score (CPS) and postoperative Inuyama fibrosis criteria were also recorded. Statistical analysis was with Spearman rank correlation analysis. Comparing MRI parameters with the reference methods, significant correlations were obtained for UR and LHL15, LU15, ICG15 (all 0.4-0.6, P < 0.05); UR and CPS (-0.64, P < 0.001); Ve and Inuyama (0.44, P < 0.05). Measures of liver function obtained by routine Gd-EOB-DTPA DCE-MRI with tracer kinetic modelling may provide a suitable method for the evaluation of liver functional reserve. (orig.)

  15. Antioxidant flavonoids bind human serum albumin

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    Kanakis, C. D.; Tarantilis, P. A.; Polissiou, M. G.; Diamantoglou, S.; Tajmir-Riahi, H. A.

    2006-10-01

    Human serum albumin (HSA) is a principal extracellular protein with a high concentration in blood plasma and carrier for many drugs to different molecular targets. Flavonoids are powerful antioxidants and prevent DNA damage. The antioxidative protections are related to their binding modes to DNA duplex and complexation with free radicals in vivo. However, flavonoids are known to inhibit the activities of several enzymes such as calcium phospholipid-dependent protein kinase, tyrosine protein kinase from rat lung, phosphorylase kinase, phosphatidylinositol 3-kinase and DNA topoisomerases that exhibit the importance of flavonoid-protein interaction. This study was designed to examine the interaction of human serum albumin (HSA) with quercetin (que), kaempferol (kae) and delphinidin (del) in aqueous solution at physiological conditions, using constant protein concentration of 0.25 mM (final) and various drug contents of 1 μM-1 mM. FTIR and UV-vis spectroscopic methods were used to determine the polyphenolic binding mode, the binding constant and the effects of flavonoid complexation on protein secondary structure. The spectroscopic results showed that flavonoids are located along the polypeptide chains through H-bonding interactions with overall affinity constant of Kque = 1.4 × 10 4 M -1, Kkae = 2.6 × 10 5 M -1 and Kdel = 4.71 × 10 5 M -1. The protein secondary structure showed no alterations at low pigment concentration (1 μM), whereas at high flavonoid content (1 mM), major reduction of α-helix from 55% (free HSA) to 42-46% and increase of β-sheet from 15% (free HSA) to 17-19% and β-anti from 7% (free HSA) to 10-20% occurred in the flavonoid-HSA adducts. The major reduction of HSA α-helix is indicative of a partial protein unfolding upon flavonoid interaction.

  16. Klotho is a serum factor related to human aging

    Institute of Scientific and Technical Information of China (English)

    肖能明; 张焱明; 郑权; 顾军

    2004-01-01

    Background Does klotho (KL) protein exist in human serum, and is there any correlation between KL protein in serum with human aging? In order to answer those questions, we identified KL protein in human serum and established the correlation between KL protein in human serum and aging.Methods We prepared a polyclonal antibody against human KL protein that was able to recognize the C-terminal of human secreted KL protein. Western blot and enzyme-linked immunosorbent assay (ELISA) were used to identify KL protein in human serum.Results In Western blot, the antibody specifically recognized a 60-kD KL protein in both human and mice serum. The population aged from 0 to 91 years screened by ELISA revealed that the level of serum KL declined while age increased, though each individual level was variable and that the trend of decreasing in serum KL had no difference in sex.Conclusion Our data suggest that KL is a serum factor related to human aging.

  17. Binding of disodium cromoglycate to human serum albumin

    Science.gov (United States)

    Ochoa de Aspuru, Eduardo; Zatón, Ana M. L.

    1998-07-01

    The binding of several benzopiranone derivatives to human serum albumin was determined. The antiallergic drug disodium cromoglycate binds weakly to serum albumin. However, its precursors, chromones of smaller size, were able to bind in a hydrophobic pocket in the protein, and are carried by serum albumin in blood.

  18. Farm Animal Serum Proteomics and Impact on Human Health

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    Francesco Di Girolamo

    2014-09-01

    Full Text Available 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.

  19. Preliminary Framework for Human-Automation Collaboration

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    Oxstrand, Johanna Helene [Idaho National Lab. (INL), Idaho Falls, ID (United States); Le Blanc, Katya Lee [Idaho National Lab. (INL), Idaho Falls, ID (United States); Spielman, Zachary Alexander [Idaho National Lab. (INL), Idaho Falls, ID (United States)

    2015-09-01

    The Department of Energy’s Advanced Reactor Technologies Program sponsors research, development and deployment activities through its Next Generation Nuclear Plant, Advanced Reactor Concepts, and Advanced Small Modular Reactor (aSMR) Programs to promote safety, technical, economical, and environmental advancements of innovative Generation IV nuclear energy technologies. The Human Automation Collaboration (HAC) Research Project is located under the aSMR Program, which identifies developing advanced instrumentation and controls and human-machine interfaces as one of four key research areas. It is expected that the new nuclear power plant designs will employ technology significantly more advanced than the analog systems in the existing reactor fleet as well as utilizing automation to a greater extent. Moving towards more advanced technology and more automation does not necessary imply more efficient and safer operation of the plant. Instead, a number of concerns about how these technologies will affect human performance and the overall safety of the plant need to be addressed. More specifically, it is important to investigate how the operator and the automation work as a team to ensure effective and safe plant operation, also known as the human-automation collaboration (HAC). The focus of the HAC research is to understand how various characteristics of automation (such as its reliability, processes, and modes) effect an operator’s use and awareness of plant conditions. In other words, the research team investigates how to best design the collaboration between the operators and the automated systems in a manner that has the greatest positive impact on overall plant performance and reliability. This report addresses the Department of Energy milestone M4AT-15IN2302054, Complete Preliminary Framework for Human-Automation Collaboration, by discussing the two phased development of a preliminary HAC framework. The framework developed in the first phase was used as the

  20. Review: Glycation of human serum albumin.

    Science.gov (United States)

    Anguizola, Jeanethe; Matsuda, Ryan; Barnaby, Omar S; Hoy, K S; Wa, Chunling; DeBolt, Erin; Koke, Michelle; Hage, David S

    2013-10-21

    Glycation involves the non-enzymatic addition of reducing sugars and/or their reactive degradation products to amine groups on proteins. This process is promoted by the presence of elevated blood glucose concentrations in diabetes and occurs with various proteins that include human serum albumin (HSA). This review examines work that has been conducted in the study and analysis of glycated HSA. The general structure and properties of HSA are discussed, along with the reactions that can lead to modification of this protein during glycation. The use of glycated HSA as a short-to-intermediate term marker for glycemic control in diabetes is examined, and approaches that have been utilized for measuring glycated HSA are summarized. Structural studies of glycated HSA are reviewed, as acquired for both in vivo and in vitro glycated HSA, along with data that have been obtained on the rate and thermodynamics of HSA glycation. In addition, this review considers various studies that have investigated the effects of glycation on the binding of HSA with drugs, fatty acids and other solutes and the potential clinical significance of these effects.

  1. 76 FR 39399 - Chlorpyrifos Registration Review; Preliminary Human Health Risk Assessment; Notice of Availability

    Science.gov (United States)

    2011-07-06

    ... AGENCY Chlorpyrifos Registration Review; Preliminary Human Health Risk Assessment; Notice of Availability... availability of EPA's preliminary human health risk assessment for the registration review of chlorpyrifos and... comprehensive preliminary human health risk assessment for all chlorpyrifos uses. After reviewing comments...

  2. 76 FR 52945 - Chlorpyrifos Registration Review; Preliminary Human Health Risk Assessment; Extension of Comment...

    Science.gov (United States)

    2011-08-24

    ... AGENCY Chlorpyrifos Registration Review; Preliminary Human Health Risk Assessment; Extension of Comment... availability of the chlorpyrifos registration review; preliminary human health risk assessment. This document... for the chlorpyrifos reregistration review, preliminary human health risk assessment, established in...

  3. Human autologous serum as a substitute for fetal bovine serum in human Schwann cell culture.

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    Parisa Goodarzi

    2014-04-01

    Full Text Available Nowadays, cell -based and tissue engineered products have opened new horizons in treatment of incurable nervous system disorders. The number of studies on the role of Schwann cells (SC in treating nervous disorders is higher than other cell types. Different protocols have been suggested for isolation and expansion of SC which most of them have used multiple growth factors, mitogens and fetal bovine sera (FBS in culture medium. Because of potential hazards of animal-derived reagents, this study was designed to evaluate the effect of replacing FBS with human autologous serum (HAS on SC's yield and culture parameters. Samples from 10 peripheral nerve biopsies were retrieved and processed under aseptic condition. The isolated cells cultured in FBS (1st group or autologous serum (2nd group. After primary culture the cells were seeded at 10000 cell/cm2 in a 12 wells cell culture plate for each group. At 100% confluency, the cell culture parameters (count, viability, purity and culture duration of 2 groups were compared using paired t-test. The average donors' age was 35.80 (SD=13.35 and except for 1 sample the others cultured successfully. In first group, the averages of cell purity, viability and culture duration were 97% (SD=1.32, 97/33% (SD=1.22 and 11.77 (SD=2.58 days respectively. This parameters were 97.33% (SD=1.00, 97.55% (SD=1.33 and 10.33 days (SD=1.65 in second group. The difference of cell count, purity and viability were not significant between 2 groups (P>0.05. The cells of second group reached to 100% confluency in shorter period of time (P=0.03. The results of this study showed that autologous serum can be a good substitute for FBS in human SC culture. This can reduce the costs and improve the safety of cell product for clinical application.

  4. Type IV collagen-degrading enzyme activity in human serum.

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    Hashimoto, Noriaki; Kobayashi, Michio; Watanabe,Akiharu; Higashi, Toshiro; Tsuji, Takao

    1988-01-01

    Type IV collagen-degrading enzyme activity was detected in human serum. Serum was preincubated with 4-aminophenylmercuric acetate and trypsin to activate the enzyme prior to assay. Type IV collagen, purified from human placentas and radiolabeled with [1-14C] acetic anhydride, was used as the substrate. The enzyme activity was measured at pH 7.5 and inhibited by treatment with ethylenediaminetetraacetic acid or heat. The assay of type IV collagen-degrading enzyme in human serum might be useful...

  5. Type IV collagen-degrading enzyme activity in human serum.

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    Hashimoto,Noriaki

    1988-02-01

    Full Text Available Type IV collagen-degrading enzyme activity was detected in human serum. Serum was preincubated with 4-aminophenylmercuric acetate and trypsin to activate the enzyme prior to assay. Type IV collagen, purified from human placentas and radiolabeled with [1-14C] acetic anhydride, was used as the substrate. The enzyme activity was measured at pH 7.5 and inhibited by treatment with ethylenediaminetetraacetic acid or heat. The assay of type IV collagen-degrading enzyme in human serum might be useful for estimating the degradation of type IV collagen.

  6. A Preliminary Analysis of Non-small Cell Lung Cancer Biomarkers in Serum

    Institute of Scientific and Technical Information of China (English)

    XUE-YUAN XIAO; YING TANG; XIU-PING WEI; DA-CHENG HE

    2003-01-01

    Objective To identify potential serum biomarkers that could be used to discriminate lungcancers from normal. Methods Proteomic spectra of twenty-eight serum samples from patientswith non-small cell lung cancer and twelve from normal individuals were generated by SELDI(Surfaced Enhanced Laser Desorption/Ionization) Mass Spectrometry. Anion-exchange columns wereused to fractionate the sera into 6 designated pH groups. Two different types of protein chip arrays,IMAC-Cu and WCX2, were employed. Samples were examined in PBSII Protein Chip Reader(Ciphergen Biosystem Inc) and the discriminatory profiling between cancer and normal samples wasanalyzed with Biomarker Pattern software. Results Five distinct potential lung cancer biomarkerswith higher sensitivity and specificity were found, with four common biomarkers in both IMAC-Cuand WCX2 chip; the remaining biomarker occurred only in WCX2 chip. Two biomarkers wereup-regulated while three biomarkers were down-regulated in the serum samples from patients withnon-small cell lung cancer. The sensitivities provided by the individual biomarkers were 75%-96.43%and specificities were 75%-100%. Conclusions The preliminary results suggest that serum is acapable resource for detecting specific non-small cell lung cancer biomarkers. SELDI massspectrometry is a useful tool for the detection and identification of new potential biomarker ofnon-small cell lung cancer in serum.

  7. Molecular basis of indomethacin-human serum albumin interaction

    DEFF Research Database (Denmark)

    Trivedi, V D; Vorum, H; Honoré, B

    1999-01-01

    Studies on the strength and extent of binding of the non-steroidal anti-inflammatory drug indomethacin to human serum albumin (HSA) have provided conflicting results. In the present work, the serum-binding of indomethacin was studied in 55 mM sodium phosphate buffer (pH 7.0) at 28 degrees C, by u...

  8. Preliminary Study of MALDI-TOF Mass Spectrometry-based Screening of Patients with the NSCLC Serum-Specific Peptides

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    Juan AN

    2013-05-01

    Full Text Available Background and objective The improved survival of patients with lung cancer depends on early diagnosis of lung cancer. However, the traditional diagnostic techniques have several limitations. Mass spectrometry (MS has been applied as a core technology for cancer diagnosis in preliminary proteomic studies. The aim of this study is to explore the differences in the serum peptide levels of patients with non-small cell lung cancer (NSCLC and healthy individuals using matrix-assisted laser desorption/ionization (MALDI-time-of-flight (TOF-MS. A NSCLC serum classification model was then established. Methods One hundred and thirty three cases of patients with NSCLC serum specimens and 132 cases of healthy human serum specimens were randomly divided into two groups in accordance with the ratio of three to one without age and gender differences. The training group was used to establish the classification model, this group included serum samples from 100 NSCLC cases and 100 healthy individuals. The test group for validating the proposed model was composed of the remaining serum samples from 33 NSCLC cases and 32 healthy individuals. Peptides were extracted from the samples using magnetic beads- immobilized metal affinity capture - copper, and their mass spectra were obtained using an automated MALDI-TOF-MS system. The MS data from the training group was analyzed using the ClinproToolTM software to identify the individual peptide fragments and establish the classification model. The sensitivity and specificity of the model were verified by blind testing with the test group. Results Among the 131 different peptide peaks, ranging from m/z 1,000 Da to 10,000 Da, 14 peaks were significantly different in the NSCLC samples of the training group, as compared with the controls (P<0.000,001; AUC≥0.9; these included 2 higher peaks and 12 lower peaks. The classification model was established, and the test group was verified for only 3 peptide peaks (7,478.59, 2

  9. Human serum provided additional values in growth factors supplemented medium for human chondrocytes monolayer expansion and engineered cartilage construction.

    Science.gov (United States)

    Chua, K H; Aminuddin, B S; Fuzina, N H; Ruszymah, B H I

    2004-05-01

    We have previously formulated an optimized human chondrocytes growth medium based on 2% fetal bovine serum supplementation. For clinical usage, the animal serum must be replaced by patient own serum. We investigated the effects of human serum concentration for human nasal septum chondrocytes monolayer culture and cartilage reconstruction. Human serum demonstrated a dose dependent manner in promoting chondrocytes growth and cartilage engineering.

  10. 99M-technetium labeled macroaggregated human serum albumin pharmaceutical

    Science.gov (United States)

    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.

  11. In vitro inhibition of the paraoxonase from human serum with ...

    African Journals Online (AJOL)

    SERVER

    2008-03-04

    Mar 4, 2008 ... Sulfonamide was an effective inhibitor on purified human serum PON1 activity for phenylacetate and paraoxon .... efficiency (kcat/KM≈10. 4 M-1, s-1). .... Directed evolution of mammalian paraoxonases PON1 and PON3 for.

  12. Radioimmunoassay for prostatic acid phosphatase in human serum. Methodologic aspects

    Energy Technology Data Exchange (ETDEWEB)

    Pradalier, N.; Canal, P.; Pujol, A.; Fregevu, Y. (Groupe de Recherches du Centre Claudius-Regaud, Toulouse (France)); Soula, G. (Faculte des Sciences Pharmaceutiques, Toulouse (France))

    1982-01-01

    We propose a double antibody radioimmunoassay for human prostatic acid phosphatase (PAP) in serum for diagnosis and management of prostatic adenocarcinoma under treatment. The antigen is purified from human prostatic fluid by a gel-filtration on Sephadex G 100 followed by affinity chromatography on Con A Sepharose. A specific antibody is raised in rabbits and purified by immunoadsorption with a female serum. The described technique offers both radioisotopic sensibility and immunologic specificity. Physiological values determined in the serum of 125 healthy males are below 2 ng/ml. No significative differences are observed with age. The proposed technique also shows significant differences between values evaluated for benign prostatic hyperplasia and prostatic adenocarcinoma.

  13. Atomic structure and chemistry of human serum albumin

    Science.gov (United States)

    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.

  14. Isoforms of murine and human serum amyloid P component

    DEFF Research Database (Denmark)

    Nybo, Mads; Hackler, R; Kold, B

    1998-01-01

    Isoelectric focusing (IEF) and immunofixation of murine serum amyloid P component (SAP), purified and in serum, showed a distinct and strain-dependent isoform pattern with up to seven bands (pI 5.1-5.7). Neuraminidase treatment caused a shift of the isoforms to more basic pI values, but did...... of isoforms of human SAP required the presence of urea and higher SAP concentrations. TEF and immunofixation of SAP monomers showed five to eight isoforms, ranging from pI 4.7-5.7. IEF of SAP in human serum resulted in a less distinct pattern and more acidic isoforms. As with murine SAP, neuraminidase...

  15. Purification of NAD(+) glycohydrolase from human serum.

    Science.gov (United States)

    Coşkun, Ozlem; Nurten, Rüstem

    2013-07-01

    In the present study, NAD(+) glycohydrolase was purified from serum samples collected from healthy individuals using ammonium sulfate fractionation, Affi-Gel blue (Cibacron Blue F3GA) affinity chromatography, Sephadex G-100 column chromatography and isoelectric focusing. The final step was followed by a second Sephadex G-100 column chromatography assay in order to remove the ampholytes from the isoelectric focusing step. In terms of enhancement of specific activity, the NAD(+) glycohydrolase protein was purified ∼480-fold, with a yield of 1% compared with the initial serum fraction. The purified fraction appeared to be homogeneous, with a molecular weight of 39 kDa, as revealed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis, and also corresponded to the soluble (monomeric) form of surface antigen CD38.

  16. Hypoxemia increases serum interleukin-6 in humans

    DEFF Research Database (Denmark)

    Klausen, T; Olsen, Niels Vidiendal; Poulsen, T D

    1997-01-01

    Serum concentrations of interleukin (IL) 1 beta, IL-1 receptor antagonist (IL-1ra), IL-6, tumor necrosis factor (TNF) alpha, and C-reactive protein (CRP) were determined in ten healthy men at sea level and during four days of altitude hypoxia (4350m above sea level). The mean (SD) arterial blood...... oxygen saturations were 78.6 (7.3)%, 82.4 (4.9)%, and 83.4 (5.3)% in the first, second, and third days at altitude, respectively. A symptom score of acute mountain sickness (AMS) revealed that the subjects had mostly light symptoms of AMS. Mean serum IL-6 increased from 1.36 (1.04) pg x ml(-1) at sea...

  17. A Preliminary Analysis of Calcifying Particles in the Serum and Prostates of Patients with Prostatic Inflammation

    Science.gov (United States)

    Jones, Jeffrey A.; Carlson, Grant; Kajander, E. Olavi; Warmflash, David; Taylor, Karen; Ayala, Gustavo; Shoskes, Daniel; Everett, Meg; Feedback, Dan; Ciftcioglu, Neva

    2006-01-01

    Chronic diseases of the prostate such as benign prostatic hyperplasia (BPH) & chronic pelvic pain syndrome (CPPS) have associated findings of chronic inflammation, despite a lack of causal relationship. Numerous attempts to define an infectious agent responsible for the clinical findings have been inconsistent. The possibility of an infectious agent, that has not been uncovered with routine culturing methods, forms the basis for this study. Serum from 940 healthy Finnish men were compared with serum from 40 Crohn's, 40 path dx prostatitis, & 40 with path dx carcinoma, using an enzyme-linked immunosorbant assay (ELISA), to detect antigens specific to Nanobacteria(NB) utilizing monoclonal antibodies (Ab) 5/3 and 8D10. This ELISA has not been validated for detecting NB-associated with clinical prostatic disease, yet cross-reactivity with other bacterial species is low. Immunohistochemistry was performed on de-paraffinized prostatic tissue slides, de-calcified with EDTA and stained with the DAKO Catalyzed Signal Amplification kit, employing 8D10 as the primary (target/antigen-detecting) Ab. The mean (plus or minus SD) & median concentrations of NB antigen (U/50 L) were 379.59 (plus or minus 219.28) & 640.00 for patients with prostatitis (BPH) vs 3.31 (plus or minus 3.55) & 2.94 for prostate adenocarcinoma, 1.88 (plus or minus 2.94) & 0.80 for Crohn's disease, & 7.43 (plus or minus 25.57) & 0.00 for patients with no clinical prostatic disease. Unpaired t-tests revealed statistically significant differences between the prostatitis (BPH) sera & each of the other groups with p less than 0.005, but no differences between the other groups themselves. Preliminary studies with immunohistochemistry & 3-D confocal microscopy reveal 16/24 tissue sections + for NB Ag in BPH vs. only 2/22 tissue sections with prostate cancer. The preliminary findings of this serum screening study suggest that NB antigen may be commonly found in the serum of patients with the pathological diagnosis

  18. Molecular sex differences in human serum.

    Directory of Open Access Journals (Sweden)

    Jordan M Ramsey

    Full Text Available BACKGROUND: Sex is an important factor in the prevalence, incidence, progression, and response to treatment of many medical conditions, including autoimmune and cardiovascular diseases and psychiatric conditions. Identification of molecular differences between typical males and females can provide a valuable basis for exploring conditions differentially affected by sex. METHODOLOGY/PRINCIPAL FINDINGS: Using multiplexed immunoassays, we analyzed 174 serum molecules in 9 independent cohorts of typical individuals, comprising 196 males and 196 females. Sex differences in analyte levels were quantified using a meta-analysis approach and put into biological context using k-means to generate clusters of analytes with distinct biological functions. Natural sex differences were established in these analyte groups and these were applied to illustrate sexually dimorphic analyte expression in a cohort of 22 males and 22 females with Asperger syndrome. Reproducible sex differences were found in the levels of 77 analytes in serum of typical controls, and these comprised clusters of molecules enriched with distinct biological functions. Analytes involved in fatty acid oxidation/hormone regulation, immune cell growth and activation, and cell death were found at higher levels in females, and analytes involved in immune cell chemotaxis and other indistinct functions were higher in males. Comparison of these naturally occurring sex differences against a cohort of people with Asperger syndrome indicated that a cluster of analytes that had functions related to fatty acid oxidation/hormone regulation was associated with sex and the occurrence of this condition. CONCLUSIONS/SIGNIFICANCE: Sex-specific molecular differences were detected in serum of typical controls and these were reproducible across independent cohorts. This study extends current knowledge of sex differences in biological functions involved in metabolism and immune function. Deviations from typical

  19. Differences between human plasma and serum metabolite profiles.

    Directory of Open Access Journals (Sweden)

    Zhonghao Yu

    Full Text Available BACKGROUND: Human plasma and serum are widely used matrices in clinical and biological studies. However, different collecting procedures and the coagulation cascade influence concentrations of both proteins and metabolites in these matrices. The effects on metabolite concentration profiles have not been fully characterized. METHODOLOGY/PRINCIPAL FINDINGS: We analyzed the concentrations of 163 metabolites in plasma and serum samples collected simultaneously from 377 fasting individuals. To ensure data quality, 41 metabolites with low measurement stability were excluded from further analysis. In addition, plasma and corresponding serum samples from 83 individuals were re-measured in the same plates and mean correlation coefficients (r of all metabolites between the duplicates were 0.83 and 0.80 in plasma and serum, respectively, indicating significantly better stability of plasma compared to serum (p = 0.01. Metabolite profiles from plasma and serum were clearly distinct with 104 metabolites showing significantly higher concentrations in serum. In particular, 9 metabolites showed relative concentration differences larger than 20%. Despite differences in absolute concentration between the two matrices, for most metabolites the overall correlation was high (mean r = 0.81±0.10, which reflects a proportional change in concentration. Furthermore, when two groups of individuals with different phenotypes were compared with each other using both matrices, more metabolites with significantly different concentrations could be identified in serum than in plasma. For example, when 51 type 2 diabetes (T2D patients were compared with 326 non-T2D individuals, 15 more significantly different metabolites were found in serum, in addition to the 25 common to both matrices. CONCLUSIONS/SIGNIFICANCE: Our study shows that reproducibility was good in both plasma and serum, and better in plasma. Furthermore, as long as the same blood preparation procedure is

  20. Serum haptoglobin: a novel marker of adiposity in humans.

    Science.gov (United States)

    Chiellini, C; Santini, F; Marsili, A; Berti, P; Bertacca, A; Pelosini, C; Scartabelli, G; Pardini, E; López-Soriano, J; Centoni, R; Ciccarone, A M; Benzi, L; Vitti, P; Del Prato, S; Pinchera, A; Maffei, M

    2004-06-01

    Haptoglobin (Hp) is a glycoprotein involved in the acute phase response to inflammation. Our previous findings indicate that Hp mRNA and protein are present in the adipose tissue of rodents and that Hp gene expression is up-regulated in obese models. The aim of the present study was to establish whether Hp could be considered a marker of obesity in humans. In 312 subjects, serum Hp was correlated directly with body mass index (BMI), leptin, C-reactive protein (CRP), and age. In a multivariate stepwise regression analysis, BMI and CRP were independent determinants of serum Hp in females, with BMI having the strongest effect. CRP and age were independent determinants of serum Hp in males, although explaining only a modest percentage of the total variability. Serum Hp was positively associated with body fat, as assessed by dual-energy x-ray absorptiometry, both in female and in male groups. The level of significance improved when serum Hp was analyzed against fat mass adjusted for lean mass. Finally, Northern and Western blot analyses performed in biopsies of sc abdominal fat from 20 obese individuals showed the presence of Hp mRNA and protein in the human adipose tissue. In conclusion, serum Hp constitutes a novel marker of adiposity in humans, and the adipose tissue likely contributes to determine its levels.

  1. Identification of a sulfate metabolite of PCB 11 in human serum.

    Science.gov (United States)

    Grimm, Fabian A; Lehmler, Hans-Joachim; Koh, Wen Xin; DeWall, Jeanne; Teesch, Lynn M; Hornbuckle, Keri C; Thorne, Peter S; Robertson, Larry W; Duffel, Michael W

    2017-01-01

    Despite increasing evidence for a major role for sulfation in the metabolism of lower-chlorinated polychlorinated biphenyls in vitro and in vivo, and initial evidence for potential bioactivities of the resulting sulfate ester metabolites, the formation of PCB sulfates in PCB exposed human populations had not been explored. The primary goal of this study was to determine if PCB sulfates, and potentially other conjugated PCB derivatives, are relevant classes of PCB metabolites in the serum of humans with known exposures to PCBs. In order to detect and quantify dichlorinated PCB sulfates in serum samples of 46 PCB-exposed individuals from either rural or urban communities, we developed a high-resolution mass spectrometry-based protocol using 4-PCB 11 sulfate as a model compound. The method also allowed the preliminary analysis of these 46 human serum extracts for the presence of other metabolites, such as glucuronic acid conjugates and hydroxylated PCBs. Sulfate ester metabolites derived from dichlorinated PCBs were detectable and quantifiable in more than 20% of analyzed serum samples. Moreover, we were able to utilize this method to detect PCB glucuronides and hydroxylated PCBs, albeit at lower frequencies than PCB sulfates. Altogether, our results provide initial evidence for the presence of PCB sulfates in human serum. Considering the inability of previously employed analytical protocols for PCBs to extract these sulfate ester metabolites and the concentrations of these metabolites observed in our current study, our data support the hypothesis that total serum levels of PCB metabolites in exposed individuals may have been underestimated in the past.

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

    Science.gov (United States)

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

    2016-03-01

    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.

  3. A low percentage of autologous serum can replace bovine serum to engineer human nasal cartilage

    Directory of Open Access Journals (Sweden)

    F Wolf

    2008-02-01

    Full Text Available For the generation of cell-based therapeutic products, it would be preferable to avoid the use of animal-derived components. Our study thus aimed at investigating the possibility to replace foetal bovine serum (FBS with autologous serum (AS for the engineering of cartilage grafts using expanded human nasal chondrocytes (HNC. HNC isolated from 7 donors were expanded in medium containing 10% FBS or AS at different concentrations (2%, 5% and 10% and cultured in pellets using serum-free medium or in Hyaff®-11 meshes using medium containing FBS or AS. Tissue forming capacity was assessed histologically (Safranin O, immunohistochemically (type II collagen and biochemically (glycosaminoglycans -GAG- and DNA. Differences among experimental groups were assessed by Mann Whitney tests. HNC expanded under the different serum conditions proliferated at comparable rates and generated cartilaginous pellets with similar histological appearance and amounts of GAG. Tissues generated by HNC from different donors cultured in Hyaff®-11 had variable quality, but the accumulated GAG amounts were comparable among the different serum conditions. Staining intensity for collagen type II was consistent with GAG deposition. Among the different serum conditions tested, the use of 2% AS resulted in the lowest variability in the GAG contents of generated tissues. In conclusion, a low percentage of AS can replace FBS both during the expansion and differentiation of HNC and reduce the variability in the quality of the resulting engineered cartilage tissues.

  4. Radioimmunoassay of calcitonin in unextracted human serum: a sensitive method

    Energy Technology Data Exchange (ETDEWEB)

    Emmertsen, K.; Marqversen, J.; Jensen, F.T.; Hansen, H.H. (Kommunehospitalet, Copenhagen (Denmark))

    1982-11-01

    A sensitive sequential direct radioimmunoassay of calcitonin (CT) is described. Human synthetic monomer CT was used for /sup 125/I-labelling. The antibody was directed against mid-portion and C-terminal parts of the human CT molecule. No significant cross reactivity with salmon or porcine CT was found. Separation of free from antibody-bound /sup 125/I-labelled CT was performed using a double antibody-polyethyleneglycol mixture. Dilution curves of sera from normal controls and patients with medullary thyroid carcinoma (MCT) were parallel to the standard curve. The intraassay coefficients of variation (CV) were 6% and 3% at serum concentrations of 90 and 230 ng/l respectively. The interassay CV were 13% and 10% at mean serum CT concentrations of 50 and 210 ng/l respectively. The lower limit of detection was 20 ng/l. Thirty-one healthy controls had serum CT levels in the range of 35-135 ng/l. Elevated basal serum CT concentrations were found in 11 patients with MCT and the serum concentrations increased markedly after pentagastrin stimulation. Subsequent thyroidectomy in eight of the 11 patients with MCT reduced basal serum CT levels, in six to within the reference range for normals.

  5. Serum chemistry alterations, including creatine kinase isoenzymes, in furazolidone toxicosis of ducklings: preliminary findings.

    Science.gov (United States)

    Webb, D M; DeNicola, D B; Van Vleet, J F

    1991-01-01

    Furazolidone induces a cardiotoxicosis when fed in toxic concentrations to newly hatched ducklings. This preliminary experiment was designed to determine if creatine kinase (CK) isoenzymic activities or other serum analytes would be useful as indicators of these cardiac alterations. Sera from 12 ducklings (six fed a control ration and six fed the control ration with 700 mg furazolidone added per kg of feed [700 ppm] for 28 days) were analyzed for CK isoenzymic activities, electrolytes, nitrogenous metabolites, hepatic enzymic activities, bilirubin, and glucose. Statistically significant differences between control and treated groups were detected for creatine kinase MB (CK-MB, cardiac muscle origin) isoenzymic activity and bilirubin, potassium, calcium, and total carbon dioxide concentrations. Differences other than CK-MB isoenzymic activity were generally explained by factors related to the toxicosis or sample handling. These findings suggest that CK-MB isoenzymic activity may be useful to detect and monitor the progress of cardiac injury in furazolidone toxicosis, thereby increasing the usefulness of this model of dilated cardiomyopathy. Our findings, analyzed on the Kodak Ektachem 700 Dry Chemistry Analyzer, are compared with serum chemistry values reported in the literature.

  6. Prospective investigation of serum anti-Müllerian hormone concentration in ovulatory intrauterine insemination patients: a preliminary study

    DEFF Research Database (Denmark)

    Freiesleben, N la Cour; Rosendahl, Mikkel; Johannsen, Trine Holm;

    2010-01-01

    This preliminary prospective study investigated serum anti-Müllerian hormone (AMH) through correlations to other basal parameters (123 patients) and according to ovarian response to 75 IU recombinant follicle-stimulating hormone (rFSH)/day (62 patients) in ovulatory patients' first rFSH treatment...

  7. Multiple isoforms of the human pentraxin serum amyloid P component

    DEFF Research Database (Denmark)

    Sørensen, Inge Juul; Andersen, Ove; Nielsen, EH;

    1995-01-01

    Human serum amyloid P component (SAP) isolated from 20 healthy individuals was analyzed by anion exchange chromatography and isoelectric focusing (IEF) in order to investigate the existence of multiple forms of SAP and interindividual structural differences. Anion exchange chromatography showed one...

  8. Competitive protein adsorption to polymer surface from human serum

    DEFF Research Database (Denmark)

    Holmberg, Maria; Jensen, Karin Bagger Stibius; Larsen, Niels Bent;

    2008-01-01

    Surface modification by "soft" plasma polymerisation to obtain a hydrophilic and non-fouling polymer surface has been validated using radioactive labelling. Adsorption to unmodified and modified polymer surfaces, from both single protein and human serum solutions, has been investigated. By using ...

  9. Isolation of human anti-serum albumin Fab antibodies with an extended serum-half life.

    Science.gov (United States)

    Kang, Hyeon-Ju; Kim, Hye-Jin; Cha, Sang-Hoon

    2016-01-01

    The serum albumin (SA) has been exploited to generate long-acting biotherapeutics by taking advantage of the FcRn-mediated recycling mechanism in a direct or an indirect way. Since Fab fragments have been proven to be clinically safe for human usage, we assumed that human anti-SA Fab antibodies could have a great potential as a carrier molecule to extend the serum half-life of therapeutic proteins. We, herein, had attempted to isolate anti-SA Fab antibodies from HuDVFab-8L antibody library via a phage display technology, and identified eight discrete human Fab antibodies. One of the Fab antibodies, SL335, showed the strongest binding reactivity to human SA with nM range of affinity at both pH 6 and pH 7.4, and cross-reacted to SAs from various species including rat, mouse, canine and monkey. The in vivo pharmacokinetic assay using a rat model indicated that SL335 has approximately 10 fold longer serum half-life and 26 to 44-fold increase in AUC0 → ∞ compared to the negative control Fab molecule in both intravenous and subcutaneous administrations. Knowing that Fabs have proven to be safe in clinics for a long time, SL335 seems to have a great potential in generating long-acting protein drugs by tagging effector molecules with either chemical conjugation or genetic fusion.

  10. Probing cocaine-antibody interactions in buffer and human serum.

    Directory of Open Access Journals (Sweden)

    Muthu Ramakrishnan

    Full Text Available BACKGROUND: Despite progress in cocaine immunotherapy, the kinetic and thermodynamic properties of antibodies which bind to cocaine and its metabolites are not well understood. It is also not clear how the interactions between them differ in a complex matrix such as the serum present in the human body. In the present study, we have used microscale thermophoresis (MST, isothermal titration calorimetry (ITC, and surface plasmon resonance (SPR we have evaluated the affinity properties of a representative mouse monoclonal (mAb08 as well as those of polyclonal antibodies purified from vaccinated mouse and human patient serum. RESULTS: MST analysis of fluorescently tagged mAb08 binding to cocaine reveals an approximately 15 fold decrease in its equilibrium dissociation constant in 20-50% human serum compared with that in saline buffer. A similar trend was also found using enriched polyclonal antibodies purified from vaccinated mice and patient serum, for which we have used fluorescently tagged bovine serum albumin conjugated to succinyl norcocaine (BSA-SNC. This conjugate closely mimics both cocaine and the hapten used to raise these antibodies. The ITC data also revealed that cocaine has a moderate affinity of about 2 µM to 20% human serum and very little interaction with human serum albumin or nonspecific human IgG at that concentration range. In a SPR inhibition experiment, the binding of mAb08 to immobilized BSA-SNC was inhibited by cocaine and benzoylecgonine in a highly competitive manner, whereas the purified polyclonal antibodies from vaccinated humans and mice, revealed preferential selectivity to pharmacologically active cocaine but not to the inactive metabolite benzoylecgonine. We have also developed a simple binding model to simulate the challenges associated with cocaine immunotherapy using the variable quantitative and kinetic properties of the antibodies. CONCLUSIONS: High sensitivity calorimetric determination of antibody binding to

  11. A Protocol for Measurement of Noncoding RNA in Human Serum

    Directory of Open Access Journals (Sweden)

    Caroline J. Taylor

    2012-01-01

    Full Text Available MicroRNAs (miRNAs are small noncoding RNAs that act as regulators of gene expression by targeting mature messenger RNAs. Following the initial report of the presence of miRNAs in serum and plasma a number of studies have successfully demonstrated the use of these miRNAs as biomarkers of disease. Currently, there are many methods of isolating total RNA from liquid samples. Here, we describe a simple, cost effective method for extraction of RNA from human serum as well as subsequent real time PCR analysis of miRNA levels.

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

    DEFF Research Database (Denmark)

    Pedersen, Helle Krogh; Gudmundsdottir, Valborg; Nielsen, Henrik Bjørn

    2016-01-01

    -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......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...

  13. Automated conductimetric assay of human serum cholinesterase activity.

    Science.gov (United States)

    Duffy, P; Wallach, J M

    1989-01-01

    Serum cholinesterase activity was determined by conductimetry using samples in the microliter range. Butyrylcholine iodide was demonstrated to be a convenient substrate for the conductimetric assay. Validation of the microassay was made by using either purified enzyme or control serum. In the range of 0-60 U/l, a linear relationship was demonstrated. Correlation with a reference spectrophotometric method was obtained with a slope of 1.18. An explanation of this value is proposed, as different hydrolysis rates were obtained with human sera, depending on the substrate used (butyrylthio- or butyryl-choline ester).

  14. Human Health Effects, Task Force Assessment, Preliminary Report.

    Science.gov (United States)

    Aronow, Wilbert S.; And Others

    Presented in this preliminary report is one of seven assessments conducted by a special task force of Project Clean Air, the Human Health Effects Task Force. The reports summarize assessments of the state of knowledge on various air pollution problems, particularly in California, and make tentative recommendations as to what the University of…

  15. Preliminary analysis of modified low density lipoproteins in the serum of healthy and obese dogs and cats

    Directory of Open Access Journals (Sweden)

    Nobuko eMori

    2015-09-01

    Full Text Available Oxidized low-density lipoprotein (LDL is thought to play an important role in the inflammatory response associated with human obesity. The purpose of this preliminary study was to determine oxidized LDL concentrations in healthy dogs and cats, and to evaluate whether obesity affects oxidized LDL concentration, using 39 cats and 19 dogs that had visited 2 different veterinary clinics in Japan. We hypothesized that oxidized LDL concentrations measured against body condition score (BCS may have a potential value in evaluating the qualities of accumulated or circulating lipids in obese dogs and cats that do not show signs of metabolic diseases. The mean oxidized LDL value in BCS3 dogs (2.4 ± 0.9 μg/dl was very similar to that of BCS5 dogs (2.2 ± 0.3 μg/dl. The mean oxidized LDL value of BCS4 dogs was 7.2 ± 10.3 μg/dl and the highest among three groups. BCS4 dogs included two dogs whose oxidized LDL values were higher than the mean oxidized LDL value of healthy humans (11.2 ± 0.3 μg/dl. On the other hand, the mean oxidized LDL value of BCS3 cats was 2.5 ± 0.9 μg/dl, and those of BCS4 and 5 cats were higher than that of BCS3, but there was no significant difference. The BCS4 cat group included one cat with a higher oxidized LDL value, and the BCS5 group also included two cats with oxidized LDL values higher than the mean oxidized LDL value of healthy humans. Interestingly, the oxidized LDL values in 2 obese dogs and 3 obese cats were indeed higher than the mean oxidized LDL value of humans with coronary artery disease (20.1 ± 1.1 μg/dl. In conclusion, this preliminary study showed reference ranges of oxidized dogs and cats against BCS. Obesity alone does not appear to have any direct effect on serum oxidized LDL values in healthy dogs and cats.

  16. The Unexplored Roles of Human Serum IgA

    Science.gov (United States)

    Leong, Ka Wai

    2014-01-01

    The roles of human serum IgA, in contrast to that of mucosal IgA, are relatively unexplored. Previous studies have shown that IgA mediates either pro- or anti-inflammatory effects in innate immune cells. Serum IgA has been shown to interact with many proteins and glycoproteins of which the functions and mechanisms are not fully characterized. Here, we present fresh perspectives into the roles of serum IgA, describing novel IgA–protein interactions, the importance of its glycosylation status in normal functions, and the plausible role of IgA as a driver and regulator of autoimmune diseases/immune overactivation. Other potential roles, including the regulation of cytokines, effector cell function, and homeostasis, are considered in view of the maintenance of immune function. We anticipate future research to uncover new anti-inflammatory or pro-inflammatory roles of human serum IgA in immune functions and dysfunctions, with implications on systemic lupus erythematosus (SLE). PMID:25188736

  17. Radioimmunoassay of aspartate aminotransferase isoenzymes in human serum

    Energy Technology Data Exchange (ETDEWEB)

    Leung, F.Y.; Niblock, A.E.; Henderson, A.R.

    1984-08-01

    A description is given of the development of a sensitive, specific radioimmunoassay for the cytoplasmic and mitochondrial isoenzymes of human aspartate aminotransferase (L-aspartate:2-oxoglutarate aminotransferase; EC 2.6.1.1). Isoenzymes from human heart tissue were purified to homogeneity and used to raise high-titer antisera in rabbits. The antisera were partly purified by selective column chromatography. The Bolton-Hunter reagent was used to radioiodinate the isoenzymes. The assay requires 100 microL of serum, includes a solid-phase second-antibody separation, and can be completed in less than 3 h. There was no cross reactivity between the two isoenzymes. As little as 5 micrograms (50 pmol) of each aspartate aminotransferase can be measured per liter of serum.

  18. [Reaction mechanism of cefotaxime with human serum albumin].

    Science.gov (United States)

    Liu, Luo-sheng; Wang, Xing-po; Zhao, Quan-qin; Zhang, Yu-yi

    2006-06-01

    The reaction mechanism of cefotaxime with human serum albumin (HSA) and the affinity between cefotaxime and beta-lactamase were investigated by spectrometry and spectrofluorimetry. The interaction dissociation constants of human serum albumin and cefotaxime were determined from a double reciprocal Lineweaver-Burk plot. The binding distance and transfer efficiency between cefotaxime and HSA were also obtained according to the theory of Förster non-radiation energy transfer. The result suggested that the main binding force between cefotaxime and HSA is electrostatic force interaction. The high beta-lactamase stability of cefotaxime may be correlative with its molecular structure. The antibiotic activity and valence are connected with transfer efficiency and dissociation constant. The effect of cefotaxime on the conformation of HSA was also analyzed using synchronous fluorescence spectrometry.

  19. The innate resistance of Trypanosoma copemani to human serum.

    Science.gov (United States)

    Austen, J M; Ryan, U; Ditcham, W G F; Friend, J A; Reid, S A

    2015-06-01

    Trypanosoma copemani is known to be infective to a variety of Australian marsupials. Characterisation of this parasite revealed the presence of stercorarian-like life-cycle stages in culture, which are similar to T. rangeli and T. cruzi. The blood incubation infectivity test (BIIT) was adapted and used to determine if T. copemani, like T. cruzi and T. rangeli, has the potential to grow in the presence of human serum. To eliminate any effects of anticoagulants on the complement system and on human high density lipoprotein (HDL), only fresh whole human blood was used. Trypanosoma copemani was observed by microscopy in all human blood cultures from day 5 to day 19 post inoculation (PI). The mechanism for normal human serum (NHS) resistance in T. copemani is not known. The results of this study show that at least one native Australian trypanosome species may have the potential to be infective for humans.

  20. Exosome enrichment of human serum using multiple cycles of centrifugation.

    Science.gov (United States)

    Kim, Jeongkwon; Tan, Zhijing; Lubman, David M

    2015-09-01

    In this work, we compared the use of repeated cycles of centrifugation at conventional speeds for enrichment of exosomes from human serum compared to the use of ultracentrifugation (UC). After removal of cells and cell debris, a speed of 110 000 × g or 40 000 × g was used for the UC or centrifugation enrichment process, respectively. The enriched exosomes were analyzed using the bicinchoninic acid assay, 1D gel separation, transmission electron microscopy, Western blotting, and high-resolution LC-MS/MS analysis. It was found that a five-cycle repetition of UC or centrifugation is necessary for successful removal of nonexosomal proteins in the enrichment of exosomes from human serum. More significantly, 5× centrifugation enrichment was found to provide similar or better performance than 5× UC enrichment in terms of enriched exosome protein amount, Western blot band intensity for detection of CD-63, and numbers of identified exosome-related proteins and cluster of differentiation (CD) proteins. A total of 478 proteins were identified in the LC-MS/MS analyses of exosome proteins obtained from 5× UCs and 5× centrifugations including many important CD membrane proteins. The presence of previously reported exosome-related proteins including key exosome protein markers demonstrates the utility of this method for analysis of proteins in human serum.

  1. Cartography of human diaphragmatic innervation: preliminary data.

    Science.gov (United States)

    Verin, Eric; Marie, Jean-Paul; Similowski, Thomas

    2011-04-30

    In humans, anatomy indicates that the phrenic nerve mainly arises from the C4 cervical root, with variable C3 and C5 contributions. How this translates into functional innervation is unknown. The diaphragm response to electrical stimulation of C3, C4 and C5 was described in three patients undergoing surgical laryngeal reinnervation with an upper phrenic root (surface chest electrodes at anterior, lateral and posterior sites; oesophageal and gastric pressures (Pes and Pga) to derive transdiaphragmatic pressure (Pdi)). Anatomically, the phrenic nerve predominantly originated from C4. Phrenic stimulation elicited motor responses at the three sites in the three patients, as did C4 stimulation. It produced Pdi values of 9, 11, and 14cmH(2)O in the three patients, respectively, vs. 9, 9, and 7cmH(2)O for C4. C3 stimulation produced modest Pdi responses, whereas C5 stimulation could produce Pdi responses close to those observed with C4 stimulation. These singular observations confirm the dominance of C4 in diaphragm innervation but suggest than C5 can be of importance. Copyright © 2010 Elsevier B.V. All rights reserved.

  2. Radioimmunoassay of pepsinogens I and Ⅱin human serum

    Institute of Scientific and Technical Information of China (English)

    XiaoZhi-Jian; JiangMeng-Jun; 等

    1998-01-01

    Radioimmunoassay(RIA) for pepsinogens I and II(PGI and II)in serum is developed by using the purified PG I and II from human gastric mucosa.The assay range was 8-2560644g/L for PG I and 2-640644g/L for PGⅡ,the sensitivity was 1.3 and 0.6μg/L,respectively.The reproducibilities of PGI-RIA(106.9%) and PGⅡ-RIA(106.7%)are quite well.The within-and detween-assay coefficient of variation(CV) of each RIA was 5.1% and 6.3% for PGI,7.2% and 8.9% for PGⅡ,The serum PG Ⅰlevel in healthy volunteer was 60.41±14.98μg/L(mean±SD).significantly higher than the serum PGⅡ level which was 20.70±9.64μg/L.Therefore,the PGI-RIA and PGⅡ-RIA are useful tools in studying the relation between serum pepsinogen levels and various peptic disorders.

  3. Beer consumption and changes in stability of human serum proteins.

    Science.gov (United States)

    Gorinstein, S; Caspi, A; Goshev, I; Moncheva, S; Zemser, M; Weisz, M; Libman, I; Lerner, H T; Trakhtenberg, S; Martín-Belloso, O

    2001-03-01

    The aim of this study was to evaluate the influence of beer consumption (BC) on the functional and structural properties of human serum proteins (HSP). Thirty-eight volunteers (after coronary bypass) were divided into two groups: experimental (EG) and control (CG). Nineteen volunteers of the EG consumed 330 mL per day of beer (about 20 g of alcohol) for 30 consecutive days. The CG volunteers consumed mineral water instead of beer. Blood samples were collected from EG and CG patients before and after the experiment. Albumin (Alb), globulin (Glo), and methanol-precipitable proteins (MPP) from human serum were denatured with 8 M urea. Fluorescence and electrophoresis were employed in order to elucidate urea-induced conformational changes and structural behavior of proteins. The measured fluorescence emission spectra were used to estimate the stability of native and denatured protein fractions before and after BC. It was found that before BC the fractions most stable to urea denaturation were Glo, Alb, and MPP fractions. After BC in most of the beer-consuming patients (EG) some changes in native and denatured protein fractions were detected: a tendency to lower stability and minor structural deviations. These qualitative changes were more profound in MPP than in Alb and Glo. Thus, Glo is more resistible to alcohol influence than Alb, which in turn is more resistible than MPP. No serum protein changes were detected in patients of CG.

  4. Relationship among serum taurine, serum adipokines, and body composition during 8-week human body weight control program.

    Science.gov (United States)

    You, Jeong Soon; Park, Ji Yeon; Zhao, Xu; Jeong, Jin Seok; Choi, Mi Ja; Chang, Kyung Ja

    2013-01-01

    Human adipose tissue is not only a storage organ but also an active endocrine organ to release adipokines. This study was conducted to investigate the relationship among serum taurine and adipokine levels, and body composition during 8-week human body weight control program in obese female college students. The program consisted of diet therapy, exercise, and behavior modification. After the program, body weight, body fat mass, percent body fat, and body mass index (BMI) were significantly decreased. Serum triglyceride (TG), total cholesterol (TC), and low-density lipoprotein cholesterol (LDL-C) levels were significantly decreased. Also serum adiponectin level was significantly increased and serum leptin level was significantly decreased. There were no differences in serum taurine and homocysteine levels. The change of serum adiponectin level was positively correlated with change of body fat mass and percent body fat. These results may suggest that body fat loss by human body weight control program is associated with an increase in serum adiponectin in obese female college students. Therefore, further study such as taurine intervention study is needed to know more exact correlation between dietary taurine intake and serum adipokines or body composition.

  5. Multiple isoforms of the human pentraxin serum amyloid P component

    DEFF Research Database (Denmark)

    Sørensen, Inge Juul; Andersen, Ove; Nielsen, EH;

    1995-01-01

    Human serum amyloid P component (SAP) isolated from 20 healthy individuals was analyzed by anion exchange chromatography and isoelectric focusing (IEF) in order to investigate the existence of multiple forms of SAP and interindividual structural differences. Anion exchange chromatography showed one...... major and several minor subpopulations of SAP. IEF of all SAP isolates showed a previously unreported degree of heterogeneity with six isoelectric forms (pKi range 5.5-6.1) and with minor interindividual differences in respect of isoelectric points. Total enzymatic deglycosylation of SAP reduced...

  6. Biomolecular Interaction Study of Cyclolinopeptide A with Human Serum Albumin

    Directory of Open Access Journals (Sweden)

    Ben Rempel

    2010-01-01

    Full Text Available The kinetics, energetics, and structure of Cyclolinopeptide A binding with Human Serum Albumin were investigated with surface plasmon resonance and circular dichroism. The complex is formed through slow recognition kinetics that is temperature sensitive in the range of 20°C–37°C. The overall reaction was observed to be endothermic (ΔH=204 kJ mol−1 and entropy driven (ΔS=746 J mol−1K−1 with overall small changes to the tertiary structure.

  7. POTENT INVITRO ANTI-HUMAN IMMUNODEFICIENCY VIRUS-1 ACTIVITY OF MODIFIED HUMAN SERUM ALBUMINS

    NARCIS (Netherlands)

    JANSEN, RW; MOLEMA, G; PAUWELS, R; SCHOLS, D; DECLERCQ, E; MEIJER, DKF

    1991-01-01

    A series of neoglycoproteins was synthesized by coupling of thiophosgene-activated p-aminophenyl derivatives [Biol. Cell. 47:95-110 (1983); J. Histochem. Cytochem. 32:1091-1094 (1984)] of various sugars to human serum albumin. The compounds were evaluated for their in vitro activity against human im

  8. Effects of Macromolecular Crowding on Human Adipose Stem Cell Culture in Fetal Bovine Serum, Human Serum, and Defined Xeno-Free/Serum-Free Conditions

    Directory of Open Access Journals (Sweden)

    Mimmi Patrikoski

    2017-01-01

    Full Text Available Microenvironment plays an important role for stem cell proliferation and differentiation. Macromolecular crowding (MMC was recently shown to assist stem cells in forming their own matrix microenvironment in vitro. The ability of MMC to support adipose stem cell (ASC proliferation, metabolism, and multilineage differentiation was studied under different conditions: fetal bovine serum- (FBS- and human serum- (HS- based media and xeno- and serum-free (XF/SF media. Furthermore, the immunophenotype of ASCs under MMC was evaluated. The proliferative capacity of ASCs under MMC was attenuated in each condition. However, osteogenic differentiation was enhanced under MMC, shown by increased deposition of mineralized matrix in FBS and HS cultures. Likewise, significantly greater lipid droplet accumulation and increased collagen IV deposition indicated enhanced adipogenesis under MMC in FBS and HS cultures. In contrast, chondrogenic differentiation was attenuated in ASCs expanded under MMC. The ASC immunophenotype was maintained under MMC with significantly higher expression of CD54. However, MMC impaired metabolic activity and differentiation capacity of ASCs in XF/SF conditions. Both the supportive and inhibitory effects of MMC on ASC are culture condition dependent. In the presence of serum, MMC maintains ASC immunophenotype and enhances adipogenic and osteogenic differentiation at the cost of reduced proliferation.

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

    Science.gov (United States)

    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.

  10. Spectrophotometric assay of creatinine in human serum sample

    Directory of Open Access Journals (Sweden)

    Avinash Krishnegowda

    2017-05-01

    Full Text Available A new spectrophotometric method for the analysis of creatinine concentration in human serum samples is developed. The method explores the oxidation of p-methylamino phenol sulfate (Metol in the presence of copper sulfate and creatinine which yields an intense violet colored species with maximum absorbance at 530 nm. The calibration graph of creatinine by fixed time assay ranged from 4.4 to 620 μM. Recovery of creatinine in human serum samples varied from 101% to 106%. Limit of detection and limit of quantification were 0.145 μM and 0.487 μM respectively. Sandell’s sensitivity was 0.112 μg cm−2 and molar absorptivity was 0.101 × 104 L mol−1 cm−1. Within day precision was 2.5–4.8% and day-to-day precision range was 3.2–7.8%. The robustness and ruggedness of the method expressed in RSD values ranged from 0.78% to 2.12% and 1.32% to 3.46% respectively, suggesting that the developed method was rugged. This method provides good sensitivity and is comparable to standard Jaffe’s method with comparatively less interference from foreign substances.

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

    Science.gov (United States)

    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.

  12. A novel immunological assay for hepcidin quantification in human serum.

    Directory of Open Access Journals (Sweden)

    Vasiliki Koliaraki

    Full Text Available BACKGROUND: Hepcidin is a 25-aminoacid cysteine-rich iron regulating peptide. Increased hepcidin concentrations lead to iron sequestration in macrophages, contributing to the pathogenesis of anaemia of chronic disease whereas decreased hepcidin is observed in iron deficiency and primary iron overload diseases such as hereditary hemochromatosis. Hepcidin quantification in human blood or urine may provide further insights for the pathogenesis of disorders of iron homeostasis and might prove a valuable tool for clinicians for the differential diagnosis of anaemia. This study describes a specific and non-operator demanding immunoassay for hepcidin quantification in human sera. METHODS AND FINDINGS: An ELISA assay was developed for measuring hepcidin serum concentration using a recombinant hepcidin25-His peptide and a polyclonal antibody against this peptide, which was able to identify native hepcidin. The ELISA assay had a detection range of 10-1500 microg/L and a detection limit of 5.4 microg/L. The intra- and interassay coefficients of variance ranged from 8-15% and 5-16%, respectively. Mean linearity and recovery were 101% and 107%, respectively. Mean hepcidin levels were significantly lower in 7 patients with juvenile hemochromatosis (12.8 microg/L and 10 patients with iron deficiency anemia (15.7 microg/L and higher in 7 patients with Hodgkin lymphoma (116.7 microg/L compared to 32 age-matched healthy controls (42.7 microg/L. CONCLUSIONS: We describe a new simple ELISA assay for measuring hepcidin in human serum with sufficient accuracy and reproducibility.

  13. Serum sialic acid and CEA concentrations in human breast cancer.

    OpenAIRE

    Hogan-Ryan, A.; Fennelly, J J; Jones, M.; Cantwell, B; Duffy, M J

    1980-01-01

    The concentration of bound sialic acid in the sera of 56 normal subjects and 65 subjects with breast cancer was measured, in order to determine (1) whether serum sialic acid concentrations are raised in breast cancer and (2) whether the concentration of sialic acid in serum reflects tumour stage. The amount of sialic acid in serum was compared to serum carcinoembryonic antigen (CEA) values. Urinary hydroxyproline and serum alkaline phosphatase concentrations were used as indicators of bone an...

  14. Serum-Free Cryopreservation of Human Amniotic Epithelial Cells

    Directory of Open Access Journals (Sweden)

    H. Niknejad

    2013-04-01

    Full Text Available Introduction & Objective: One of the important issues in long term storage of cells is removal of animal serum from cell culture environments. The aim of this study was to evaluate amni-otic fluid (AF, which is full of growth factors, as substitute for fetal bovine serum (FBS in the cryopreservation protocol. Materials & Methods: In this experimental study human amniotic epithelial cells were isolated from placentas which were seronegative for microbial infections. The cells were preserved in 24 different patterns for 12 months in -196 ?C (liquid nitrogen and viability of cells were determined before and after cryopreservation by trypan blue and MTT assay. Moreover, Oct-4 expression was studied to determine pluripotency before and after cryopreservation with immunocytochemistry. Results were compared between groups with ANOVA (Tukey Post-Test. P.value under 0.01 and 0.05 was considered statistically significant. Results: The presence of DMEM, FBS or AF is necessary for amniotic cell cryopreservation. Trypan-blue, MTT and immunocytochemistry showed that there isn’t significant difference between using AF and FBS in viability and pluripotency of cells. Moreover, results showed that DMSO is a better cryoprotectant compared to glycerol. Conclusion : Results showed that amniotic fluid can be a proper substitute for FBS in amniotic epithelial cells cryopreservation. (Sci J Hamadan Univ Med Sci 2013; 20 (1:15-24

  15. Preliminary Study on Serum Lactate Dehydrogenase (LDH)-Prognostic Biomarker in Carcinoma Breast

    Science.gov (United States)

    Gandhe, Mahendra Bhauraoji; Gupta, Dilip; Reddy, M.V.R.

    2016-01-01

    Introduction Serum Lactate Dehydrogenase (LDH) is one of the biochemical markers for breast cancer. Serum LDH is enzyme required for anaerobic glycolysis. One of its isoenzyme is increased in breast cancer due to up-regulation in its gene. It leads to increase in serum LDH level in breast cancer patients. Serum LDH is economical, easily available and easy to estimate. Aim In the present study, we evaluated the LDH levels in circulation of newly diagnosed patients of breast cancer and tried to correlate it with different TNM staging of carcinoma breast before interventions and after adjuvant therapy of these patients. Materials and Methods This prospective study was done on 83 diagnosed patients of breast cancer was conducted among poor patients in rural area. This study was conducted in the Department of Surgery between October 2008 to October 2010, at MGIMS, Sevagram, Maharashtra, a rural medical college located in Central India. Out of total 83 participants, 10 participants were having adverse events following surgery and remaining 73 participants were without adverse events following surgery. The significant difference in serum LDH levels between two groups, with and without adverse surgical outcome was calculated by Mann-Whitney U test. Results Patients with higher clinical TNM staging were having higher serum LDH levels. The serum LDH levels at sixth months following surgery showed a trend of statistically significant difference between patients with and without adverse events. As increased serum LDH levels in breast cancer patients shows poor prognosis, surgical outcome or advanced metastases. Conclusion Serum LDH monitoring can be used as a prognostic biomarker in patients of breast cancer. For confirmation of this finding, we require further more studies on larger sample size and long-term follow-up in patients specifically with higher serum LDH levels. PMID:27134855

  16. The effect of smoking on serum human placental lactogen levels.

    Science.gov (United States)

    Spellacy, W N; Buhi, W C; Birk, S A

    1977-02-01

    Serial serum samples (162) were drawn weekly from normal pregnant women (53) during the last month of gestation and measurements were made of the human placental lactogen (HPL) content. The women were interviewed as to their smoking habits and divided into nonsmokers (32) and smokers of from one to two packages of cigarettes per day (21). The infant birth weight and placental weights were not significantly different. The HPL levels were elevated in the women who smoked and the differences were significant at the thirty-sixth and thirty-eighth weeks. The importance of this in interpreting HPL as a placental function test and in terms of the biology of placental function and the control of protein hormone synthesis is emphasized.

  17. Interaction of aloe-emodin with human serum albumin

    Institute of Scientific and Technical Information of China (English)

    DU JinFeng; LI Ying; ZHANG Qi; YAO XiaoJun

    2007-01-01

    The presence of several high affinity binding sites on human serum albumin (HAS) makes it a possible target for many drugs. This study is designed to examine the effect of aloe-emodin on HAS by fluorescence, CD spectroscopy and molecular modeling. The results of fluorescence measurements suggested that the hydrophobic interaction was the predominant intermolecular force stabilizing the AE-HAS complex, which was in good agreement with the result of molecular modeling study. And the enthalpy change ΔH0 and the entropy change ΔS0 were calculated to be -7.041 kJ·mol-1 and 76.619 J·mol-1·K-1 according to the Van't Hoff equation. The alterations of protein secondary structure in the presence of AE in aqueous solution were quantitatively calculated from CD spectra, and the content of α-helices obviously increased.

  18. Solution behaviour of Human Serum Albumin and GLP-1variants

    DEFF Research Database (Denmark)

    Sønderby, Pernille

    interaction is critical for the long term stability of a pharmaceutical. Protein complex formation is important for extended half-life in vivo and is essential to cellular communication such as the induction of the insulin response. This thesis focuses on human serum albumin (HSA) as a central player...... approach to half-life extension is conjugation of molecules to HSA. In this part of the thesis, novel GLP-1-albumin conjugates developed by Albumedix A/S where examined by a combined approach of pharmacokinetic studies and solution structure determination with SAXS. GLP-1 was conjugated to Cys34...... of recombinant HSA (rHSA) and two rHSA variants with lower (NB) and higher binding (HB) affinity to the neonatal Fc receptor (FcRn). Binding kinetics showed that the conjugation had limited effect on the binding properties of the conjugates to FcRn compared to the respective rHSA variants. Increased in-vivo half...

  19. Pharmacokinetics and anti-HIV-1 efficacy of negatively charged human serum albumins in mice

    NARCIS (Netherlands)

    Kuipers, M E; Swart, P J; Schutten, M; Smit, C; Proost, J H; Osterhaus, A D; Meijer, D K

    1997-01-01

    Negatively charged albumins (NCAs, with the prototypes succinylated human serum albumin (Suc-HSA) and aconitylated human serum albumin (Aco-HSA)), modified proteins with a potent anti-human immunodeficiency virus type 1 (anti-HIV-1) activity in vitro, were studied for their pharmacokinetic behaviour

  20. Serum sialic acid and CEA concentrations in human breast cancer.

    Science.gov (United States)

    Hogan-Ryan, A; Fennelly, J J; Jones, M; Cantwell, B; Duffy, M J

    1980-04-01

    The concentration of bound sialic acid in the sera of 56 normal subjects and 65 subjects with breast cancer was measured, in order to determine (1) whether serum sialic acid concentrations are raised in breast cancer and (2) whether the concentration of sialic acid in serum reflects tumour stage. The amount of sialic acid in serum was compared to serum carcinoembryonic antigen (CEA) values. Urinary hydroxyproline and serum alkaline phosphatase concentrations were used as indicators of bone and liver involvement. Erythrocyte sedimentation rate (ESR) was also measured. Significantly elevated serum sialic acid concentrations were found in breast cancer, and showed correlation with tumour stage. Serum sialic acid values did not correlate with CEA values. The results suggest that measurement of serum sialic acid concentrations may be of adjunctive value in assessing tumour stage.

  1. Crystallization and preliminary crystallographic analysis of recombinant human galectin-1

    Energy Technology Data Exchange (ETDEWEB)

    Scott, Stacy A. [Institute for Glycomics, Gold Coast Campus, Griffith University, Queensland 4222 (Australia); Scott, Ken [School of Biological Sciences, University of Auckland, Auckland (New Zealand); Blanchard, Helen, E-mail: h.blanchard@griffith.edu.au [Institute for Glycomics, Gold Coast Campus, Griffith University, Queensland 4222 (Australia)

    2007-11-01

    Human galectin-1 has been cloned, expressed in E. coli, purified and crystallized in the presence of both lactose (ligand) and β-mercaptoethanol under six different conditions. The X-ray diffraction data obtained have enabled the assignment of unit-cell parameters for two novel crystal forms of human galectin-1. Galectin-1 is considered to be a regulator protein as it is ubiquitously expressed throughout the adult body and is responsible for a broad range of cellular regulatory functions. Interest in galectin-1 from a drug-design perspective is founded on evidence of its overexpression by many cancers and its immunomodulatory properties. The development of galectin-1-specific inhibitors is a rational approach to the fight against cancer because although galectin-1 induces a plethora of effects, null mice appear normal. X-ray crystallographic structure determination will aid the structure-based design of galectin-1 inhibitors. Here, the crystallization and preliminary diffraction analysis of human galectin-1 crystals generated under six different conditions is reported. X-ray diffraction data enabled the assignment of unit-cell parameters for crystals grown under two conditions, one belongs to a tetragonal crystal system and the other was determined as monoclinic P2{sub 1}, representing two new crystal forms of human galectin-1.

  2. Studying the proliferation of human peripheral blood T lymphocytes in serum-free medium.

    Science.gov (United States)

    Tabakov, V U; Litvina, M M; Schepkina, J V; Jarilin, A A; Chestkov, V V

    2009-01-01

    We compared the cultivation of human peripheral blood lymphocytes in serum-free medium Hybris-2 and RPMI 1640 medium with 10% fetal bovine serum in the presence of phytohemagglutinin and interleukin-2. The optimal concentration of phytohemagglutinin significantly differed in serum-free and serum-containing media (0.5 and 5 microg/ml, [corrected] respectively). Both mitogens were more potent in stimulating the proliferation of lymphocytes in serum-free medium than in serum-containing medium. Strong proliferation of CD3(+) and CD4(+) T lymphocytes was observed in both media. The dynamics of other markers was similar in serum-free and serum-containing media. However, significant differences were revealed between individual donors. Our results indicate that the developed serum-free medium may be used in lymphocyte cultivation for scientific, diagnostic, and therapeutic purposes.

  3. Human Serum Albumin Complexed with Myristate and AZT

    Energy Technology Data Exchange (ETDEWEB)

    Zhu, Lili; Yang, Feng; Chen, Liqing; Meehan, Edward J.; Huang, Mingdong (UGA); (UAH)

    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.

  4. Preliminary observations on the microarchitecture of the human abdominal muscles.

    Science.gov (United States)

    Woodley, Stephanie J; Duxson, Marilyn J; Mercer, Susan R

    2007-10-01

    Precise knowledge of muscle architecture and innervation patterns is essential for the development of accurate clinical and biomechanical models. Although the gross anatomy of the human abdominal muscles has been investigated, the finer details of their microanatomy are not well described. Fascicles were systematically sampled from each of the human abdominal muscles, and small fiber bundles from selected fascicles stained with acetylcholinesterase to determine the location of motor endplate bands, myomyonal junctions, and myotendinous junctions. Statistical analysis was used to ascertain the association between fascicular length and number of endplate bands. The number of endplate bands along a fascicle was variable between different portions of each muscle, but was strongly correlated with fascicular length (r = 0.814). In fascicles less than 50 millimeters (mm) in length, only a single endplate band was generally present, while multiple endplate bands (usually two or three) were found in fascicles longer than 50 mm. The presence of myomyonal junctions throughout the longer (>50 mm) fascicles verified that they were composed of short, intrafascicularly terminating fibers, while shorter fascicles comprised fibers spanning the entire fascicular length. This preliminary study provides evidence that multiple endplate bands are contained in some regions of the abdominal muscles, an arrangement that differs from most human appendicular muscles. It is not clear whether the variations in the described fine architectural features reflect regional differences in muscle function.

  5. GM-CSF production from human airway smooth muscle cells is potentiated by human serum

    Directory of Open Access Journals (Sweden)

    Maria B. Sukkar

    2000-01-01

    Full Text Available Recent evidence suggests that airway smooth muscle cells (ASMC actively participate in the airway inflammatory process in asthma. Interleukin–1β (IL–1β and tumour necrosis factor–α (TNF–α induce ASMC to release inflammatory mediators in vitro. ASMC mediator release in vivo, however, may be influenced by features of the allergic asthmatic phenotype. We determined whether; (1 allergic asthmatic serum (AAS modulates ASMC mediator release in response to IL–1β and TNF–α, and (2 IL–1β/TNF–α prime ASMC to release mediators in response to AAS. IL–5 and GMCSF were quantified by ELISA in culture supernatants of; (1 ASMC pre-incubated with either AAS, non-allergic non-asthmatic serum (NAS or MonomedTM (a serum substitute and subsequently stimulated with IL–1β and TNF–α and (2 ASMC stimulated with IL–1β/TNF–α and subsequently exposed to either AAS, NAS or MonomedTM. IL-1g and TNF–α induced GM-CSF release in ASMC pre-incubated with AAS was not greater than that in ASMC pre-incubated with NAS or MonomedTM. IL–1β and TNF–α, however, primed ASMC to release GM-CSF in response to human serum. GM-CSF production following IL–1β/TNF–α and serum exposure (AAS or NAS was significantly greater than that following IL–1β /TNF–α and MonomedTM exposure or IL–1β/TNF–α exposure only. Whilst the potentiating effects of human serum were not specific to allergic asthma, these findings suggest that the secretory capacity of ASMC may be up-regulated during exacerbations of asthma, where there is evidence of vascular leakage.

  6. Detecting alterations of glucose and lipid components in human serum by near-infrared Raman spectroscopy

    OpenAIRE

    Borges,Rita de Cássia Fernandes; NAVARRO, Ricardo Scarparo; Giana,Hector Enrique; Tavares,Fernanda Grubisich; Fernandes,Adriana Barrinha; Silveira Junior, Landulfo

    2015-01-01

    Introduction Raman spectroscopy may become a tool for the analysis of glucose and triglycerides in human serum in real time. This study aimed to detect spectral differences in lipid and glucose components of human serum, thus evaluating the feasibility of Raman spectroscopy for diagnostic purposes. Methods A total of 44 samples of blood serum were collected from volunteers and submitted for clinical blood biochemical analysis. The concentrations of glucose, cholesterol, triglycerides, and low...

  7. Serum zinc levels in 368 patients with oral mucosal diseases: A preliminary study

    Science.gov (United States)

    Bao, Zhe-Xuan; Yang, Xiao-Wen; Shi, Jing

    2016-01-01

    Background The aim of this study was to assess the serum zinc levels in patients with common oral mucosal diseases by comparing these to healthy controls. Material and Methods A total of 368 patients, which consisted of 156 recurrent aphthous stomatitis (RAS) patients, 57 oral lichen planus (OLP) patients, 55 burning mouth syndrome (BMS) patients, 54 atrophic glossitis (AG) patients, 46 xerostomia patients, and 115 sex-and age-matched healthy control subjects were enrolled in this study. Serum zinc levels were measured in all participants. Statistical analysis was performed using a one-way ANOVA, t-test, and Chi-square test. Results The mean serum zinc level in the healthy control group was significantly higher than the levels of all other groups (p < 0.001). No individual in the healthy control group had a serum zinc level less than the minimum normal value. However, up to 24.7% (13/54) of patients with AG presented with zinc deficiency, while 21.2% (33/156) of patients with RAS, 16.4% (9/55) of patients with BMS, 15.2% (7/46) of patients with xerostomia, and 14.0% (8/57) of patients with OLP were zinc deficient. Altogether, the zinc deficiency rate was 19.02% (70/368) in the oral mucosal diseases (OMD) group (all patients with OMD). The difference between the OMD and healthy control group was significant (p <0.001). Gender differences in serum zinc levels were also present, although not statistically significant. Conclusions Zinc deficiency may be involved in the pathogenesis of common oral mucosal diseases. Zinc supplementation may be a useful treatment for oral mucosal diseases, but this requires further investigation; the optimal serum level of zinc, for the prevention and treatment of oral mucosal diseases, remains to be determined. Key words:Oral mucosal diseases, Zinc deficiency, pathogenesis. PMID:27031065

  8. Serum β-glucuronidase as a potential colon cancer marker: a preliminary study.

    Science.gov (United States)

    Waszkiewicz, Napoleon; Szajda, Sławomir Dariusz; Konarzewska-Duchnowska, Emilia; Zalewska-Szajda, Beata; Gałązkowski, Robert; Sawko, Anna; Nammous, Halim; Buko, Vyacheslav; Szulc, Agata; Zwierz, Krzysztof; Ładny, Jerzy Robert

    2015-04-08

    Colorectal cancer is characterized by high morbidity and mortality in developed countries. The lack of low-cost, easy-to-use screening diagnostic methods is one of the causes of late diagnosis of colorectal cancer. Beta-glucuronidase (GLU) is a lysosomal exoglycosidase involved in degradation of glycosaminoglycans of the cell membranes and extracellular matrix of normal and cancerous colon tissues. The aim of our research was to evaluate the activity of GLU in the serum of colorectal cancer and estimate its potential value in the diagnosis of colorectal cancer. Blood samples were collected from 21 patients with colorectal adenocarcinoma and 17 healthy subjects. GLU activity was determined by the colorimetric method of Marciniak et al. by measuring the amount of p-nitrophenol released from 4-nitrophenyl-beta-D-glucuronide, at λ = 405 nm. We found significantly greater activity of GLU (p<0.0001) in the serum of patients with colorectal cancer, as compared to the healthy subjects. The serum GLU activity significantly differentiates patients with colorectal cancer from healthy individuals. Serum GLU activity has diagnostic value and may be used in the diagnosis of colon adenocarcinoma.

  9. Serum β-glucuronidase as a potential colon cancer marker: a preliminary study

    Directory of Open Access Journals (Sweden)

    Napoleon Waszkiewicz

    2015-04-01

    Full Text Available Colorectal cancer is characterized by high morbidity and mortality in developed countries. The lack of low-cost, easy-to-use screening diagnostic methods is one of the causes of late diagnosis of colorectal cancer. Beta-glucuronidase (GLU is a lysosomal exoglycosidase involved in degradation of glycosaminoglycans of the cell membranes and extracellular matrix of normal and cancerous colon tissues. The aim of our research was to evaluate the activity of GLU in the serum of colorectal cancer and estimate its potential value in the diagnosis of colorectal cancer.Blood samples were collected from 21 patients with colorectal adenocarcinoma and 17 healthy subjects. GLU activity was determined by the colorimetric method of Marciniak et al. by measuring the amount of p-nitrophenol released from 4-nitrophenyl-beta-D-glucuronide, at λ = 405 nm.We found significantly greater activity of GLU (p<0.0001 in the serum of patients with colorectal cancer, as compared to the healthy subjects. The serum GLU activity significantly differentiates patients with colorectal cancer from healthy individuals.Serum GLU activity has diagnostic value and may be used in the diagnosis of colon adenocarcinoma.

  10. Virus-Enabled Biosensor for Human Serum Albumin.

    Science.gov (United States)

    Ogata, Alana F; Edgar, Joshua M; Majumdar, Sudipta; Briggs, Jeffrey S; Patterson, Shae V; Tan, Ming X; Kudlacek, Stephan T; Schneider, Christine A; Weiss, Gregory A; Penner, Reginald M

    2017-01-17

    The label-free detection of human serum albumin (HSA) in aqueous buffer is demonstrated using a simple, monolithic, two-electrode electrochemical biosensor. In this device, both millimeter-scale electrodes are coated with a thin layer of a composite containing M13 virus particles and the electronically conductive polymer poly(3,4-ethylenedioxy thiophene) or PEDOT. These virus particles, engineered to selectively bind HSA, serve as receptors in this biosensor. The resistance component of the electrical impedance, Zre, measured between these two electrodes provides electrical transduction of HSA binding to the virus-PEDOT film. The analysis of sample volumes as small as 50 μL is made possible using a microfluidic cell. Upon exposure to HSA, virus-PEDOT films show a prompt increase in Zre within 5 s and a stable Zre signal within 15 min. HSA concentrations in the range from 100 nM to 5 μM are detectable. Sensor-to-sensor reproducibility of the HSA measurement is characterized by a coefficient-of-variance (COV) ranging from 2% to 8% across this entire concentration range. In addition, virus-PEDOT sensors successfully detected HSA in synthetic urine solutions.

  11. Interaction of perfluorooctanoic acid with human serum albumin

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    Chen Fang-Fang

    2009-05-01

    Full Text Available Abstract Background Recently, perfluorooctanoic acid (PFOA has become a significant issue in many aspects of environmental ecology, toxicology, pathology and life sciences because it may have serious effects on the endocrine, immune and nervous systems and can lead to embryonic deformities and other diseases. Human serum albumin (HSA is the major protein component of blood plasma and is called a multifunctional plasma carrier protein because of its ability to bind an unusually broad spectrum of ligands. Results The interaction of PFOA with HSA was investigated in the normal physiological condition by equilibrium dialysis, fluorospectrometry, isothermal titration calorimetry (ITC and circular dichroism (CD. The non-covalent interaction is resulted from hydrogen bond, van der Waals force and hydrophobic stack. PFOA binding to HSA accorded with two-step binding model with the saturation binding numbers of PFOA, only 1 in the hydrophobic intracavity of HSA and 12 on the exposed outer surface. The interaction of PFOA with HSA is spontaneous and results in change of HSA conformation. The possible binding sites were speculated. Conclusion The present work suggested a characterization method for the intermolecular weak interaction. It is potentially useful for elucidating the toxigenicity of perfluorochemicals when combined with biomolecular function effect, transmembrane transport, toxicological testing and the other experiments.

  12. Cooperative binding of drugs on human serum albumin

    Science.gov (United States)

    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.

  13. HPLC determination of valproic acid in human serum.

    Science.gov (United States)

    Kishore, P; Rajani Kumar, V; Satyanarayana, V; Krishna, D R

    2003-06-01

    A HPLC method for the determination of valproic acid (VA) in human serum using diazepam as internal standard (I.S.) is described. The eluates were separated with a C18 250 x 4.6 mm internal diameter reversed phase column maintained at a temperature of 50 degrees C. A mobile phase consisting of acetonitrile and 0.05 M phosphate buffer (pH 3.0) 45:55 v/v was used at a flow rate of 1.2 ml/min. Wavelength was switched from 360 nm to 210 nm during valproic acid retention. The method was linear over a concentration range of 20 to 160 microg/ml for valproic acid. Recovery was greater than 94% over a concentration range of 20 to 120 microg/ml and the limit of quantitation was 1 microg/ml. The intra day and inter day relative standard deviation (R.S.D.) measured at 20, 60, 80 and 120 microg/ml ranged from 1.46 to 5.34% and 0.83 to 5.03% respectively. The method is simple, rapid, accurate and sensitive and it was used for Therapeutic Drug Monitoring (TDM) in Indian epileptic patient population. The results obtained with this method correlated well with clinical practice.

  14. Interactions of human serum albumin with doxorubicin in different media

    Science.gov (United States)

    Gun'ko, Vladimir M.; Turov, Vladimir V.; Krupska, Tetyana V.; Tsapko, Magdalina D.

    2017-02-01

    Interactions of human serum albumin (10 wt% H2O and 0.3 wt% sodium caprylate) with doxorubicin hydrochloride (1 wt%) were studied alone or with addition of HCl (3.6 wt% HCl) using 1H NMR spectroscopy. A model of hydrated HSA/12DOX was calculated using PM7 method with COSMO showing large variations in the binding constant depending on structural features of DOX/HSA complexes. DOX molecules/ions displace bound water from narrow intramolecular voids in HSA that leads to diminution of freezing-melting point depression of strongly bound water (SBW). Structure of weakly bound water (WBW) depends much weaker on the presence of DOX than SBW because a major fraction of DOX is bound to adsorption sites of HSA. Addition of HCl results in strong changes in structure of macromolecules and organization of water in hydration shells of HSA (i.e., mainly SBW) and in the solution (i.e., WBW + non-bound bulk water).

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

    Science.gov (United States)

    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.

  16. Complexation of insecticide chlorantraniliprole with human serum albumin: Biophysical aspects

    Energy Technology Data Exchange (ETDEWEB)

    Ding Fei [Department of Chemistry, China Agricultural University, No. 2 Yuanmingyuan Xi Road, Haidian District, Beijing 100193 (China); Liu Wei [College of Economics and Management, China Agricultural University, Beijing 100083 (China); Diao Jianxiong [Department of Chemistry, China Agricultural University, No. 2 Yuanmingyuan Xi Road, Haidian District, Beijing 100193 (China); Yin Bin [Key Laboratory of Pesticide Chemistry and Application Technology, Ministry of Agriculture, Department of Applied Chemistry, China Agricultural University, Beijing 100193 (China); Zhang Li, E-mail: zhli.work@gmail.co [Key Laboratory of Pesticide Chemistry and Application Technology, Ministry of Agriculture, Department of Applied Chemistry, China Agricultural University, Beijing 100193 (China); Sun Ying, E-mail: sunying@cau.edu.c [Department of Chemistry, China Agricultural University, No. 2 Yuanmingyuan Xi Road, Haidian District, Beijing 100193 (China)

    2011-07-15

    Chlorantraniliprole is a novel insecticide belonging to the diamide class of selective ryanodine receptor agonists. A biophysical study on the binding interaction of a novel diamide insecticide, chlorantraniliprole, with staple in vivo transporter, human serum albumin (HSA) has been investigated utilizing a combination of steady-state and time-resolved fluorescence, circular dichroism (CD), and molecular modeling methods. The interaction of chlorantraniliprole with HSA gives rise to fluorescence quenching through static mechanism, this corroborates the fluorescence lifetime outcomes that the ground state complex formation and the predominant forces in the HSA-chlorantraniliprole conjugate are van der Waals forces and hydrogen bonds, as derived from thermodynamic analysis. The definite binding site of chlorantraniliprole in HSA has been identified from the denaturation of protein, competitive ligand binding, and molecular modeling, subdomain IIIA (Sudlow's site II) was designated to possess high-affinity binding site for chlorantraniliprole. Moreover, using synchronous fluorescence, CD, and three-dimensional fluorescence we testified some degree of HSA structure unfolding upon chlorantraniliprole binding. - Highlights: {yields} Our study highlights for the first time how binding dynamics can predominate for the new diamide insecticide, chlorantraniliprole. {yields} Chlorantraniliprole is situated within subdomain IIIA, Sudlow's site II, which is the same as that of indole-benzodiazepine site. {yields} Biophysical and molecular modeling approaches are useful to resolve the ligand interaction with biomacromolecule. {yields} It serves as a protective device in binding and in inactivating potential toxic compounds to which the body is exposed.

  17. [Preparation of Human Serum Albumin Micro/Nanotubes].

    Science.gov (United States)

    Jiao, Pei-pei; Guo, Yan-li; Niu, Ai-hua; Kang, Xiao-feng

    2016-01-01

    In this research, protein micro/nanotubes were fabricated by alternate layer-by-layer (LbL) assembly of human serum albumin (HSA) and polyethyleneimine (PEI) into polycarbonate (PC) membranes. The experimental conditions of pH values, ionic strength, the depositions cycles and the diameter of porous membrane were discussed. The morphology and composition of tubes were characterized by scanning electron microscope (SEM), transmission electron microscope (TEM), fourier transform infrared spectroscopy (FTIR) and energy dispersive spectroscopy (EDS). The results show that pH and ionic strength of the solution are the key factors that influence the effect of assembly. Micro/nanotubes with good opening hollow tubular structure were obtained when pH 7.4 HSA solution and pH 10.3 PEI solution without NaCl were used in synthesis procedure. The outer diameter of tube was dependent on the PC template, thus the micro/nanotubes size was controlled by the wall thickness, which can be adjusted by the number of layers of the HSA and PEI deposited along the pore walls. To avoid the thin wall being damaged in dissolving the template and vacuum drying, the PEI/HSA bilayer number should not be less than 3. The polar solvent N,N-dimethylformamide (DMF) can dissolve PC template to release the micro/nanotubes.

  18. Elevated Serum Tryptase and Endothelin in Patients with ST Segment Elevation Myocardial Infarction: Preliminary Report

    Directory of Open Access Journals (Sweden)

    Lukasz Lewicki

    2015-01-01

    Full Text Available An inflammatory response plays a crucial role in myocardial damage after an acute myocardial infarction. Objectives. To measure serum concentrations of several mediators in patients with an acute myocardial infarction (STEMI and to assess their potential relationship with a risk of coronary instability. Patients and Methods. The 33 patients with STEMI and 19 healthy volunteers were analyzed. The clinical data were obtained; as well serum concentrations of tryptase, endothelin (ET-1, angiogenin, soluble c-kit, and PDGF were measured. Results. Patients with STEMI had higher serum tryptase and ET-1 than healthy volunteers (2,5 ± 0,4 ng/mL versus 1,1 ± 0,4 ng/mL and 0,7 ± 0,1 ng/mL versus 0,3 ± 0,1 ng/mL, resp.. Subjects with significant lesion in left anterior descending artery (LAD had lower serum ET-1 compared to those with normal LAD (0,6 ± 0,2 pg/mL versus 0,9 ± 0,4 pg/mL. Patients with three-vessel coronary artery disease (CAD had higher level of soluble c-kit compared to those with one- or two-vessel CAD: 19,9 ± 24,1 ng/mL versus 5,6 ± 1,9 ng/mL. Conclusions. Elevated serum tryptase and ET-1 may be markers of increased coronary instability; some cytokines may be related to the extension of CAD.

  19. Human serum albumin complexes with chlorophyll and chlorophyllin.

    Science.gov (United States)

    Ouameur, A Ahmed; Marty, R; Tajmir-Riahi, H A

    2005-02-15

    Porphyrins and their metal derivatives are strong protein binders. Some of these compounds have been used for radiation sensitization therapy of cancer and are targeted to interact with cellular DNA and protein. The presence of several high-affinity binding sites on human serum albumin (HSA) makes it possible target for many organic and inorganic molecules. Chlorophyll a and chlorophyllin (a food-grade derivative of chlorophyll), the ubiquitous green plant pigment widely consumed by humans, are potent inhibitors of experimental carcinogenesis and interact with protein and DNA in many ways. This study was designed to examine the interaction of HSA with chlorophyll (Chl) and chlorophyllin (Chln) in aqueous solution at physiological conditions. Fourier transform infrared, UV-visible, and CD spectroscopic methods were used to determine the pigment binding mode, the binding constant, and the effects of porphyrin complexation on protein secondary structure. Spectroscopic results showed that chlorophyll and chlorophyllin are located along the polypeptide chains with no specific interaction. Stronger protein association was observed for Chl than for Chln, with overall binding constants of K(Chl) = 2.9 x 10(4)M(-1) and K(Chln) = 7.0 x 10(3)M(-1). The protein conformation was altered (infrared data) with reduction of alpha-helix from 55% (free HSA) to 41-40% and increase of beta-structure from 22% (free HSA) to 29-35% in the pigment-protein complexes. Using the CDSSTR program (CD data) also showed major reduction of alpha-helix from 66% (free HSA) to 58 and 55% upon complexation with Chl and Chln, respectively.

  20. Pathogen inactivation of human serum facilitates its clinical use for islet cell culture and subsequent transplantation.

    Science.gov (United States)

    Ståhle, Magnus U; Brandhorst, Daniel; Korsgren, Olle; Knutson, Folke

    2011-01-01

    Serum is regarded as an essential supplement to promote survival and growth of cells during culture. However, the potential risk of transmitting diseases disqualifies the use of serum for clinical cell therapy in most countries. Hence, most clinical cell therapy programs have replaced human serum with human serum albumin, which can result in inferior quality of released cell products. Photochemical treatment of different blood products utilizing Intercept® technology has been shown to inactivate a broad variety of pathogens of RNA and DNA origin. The present study assesses the feasibility of using pathogen-inactivated, blood group-compatible serum for use in human pancreatic islet culture. Isolated human islets were cultured at 37°C for 3-4 days in CMRL 1066 supplemented with 10% of either pathogen-inactivated or nontreated human serum. Islet quality assessment included glucose-stimulated insulin release (perifusion), ADP/ATP ratio, cytokine expression, and posttransplant function in diabetic nude mice. No differences were found between islets cultured in pathogen-inactivated or control serum regarding stimulated insulin release, intracellular insulin content, and ADP/ATP ratio. Whether media was supplemented with treated or nontreated serum, islet expression of IL-6, IL-8, MCP-1, or tissue factor was not affected. The final diabetes-reversal rate of mice receiving islets cultured in pathogen-inactivated or nontreated serum was 78% and 87%, respectively (NS). As reported here, pathogen-inactivated human serum does not affect viability or functional integrity of cultured human islets. The implementation of this technology for RNA- and DNA-based pathogen inactivation should enable reintroduction of human serum for clinical cell therapy.

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

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    Guo, Shihui; Shi, Xiaoli; Yang, Feng; Chen, Liqing; Meehan, Edward J.; Bian, Chuanbing; Huang, Mingdong; (UAH); (Chinese Aca. Sci.)

    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.

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

    Energy Technology Data Exchange (ETDEWEB)

    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.

  3. Interaction of VO2+ ion with human serum transferrin and albumin.

    Science.gov (United States)

    Sanna, Daniele; Garribba, Eugenio; Micera, Giovanni

    2009-04-01

    The complexation of VO(2+) ion with the high molecular mass components of the blood serum, human serum transferrin (hTf) and albumin (HSA), has been re-examined using EPR spectroscopy. In the case of transferrin, the results confirm those previously obtained, showing that VO(2+) ion occupies three different binding sites, A, B(1) and B(2), distinguishable in the X-band anisotropic spectrum recorded in D(2)O. With albumin the results show that a dinuclear complex (VO)(2)(d)HSA is formed in equimolar aqueous solutions or with an excess of protein; in the presence of an excess of VO(2+), the multinuclear complex (VO)(x)(m)HSA is the prevalent species, where x=5-6 indicates the equivalents of metal ion coordinated by HSA. The structure of the dinuclear species is discussed and the donor atoms involved in the metal coordination are proposed on the basis of the measured EPR parameters. Two different binding modes of albumin can be distinguished varying the pH, with only one species being present at the physiological value. The results show that the previously named "strong" site is not the N-terminal copper binding site, and some hypothesis on the metal coordination is discussed, with the (51)V A(z) values for the proposed donor sets obtained by DFT (density functional theory) calculations. Finally, preliminary results obtained in the ternary system VO(2+)/hTf/HSA are shown in order to determine the different binding strength of the two proteins. Due to the low VO(2+) concentration used, the recording of the EPR spectra through the repeated acquisition of the weak signals is essential to obtain a good signal to noise ratio in these systems.

  4. The relationship between levels of PCBs and pesticides in human hair and blood: preliminary result.

    Science.gov (United States)

    Altshul, Larisa; Covaci, Adrian; Hauser, Russ

    2004-08-01

    Human hair as a biologic measure of exposure to persistent organic pollutants (POPs) has some advantages over the more commonly used blood and adipose tissue samples. However, one of the primary limitations is the difficulty in distinguishing between exogenous and endogenous contamination. In addition, there are currently no standardized methods for hair sample collection, washing, and chemical analysis. There is also very limited information describing the correlation between levels of organic contaminants in hair and other body compartments. To explore levels of POPs in blood and hair, samples from 10 volunteers were collected and analyzed for select organochlorine pesticides and 57 individual polychlorinated biphenyl (PCB) congeners. We demonstrated that the method for analyzing organic contaminants in human hair was reliable and reproducible. Washing hair with shampoo decreased levels of PCBs, pesticides, and lipids by 25-33% on average and up to 62% for low-chlorinated congeners. The percentage of lipids and the levels of organochlorines in hair were higher than in serum. We found strong correlation (r = 0.8) between p,p -DDE (dichlorodiphenyldichloroethylene) levels in hair and blood and moderate correlations for the more persistent PCB congeners, but no correlations or weak correlations for other organochlorines. The present study provides preliminary evidence on the utility of hair analysis for POPs; however, further larger studies are recommended before hair analysis can be successfully applied in epidemiologic studies on POPs.

  5. Influence of sitgliptin and metformin treatment on glucose fluctuation and serum inflammatory factors in preliminary diagnosed type 2 diabetes mellitus

    Institute of Scientific and Technical Information of China (English)

    Qin Xiang; Xun Wu

    2016-01-01

    Objective:To investigate the influence of stigliptin and metformin treatment on glucose fluctuation and serum inflammatory factors in preliminary diagnosed Type 2 diabetes mellitus. Methods: From January 2014 to December 2015, 168 cases of patients with newly diagnosed Type 2 diabetes mellitus were recruited and divided randomly into observation group and control group. The observation group was given sitgliptin (Januvia, 100 mg per d), while the control group was given metformin (Glucophage tablet, initial dose 0.5 g bid, twice per d), and the dosage of drugs was adjusted every 2 weeks according to glucose level. If the glucose level still wasn’t controlled to reach the normal standard by maximum dosage of drugs, 0.5 g bid of acarbose (Glucobay) was applied for the treatment 3 times one day till the glucose level reached the normal standard and the observation was kept for 6 months. The HbA1c, BMI, fluctuation indexes, and serum inflammatory factors of two groups were compared. Results: After the treatment of control group and observation group, the differences of PPGE (t=8.425,P=0.012), MAGE (t=7.348,P=0.014) and MODD (t=9.327,P=0.010) between two groups were significant (P<0.05). The differences of IL-6 (t=6.337,P=0.010) and TNF-α(t=6.521,P=0.011) level of observation group and control group after treatment were statistical significant (P<0.05).Conclusions:The sitgliptin could not only achieve glycemic control goal as metformin, but also induce glucose fluctuation and inhibit serum inflammation better.

  6. Binding interactions of pefloxacin mesylate with bovine lactoferrin and human serum albumin

    Institute of Scientific and Technical Information of China (English)

    FAN Ji-cai; CHEN Xiang; WANG Yun; FAN Cheng-ping; SHANG Zhi-cai

    2006-01-01

    The binding of pefloxacin mesylate (PFLX) to bovine lactoferrin (BLf) and human serum albumin (HSA) in dilute aqueous solution was studied using fluorescence spectra and absorbance spectra. The binding constant K and the binding sites n were obtained by fluorescence quenching method. The binding distance r and energy-transfer efficiency E between pefloxacin mesylate and bovine lactoferrin as well as human serum albumin were also obtained according to the mechanism of Forster-type dipole-dipole nonradiative energy-transfer. The effects of pefloxacin mesylate on the conformations of bovine lactoferrin and human serum albumin were also analyzed using synchronous fluorescence spectroscopy.

  7. Immunomagnetic separation and quantification of butyrylcholinesterase nerve agent adducts in human serum

    NARCIS (Netherlands)

    Sporty, J.L.S.; Lemire, S.W.; Jakubowski, E.M.; Renner, J.A.; Evans, R.A.; Williams, R.F.; Schmidt, J.G.; Schans, M.J. van der; Noort, D.; Johnson, R.C.

    2010-01-01

    A novel method for extracting butyrylcholinesterase (BuChE) from serum as a means of identifying and measuring nerve agent adducts to human BuChE is presented here. Antibutyrylcholinesterase monoclonal antibodies were conjugated to protein-G ferromagnetic particles and mixed with 500 μL serum sample

  8. Serum cholesterol concentration associated with aspirin esterase activity in older people: preliminary data

    Directory of Open Access Journals (Sweden)

    Kazuhiko Kotani, Russell Caccavello, Ricardo Hermo, Toshiyuki Yamada, Nobuyuki Taniguchi, Alejandro Gugliucci

    2010-01-01

    Full Text Available OBJECTIVE: Metabolism of aspirin (acetylsalicylic acid, commonly used in older people for the prevention of cardiovascular disease, is important to the effectiveness of this drug. Whereas part of aspirin hydrolysis occurs in blood, there is a paucity of information in regards to circulating aspirin esterase activity in various physiological and pathological conditions. High aspirin esterase activity, corresponding to faster aspirin hydrolysis (thus aspirin non-responsiveness, may occur in cardiovascular disease-prone states. The objective of this study was to investigate the effects of cardio-metabolic variables such as cholesterol on serum aspirin esterase activity in older people who participated in an intervention study on physical activity. METHODS: A total of 18 non-medicated subjects (7 men/11 women, mean age 67.8 years, body mass index = 23.4 ± 3.3 kg/m2, who completed a 3-month interventional program for a mild-to-moderate increase in physical activity, were analyzed. The body mass index, plasma glucose, serum total cholesterol and aspirin esterase activity were measured in the pre- and post-interventional phases of the study. RESULTS: During the interventional period, the changes in aspirin esterase activity correlated significantly and positively with those of total cholesterol concentrations (r = 0.542, P = 0.020; β = 0.609, P = 0.035 in a multiple linear regression analysis after adjusting for all the measured variables. CONCLUSION: The results suggest that cholesterol metabolism alterations may be associated with aspirin metabolism in older people.

  9. Analysis of human serum from women affected by cervical lesions.

    Science.gov (United States)

    Barba de la Rosa, Ana P; Lugo-Melchor, Ofelia Y; Briones-Cerecero, Erika P; Chagolla-López, Alicia; De León-Rodríguez, Antonio; Santos, Leticia; Vázquez-Ortiz, Guelaguetza; Salcedo, Mauricio

    2008-01-01

    Cervical cancer is one of the first causes of death in Mexican women population. The plasma proteome has a wide dynamic range concentrations of different protein and their alterations reflect the physiological state of the individual's health. The aim of this study was to characterize the 2D-PAGE serum patterns from healthy women and with different levels of cervical lesions. Changes in haptoglobin, apolipoproteins, and transthyretin, when comparing the serum from healthy women and serum from patients with different levels of cervical lesion were found. The Western blot analysis showed increasing concentrations of metalloproteinases (MMP's), proteins with important biological roles in tumor development and metastasis. Protein profiles in conjunction with MS, bioinformatics, and Western blot analysis, allow us to compile information for the acquisition of results to proposed candidates biomarkers of cervical cancer among Mexican women population.

  10. Native human serum amyloid P component is a single pentamer

    DEFF Research Database (Denmark)

    Sørensen, Inge Juul; Andersen, Ove; Nielsen, EH;

    1995-01-01

    Serum amyloid P component (SAP) and C-reactive protein (CRP) are members of the pentraxin protein family. SAP is the precursor protein to amyloid P component present in all forms of amyloidosis. The prevailing notion is that SAP in circulation has the form of a double pentameric molecule (decamer...... by rocket immunoelectrophoresis and electron microscopy. Thus, electron micrographs of purified SAP showed a predominance of decamers. However, the decamer form of SAP reversed to single pentamers when purified SAP was incorporated into SAP-depleted serum....

  11. Gene expression profiling of human neural progenitor cells following the serum-induced astrocyte differentiation.

    Science.gov (United States)

    Obayashi, Shinya; Tabunoki, Hiroko; Kim, Seung U; Satoh, Jun-ichi

    2009-05-01

    Neural stem cells (NSC) with self-renewal and multipotent properties could provide an ideal cell source for transplantation to treat spinal cord injury, stroke, and neurodegenerative diseases. However, the majority of transplanted NSC and neural progenitor cells (NPC) differentiate into astrocytes in vivo under pathological environments in the central nervous system, which potentially cause reactive gliosis. Because the serum is a potent inducer of astrocyte differentiation of rodent NPC in culture, we studied the effect of the serum on gene expression profile of cultured human NPC to identify the gene signature of astrocyte differentiation of human NPC. Human NPC spheres maintained in the serum-free culture medium were exposed to 10% fetal bovine serum (FBS) for 72 h, and processed for analyzing on a Whole Human Genome Microarray of 41,000 genes, and the microarray data were validated by real-time RT-PCR. The serum elevated the levels of expression of 45 genes, including ID1, ID2, ID3, CTGF, TGFA, METRN, GFAP, CRYAB and CSPG3, whereas it reduced the expression of 23 genes, such as DLL1, DLL3, PDGFRA, SOX4, CSPG4, GAS1 and HES5. Thus, the serum-induced astrocyte differentiation of human NPC is characterized by a counteraction of ID family genes on Delta family genes. Coimmunoprecipitation analysis identified ID1 as a direct binding partner of a proneural basic helix-loop-helix (bHLH) transcription factor MASH1. Luciferase assay indicated that activation of the DLL1 promoter by MASH1 was counteracted by ID1. Bone morphogenetic protein 4 (BMP4) elevated the levels of ID1 and GFAP expression in NPC under the serum-free culture conditions. Because the serum contains BMP4, these results suggest that the serum factor(s), most probably BMP4, induces astrocyte differentiation by upregulating the expression of ID family genes that repress the proneural bHLH protein-mediated Delta expression in human NPC.

  12. Subnanosecond fluorescence spectroscopy of human serum albumin as a method to estimate the efficiency of the depression therapy

    Science.gov (United States)

    Syrejshchikova, T. I.; Gryzunov, Yu. A.; Smolina, N. V.; Komar, A. A.; Uzbekov, M. G.; Misionzhnik, E. J.; Maksimova, N. M.

    2010-05-01

    The efficiency of the therapy of psychiatric diseases is estimated using the fluorescence measurements of the conformational changes of human serum albumin in the course of medical treatment. The fluorescence decay curves of the CAPIDAN probe (N-carboxyphenylimide of the dimethylaminonaphthalic acid) in the blood serum are measured. The probe is specifically bound to the albumin drug binding sites and exhibits fluorescence as a reporter ligand. A variation in the conformation of the albumin molecule substantially affects the CAPIDAN fluorescence decay curve on the subnanosecond time scale. A subnanosecond pulsed laser or a Pico-Quant LED excitation source and a fast photon detector with a time resolution of about 50 ps are used for the kinetic measurements. The blood sera of ten patients suffering from depression and treated at the Institute of Psychiatry were preliminary clinically tested. Blood for analysis was taken from each patient prior to the treatment and on the third week of treatment. For ten patients, the analysis of the fluorescence decay curves of the probe in the blood serum using the three-exponential fitting shows that the difference between the amplitudes of the decay function corresponding to the long-lived (9 ns) fluorescence of the probe prior to and after the therapeutic procedure reliably differs from zero at a significance level of 1% ( p < 0.01).

  13. Identification and quantification of serum proteins secreted into the normal human jejunum

    DEFF Research Database (Denmark)

    Andersen, Vibeke; Hegnhøj, J H

    1990-01-01

    The in vivo transfer of serum proteins to the human intestinal lumen was characterized by crossed immunoelectrophoretic analyses of intestinal perfusates from four healthy volunteers. Serum proteins with molecular masses below 100 kDa and the immunoglobulins were found in human jejunal perfusates....... Larger serum proteins were either absent (alpha and beta lipoproteins) or present in small amounts (alpha 2-macroglobulin, haptoglobulin and ceruloplasmin). These results demonstrate the existence of a selective transfer of serum proteins to the intestinal lumen under physiological conditions....... The intestinal clearance rate was 0.1 ml serum per hour per 10 cm jejunum for albumin, prealbumin, alpha 1-antitrypsin, orosomucoid, transferrin and haemopexin. The rate of secretion of total protein to the jejunal lumen was 100 mg protein per hour per 10 cm jejunum. About 45% was due to immunoglobulins...

  14. Native human serum amyloid P component is a single pentamer

    DEFF Research Database (Denmark)

    Sørensen, Inge Juul; Andersen, Ove; Nielsen, EH;

    1995-01-01

    Serum amyloid P component (SAP) and C-reactive protein (CRP) are members of the pentraxin protein family. SAP is the precursor protein to amyloid P component present in all forms of amyloidosis. The prevailing notion is that SAP in circulation has the form of a double pentameric molecule (decamer...

  15. Inverse correlation among organochlorine pesticide levels to total lipid serum contents: a preliminary study in Veracruz, México.

    Science.gov (United States)

    Caba, Mario; Meza, Enrique; Waliszewski, Stefan M; Martínez-Valenzuela, Carmen

    2015-07-01

    Organochlorine pesticides, due to their hydrophobic nature and persistence, accumulate in tissues rich in lipids, which had been used as a biomarker for environmental pollution. In humans, organochlorine pesticides are continuously circulating and equilibrating among body compartments. The objective of the study was to evaluate the concentrations of organochlorine pesticides in blood serum and compare their levels to the total lipid contents in Veracruz, México inhabitants. Our hypothesis is that concentrations of organochlorine pesticides will increase just as lipid concentrations. Levels of organochlorine pesticides were divided in ascending tertils according to their total lipid content. The linear trend model applied surprisingly reveals that the average level of all organochlorine pesticides decreases as the lipid concentration increases. From one tertil to the next β-HCH, it shows a decrease of -3.19 mg kg(-1) on lipid basis, pp.'DDE levels decrease by -3.70 mg kg(-1) on lipid basis and pp.'DDT levels decrease -1.13 mg kg(-1) on lipid basis. We conclude that the levels and the orderly sequence of organochlorine pesticide distributions in the blood serum maintain an inverse relationship to total lipid blood serum concentrations.

  16. Behavior of human serum albumin on strong cation exchange resins: I. experimental analysis.

    Science.gov (United States)

    Voitl, Agnes; Butté, Alessandro; Morbidelli, Massimo

    2010-08-20

    Experiments with human serum albumin on the strong cation exchange resin Fractogel EMD SE Hicap (M) were carried out. Even though human serum albumin was used at high purity, two peaks in gradient elution experiments occurred. The obtained data can be explained by considering that human serum albumin binds to Fractogel EMD SE Hicap (M) in two different binding conformations: the protein adsorbs instantaneously in the first conformation and then changes into the second one with a kinetic limitation. The two-peak behavior of human serum albumin was analyzed in detail, especially at various gradient lengths, concentrations and temperatures. Breakthrough curves were performed at four modifier concentrations and three velocities. The characteristic adsorption behavior, found for gradient experiments, was confirmed by the breakthrough curves. The two-peak elution pattern of human serum albumin was also found for other strong cation exchange resins, but not for weak cation exchange resins. It is concluded that the described behavior is peculiar for the interaction of human serum albumin with the strong cation exchange ligand of the resin.

  17. Human Umbilical Cord Blood Serum: Effective Substitute of Fetal Bovine Serum for Culturing of Human Multipotent Mesenchymal Stromal Cells.

    Science.gov (United States)

    Romanov, Yu A; Balashova, E E; Volgina, N E; Kabaeva, N V; Dugina, T N; Sukhikh, G T

    2017-02-01

    Optimal conditions for culturing of multipotent mesenchymal stromal cells in the presence of pooled umbilical cord blood serum were determined. It was found that umbilical cord blood serum in a concentration range of 1-10% effectively supported high viability and proliferative activity of cells with unaltered phenotype and preserved multilineage differentiation capacity. The proposed approach allows avoiding the use of xenogenic animal sera for culturing of multipotent mesenchymal stromal cells and creates prerequisites for designing and manufacturing safe cellular and/or acellular products for medical purposes.

  18. Binding of anthracycline derivatives to human serum lipoproteins.

    Science.gov (United States)

    Chassany, O; Urien, S; Claudepierre, P; Bastian, G; Tillement, J P

    1994-01-01

    The binding of eight anthracycline analogues (including mitoxantrone) to isolated serum lipoproteins (high, low and very low density lipoproteins) was studied in order to elucidate some determinants of their interaction with lipidic structures. Serum lipoproteins were isolated by ultracentrifugation. Drug binding experiments were run by ultrafiltration at 37 degrees C and pH 7.4. Anthracycline concentrations (total and free) were determined by HPLC with fluorometric detection. All the ligands were significantly bound to the three lipoprotein classes, and for each ligand the binding increased as the lipidic fraction of lipoprotein increased. From doxorubicin to iododoxorubicin, there was a tenfold increase in lipoprotein binding (doxorubicin < mitoxantrone < epirubicin < daunorubicin < pirarubicin < aclarubicin < zorubicin < iododoxorubicin). For all the ligands studied, the extent of lipoprotein binding appears to be related to chemical determinants of lipophilicity.

  19. The Effect of PRF on Serum Starved Human Dermal Fibroblast.

    Directory of Open Access Journals (Sweden)

    Sunardi Radiono

    2016-12-01

    Hasil: FDM yang berpuasa serum ternyata secara signifikan (p < 0,05 mengalami penurunan kemampuan menimbun kolagen sebesar 10%, proliferasi sebesar 20% dan penurunan kemampuan migrasi sebesar 25%. Penambahan lisat FKP ternyata dapat memulihkan aktivitas selular tersebut. Dari eksperimen ini dapat disimpulkan bahwa FKP merupakan material biologis yang dapat dikembangkan untuk memacu penyembuhan ulkus khronis. Bagaimanapun juga, untuk memperoleh bukti-bukti klinis yang baik, uji klinis tetap diperlukan.

  20. MMP-1 serum levels predict coronary atherosclerosis in humans

    Directory of Open Access Journals (Sweden)

    Reiser Maximilian

    2009-09-01

    Full Text Available Background Myocardial infarction results as a consequence of atherosclerotic plaque rupture, with plaque stability largely depending on the lesion forming extracellular matrix components. Lipid enriched non-calcified lesions are considered more instable and rupture prone than calcified lesions. Matrix metalloproteinases (MMPs are extracellular matrix degrading enzymes with plaque destabilisating characteristics which have been implicated in atherogenesis. We therefore hypothesised MMP-1 and MMP-9 serum levels to be associated with non-calcified lesions as determined by CT-angiography in patients with coronary artery disease. Methods 260 patients with typical or atypical chest pain underwent dual-source multi-slice CT-angiography (0.6-mm collimation, 330-ms gantry rotation time to exclude coronary artery stenosis. Atherosclerotic plaques were classified as calcified, mixed or non-calcified. Results In multivariable regession analysis, MMP-1 serum levels were associated with total plaque burden (OR: 1.37 (CI: 1.02-1.85; p Conclusion MMP-1 serum levels are associated with total plaque burden but do not allow a specification of plaque morphology.

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

    Energy Technology Data Exchange (ETDEWEB)

    Singaravelu, Ragunath [Department of Biochemistry, Microbiology and Immunology, University of Ottawa, Ottawa, Ontario K1N 6N5 (Canada); National Research Council of Canada, Ottawa, Ontario K1A 0R6 (Canada); Lyn, Rodney K. [Department of Chemistry, University of Ottawa, Ottawa, Ontario K1N 6N5 (Canada); National Research Council of Canada, Ottawa, Ontario K1A 0R6 (Canada); Srinivasan, Prashanth [National Research Council of Canada, Ottawa, Ontario K1A 0R6 (Canada); Delcorde, Julie [Department of Biochemistry, Microbiology and Immunology, University of Ottawa, Ottawa, Ontario K1N 6N5 (Canada); National Research Council of Canada, Ottawa, Ontario K1A 0R6 (Canada); Steenbergen, Rineke H.; Tyrrell, D. Lorne [Department of Medical Microbiology and Immunology, University of Alberta (Canada); Li Ka Shing Institute of Virology, Katz Centre for Pharmacy and Health Research, Edmonton, Alberta T6G 2S2 (Canada); Pezacki, John P., E-mail: John.Pezacki@nrc-cnrc.gc.ca [Department of Chemistry, University of Ottawa, Ottawa, Ontario K1N 6N5 (Canada); National Research Council of Canada, Ottawa, Ontario K1A 0R6 (Canada)

    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

    Directory of Open Access Journals (Sweden)

    Katherine E. Chetta

    2015-04-01

    Full Text Available 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.

  3. The susceptibility of Campylobacter concisus to the bactericidal effects of normal human serum.

    Science.gov (United States)

    Kirk, Karina Frahm; Nielsen, Hans Linde; Nielsen, Henrik

    2015-03-01

    Campylobacter concisus is an emerging pathogen of the gastrointestinal tract that has been associated with Barrett's oesophagus, enteritis and inflammatory bowel disease. Despite having invasive potential in intestinal epithelial cells in-vitro, bacteraemic cases with C. concisus are extremely scarce, having only been reported once. Therefore, we conducted a serum resistance assay to investigate the bactericidal effects of human complement on C. concisus in comparison to some other Campylobacter species. In total, 22 Campylobacter strains were tested by incubation with normal human serum and subsequent cultivation in microaerobic conditions for 48 hours. Killing time was evaluated by decrease in total CFU over time for incubation with different serum concentrations. Faecal isolates of C. concisus showed inoculum reduction to less than 50% after 30 min. Campylobacter jejuni was sensitive to serum, but killing was delayed and a bacteraemic Campylobacter fetus subsp. fetus isolate was completely serum resistant. Interestingly, sensitivity of enteric C. concisus to human serum was not associated to different faecal-calprotectin levels. We find that faecal isolates of C. concisus are sensitive to the bactericidal effects of serum, which may explain why C. concisus is not associated to bacteraemia.

  4. Preliminary evaluation of total protein concentration and electrophoretic protein fractions in fresh and frozen serum from wild Horned Vipers (Vipera ammodytes ammodytes).

    Science.gov (United States)

    Proverbio, Daniela; de Giorgi, Giada Bagnagatti; Della Pepa, Alessandra; Baggiani, Luciana; Spada, Eva; Perego, Roberta; Comazzi, Carlo; Belloli, Angelo

    2012-12-01

    Determination of the health status of reptiles is based on physical examination and evaluation of hematologic and biochemical values. Evaluation of serum total protein (TP) concentration and protein fractions plays an important role in health assessment; however, little is known about references value for these analytes in wild viperoid snakes. In addition, studies evaluating the stability of proteins in frozen viperoid serum are lacking. The aims of this study were to establish preliminary reference values for concentrations of TP and protein fractions in serum from wild vipers and to evaluate the stability of serum proteins in frozen serum samples from viperoid snakes. Blood samples were collected from wild Horned Vipers (Vipera ammodytes ammodytes). Using fresh serum, TP concentrations were determined using the biuret method and protein fractions were analyzed using agarose gel electrophoresis (AGE); albumin/globulin ratios were calculated. Analyses were also performed on serum frozen at -20°C for 70 days and then thawed. Pre- and post-storage results were compared using the Mann-Whitney U-test. Five adult wild Horned Vipers were sampled and comprised 4 males and 1 female. The female snake had higher TP concentrations than the male snakes. The electrophoretic patterns demonstrated 6 protein fractions that were similar for all 5 snakes. There were no significant changes in the concentrations of the 6 protein fractions post-storage; the percentage of the alpha-1 fraction was increased in frozen/thawed serum. Total protein concentrations in serum from Vipera ammodytes ammodytes were in agreement with published reference intervals for healthy reptiles and viperoid snakes. Serum protein fractions were easy to identify using AGE electrophoresis. © 2012 American Society for Veterinary Clinical Pathology.

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

    Science.gov (United States)

    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. DTPA complexation of bismuth in human blood serum.

    Science.gov (United States)

    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.

  7. Serum inflammatory mediators as markers of human Lyme disease activity.

    Directory of Open Access Journals (Sweden)

    Mark J Soloski

    Full Text Available Chemokines and cytokines are key signaling molecules that orchestrate the trafficking of immune cells, direct them to sites of tissue injury and inflammation and modulate their states of activation and effector cell function. We have measured, using a multiplex-based approach, the levels of 58 immune mediators and 7 acute phase markers in sera derived from of a cohort of patients diagnosed with acute Lyme disease and matched controls. This analysis identified a cytokine signature associated with the early stages of infection and allowed us to identify two subsets (mediator-high and mediator-low of acute Lyme patients with distinct cytokine signatures that also differed significantly (p<0.0005 in symptom presentation. In particular, the T cell chemokines CXCL9 (MIG, CXCL10 (IP-10 and CCL19 (MIP3B were coordinately increased in the mediator-high group and levels of these chemokines could be associated with seroconversion status and elevated liver function tests (p = 0.027 and p = 0.021 respectively. There was also upregulation of acute phase proteins including CRP and serum amyloid A. Consistent with the role of CXCL9/CXCL10 in attracting immune cells to the site of infection, CXCR3+ CD4 T cells are reduced in the blood of early acute Lyme disease (p = 0.01 and the decrease correlates with chemokine levels (p = 0.0375. The levels of CXCL9/10 did not relate to the size or number of skin lesions but elevated levels of serum CXCL9/CXCL10 were associated with elevated liver enzymes levels. Collectively these results indicate that the levels of serum chemokines and the levels of expression of their respective chemokine receptors on T cell subsets may prove to be informative biomarkers for Lyme disease and related to specific disease manifestations.

  8. Serum inflammatory mediators as markers of human Lyme disease activity.

    Science.gov (United States)

    Soloski, Mark J; Crowder, Lauren A; Lahey, Lauren J; Wagner, Catriona A; Robinson, William H; Aucott, John N

    2014-01-01

    Chemokines and cytokines are key signaling molecules that orchestrate the trafficking of immune cells, direct them to sites of tissue injury and inflammation and modulate their states of activation and effector cell function. We have measured, using a multiplex-based approach, the levels of 58 immune mediators and 7 acute phase markers in sera derived from of a cohort of patients diagnosed with acute Lyme disease and matched controls. This analysis identified a cytokine signature associated with the early stages of infection and allowed us to identify two subsets (mediator-high and mediator-low) of acute Lyme patients with distinct cytokine signatures that also differed significantly (p<0.0005) in symptom presentation. In particular, the T cell chemokines CXCL9 (MIG), CXCL10 (IP-10) and CCL19 (MIP3B) were coordinately increased in the mediator-high group and levels of these chemokines could be associated with seroconversion status and elevated liver function tests (p = 0.027 and p = 0.021 respectively). There was also upregulation of acute phase proteins including CRP and serum amyloid A. Consistent with the role of CXCL9/CXCL10 in attracting immune cells to the site of infection, CXCR3+ CD4 T cells are reduced in the blood of early acute Lyme disease (p = 0.01) and the decrease correlates with chemokine levels (p = 0.0375). The levels of CXCL9/10 did not relate to the size or number of skin lesions but elevated levels of serum CXCL9/CXCL10 were associated with elevated liver enzymes levels. Collectively these results indicate that the levels of serum chemokines and the levels of expression of their respective chemokine receptors on T cell subsets may prove to be informative biomarkers for Lyme disease and related to specific disease manifestations.

  9. Interaction of cyclodextrins with human and bovine serum albumins: A combined spectroscopic and computational investigation

    Indian Academy of Sciences (India)

    Saptarshi Ghosh; Bijan Kumar Paul; Nitin Chattopadhyay

    2014-07-01

    Interaction of cyclodextrins (CDs) with the two most abundant proteins, namely human serum albumin (HSA) and bovine serum albumin (BSA), has been investigated using steady-state and time-resolved fluorometric techniques, circular dichroism measurements and molecular docking simulation. The study reveals that the three CDs interact differently on the fluorescence and fluorescence lifetimes of the serum albumins. However, fluorescence anisotropy and circular dichroism are not affected. Depending on their size, different CDs bind to the serum albumins in different positions, resulting in changes in the spectral behaviour of the proteins. Docking study suggests the probable binding sites of the three CDs with the proteins. Combined experimental and computational studies imply that sufficiently high concentration of CDs causes loosening of the rigid structures of these transport proteins, although their secondary structures remain intact. Thus, CDs are found to be safe for the serum proteins from the structural point of view.

  10. Ligand-binding sites in human serum amyloid P component

    DEFF Research Database (Denmark)

    Heegaard, N.H.H.; Heegaard, Peter M. H.; Roepstorff, P.;

    1996-01-01

    Amyloid P component (AP) is a naturally occurring glycoprotein that is found in serum and basement membranes, AP is also a component of all types of amyloid, including that found in individuals who suffer from Alzheimer's disease and Down's syndrome. Because AP has been found to bind strongly...... of 25 mu M, while the IC50 of AP-(27-38)-peptide and AP-(33-38)-peptide are 10 mu M and 2 mu M, respectively, The understanding of the structure and function of active AP peptides will be useful for development of amyloid-targeted diagnostics and therapeutics....

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

    Science.gov (United States)

    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.

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

    Science.gov (United States)

    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.

  13. Serum Inflammatory Mediators as Markers of Human Lyme Disease Activity

    Science.gov (United States)

    Soloski, Mark J.; Crowder, Lauren A.; Lahey, Lauren J.; Wagner, Catriona A.

    2014-01-01

    Chemokines and cytokines are key signaling molecules that orchestrate the trafficking of immune cells, direct them to sites of tissue injury and inflammation and modulate their states of activation and effector cell function. We have measured, using a multiplex-based approach, the levels of 58 immune mediators and 7 acute phase markers in sera derived from of a cohort of patients diagnosed with acute Lyme disease and matched controls. This analysis identified a cytokine signature associated with the early stages of infection and allowed us to identify two subsets (mediator-high and mediator-low) of acute Lyme patients with distinct cytokine signatures that also differed significantly (pLyme disease (p = 0.01) and the decrease correlates with chemokine levels (p = 0.0375). The levels of CXCL9/10 did not relate to the size or number of skin lesions but elevated levels of serum CXCL9/CXCL10 were associated with elevated liver enzymes levels. Collectively these results indicate that the levels of serum chemokines and the levels of expression of their respective chemokine receptors on T cell subsets may prove to be informative biomarkers for Lyme disease and related to specific disease manifestations. PMID:24740099

  14. Serum human placental lactogen levels in intra-uterine fetal growth retardation.

    Science.gov (United States)

    Zail, S S; Safro, I L

    1975-11-12

    Serum human placental lactogen (HPL) levels were measured in the last trimester of pregnancy in 16 mothers who delivered small-for-gestational-age babies. Only 3 patients had levels which were below the normal range, while 4 others had levels close to the lower limit of the normal range. The finding of a normal serum HPL level therefore does not exclude the possibility of intra-uterine fetal growth retardation. No correlation was found between serum HPL levels at 37-39 weeks and infant or placental weights in full-term normal deliveries.

  15. Lectins in extracts of certain Polygonaceae seed precipitate animal and human serums.

    Science.gov (United States)

    Hanan, E B; Spindler, J W

    1968-06-28

    Seeds of four species of Polygonaceae were tested for lectins that precipitate human and animal serums. Rumex crispus, Polygonum convolvulus, and Polygonum pennsylvanicum developed specific precipitate bands on double diffusion on agar gel plates. These bands were enhanced and increased in number when extracts were tested against serums from patients with certain diseases. When tested against lyophilized serum, no precipitate bands developed. The active substance cannot be dialyzed through cellulose membrane against running tap water for 16 hours, and it is heat stable. Extracts from Fagopyrum esculentum developed no precipitate bands.

  16. ASTHMATIC HUMAN SERUM IGE-REACTIVITY WITH MOLD EXTRACTS

    Science.gov (United States)

    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...

  17. Covalent binding of the flavonoid quercetin to human serum albumin

    NARCIS (Netherlands)

    Kaldas, M.I.; Walle, U.K.; Woude, van der H.; McMillan, J.M.; Walle, T.

    2005-01-01

    Quercetin is an abundant flavonoid in the human diet with numerous biological activities, which may contribute to the prevention of human disease but also may be potentially harmful. Quercetin is oxidized in cells to products capable of covalently binding to cellular proteins, a process that may be

  18. Direct microcomputer controlled determination of zinc in human serum by flow injection atomic absorption spectrometry

    DEFF Research Database (Denmark)

    Simonsen, Kirsten Wiese; Nielsen, Bent; Jensen, Arne

    1986-01-01

    A procedure is described for the direct determination of zinc in human serum by fully automated, microcomputer controlled flow injection atomic absorption spectrometry (Fl-AAS). The Fl system is pumpless, using the negative pressure created by the nebuliser. It only consists of a three-way valve......, programmable from the microcomputer, to control the sample volume. No pre-treatment of the samples is necessary. The limit of detection is 0.14 mg l–1, and only small amounts of serum (

  19. Calcium Phosphate Crystals from Uremic Serum Promote Osteogenic Differentiation in Human Aortic Smooth Muscle Cells.

    Science.gov (United States)

    Liu, Yaorong; Zhang, Lin; Ni, Zhaohui; Qian, Jiaqi; Fang, Wei

    2016-11-01

    Recent study demonstrated that calcium phosphate (CaP) crystals isolated from high phosphate medium were a key contributor to arterial calcification. The present study further investigated the effects of CaP crystals induced by uremic serum on calcification of human aortic smooth muscle cells. This may provide a new insight for the development of uremic cardiovascular calcification. We tested the effects of uremic serum or normal serum on cell calcification. Calcification was visualized by staining and calcium deposition quantified. Expression of various bone-calcifying genes was detected by real-time PCR, and protein levels were quantified by western blotting or enzyme-linked immunosorbent assays. Pyrophosphate was used to investigate the effects of CaP crystals' inhibition. Finally, CaP crystals were separated from uremic serum to determine its specific pro-calcification effects. Uremic serum incubation resulted in progressively increased calcification staining and increased calcium deposition in HASMCs after 4, 8 and 12 days (P vs 0 day crystals with pyrophosphate incubation prevented calcium deposition and bone-calcifying gene over-expression increased by uremic serum. CaP crystals, rather than the rest of uremic serum, were responsible for these effects. Uremic serum accelerates arterial calcification by mediating osteogenic differentiation. This effect might be mainly attributed to the CaP crystal content.

  20. (99m) Tc-labelled human serum albumin cannot replace (125) I-labelled human serum albumin to determine plasma volume in patients with liver disease

    DEFF Research Database (Denmark)

    Henriksen, Ulrik Lütken; Henriksen, Jens H; Bendtsen, Flemming

    2013-01-01

    -labelled human serum albumin (99mTc-HSA) and iodine-labelled human serum albumin (125I-HSA), as the former may have advantages at repeated measurements and the latter is the classical gold standard. Study population and methods In 88 patients, (64 with liver disease, mainly cirrhosis, and 24 patients without......Summary Background and aims Determination of plasma volume (PV) is important in several clinical situations. Thus, patients with liver disease often have augmented PV as part of their sodium–water retention. This study was undertaken to compare PV determination by two indicators: technetium...... liver disease), simultaneous measurements of PV were taken with 99mTc-HSA and 125I-HSA after 1 h in the supine position. Blood samples were obtained before and 10 min after quantitative injection of the two indicators. In a subset of patients (n = 32), the measurements were repeated within 1 h. Results...

  1. Determination of neomycin and bacitracin in human or rabbit serum by HPLC-MS/MS.

    Science.gov (United States)

    Mascher, Daniel G; Unger, Christian P; Mascher, Hermann J

    2007-01-17

    The method for the simultaneous determination of neomycin and bacitracin in human or rabbit serum was developed by using ion pairing reversed phase chromatography and tandem mass spectrometry (MS/MS) detection with electrospray (ESI) in positive mode. Both substances elute under these conditions at the same time and also kanamycin as internal standard elutes almost at the same time. The sample preparation was simple-only using 0.1 mL serum by protein precipitation with acetonitrile. Neomycin and bacitracin were detected as two-fold charged ions as well as the internal standard. The calibration range of these quite difficult detectable substances was 0.2-50 microg/mL of serum. The method was validated for both human or rabbit serum. The inter batch precision of quality control samples in human serum for neomycin ranged from 4.46% to 8.99% and for bacitracin from 6.85% to 11.17%. The inter batch accuracy for neomycin ranged from 98.7% to 100.7% and for bacitracin from 99.2% to 103.0%. At lower limit of quantitation (LLOQ) level of 0.2 microg/mL inter batch precision in human serum for neomycin was 12.05% and for bacitracin 11.91%, whereas accuracies were 99.9% for neomycin and 102.7% for bacitracin. Bench top stability in human or rabbit serum was given over three freeze thaw cycles and 4h at room temperature. The method can be considered to be specific and recoveries for sample preparation were high.

  2. Identification of cyclopropaneoctanoic acid 2-hexyl in human adipose tissue and serum.

    Science.gov (United States)

    Sledzinski, Tomasz; Mika, Adriana; Stepnowski, Piotr; Proczko-Markuszewska, Monika; Kaska, Lukasz; Stefaniak, Tomasz; Swierczynski, Julian

    2013-08-01

    Fatty acids containing a cyclopropane ring in their structure (cyclopropane FA) have been found in a wide variety of bacteria, a number of protozoa, and Myriapoda. Little is known about cyclopropane FA in mammal, especially in human tissues. The present study deals with the identification of cyclopropane FA in adipose tissue and serum of humans and rats. Fatty acids extracted from the adipose tissue and serum obtained from obese women during bariatric surgery were methylated and analyzed on GC-MS. We have identified: cyclopropaneoctanoic acid 2-hexyl, cyclopropaneoctanoic acid 2-octyl, cyclopropanenonanoic acid, and 2-[[2-[(2-ethylcyclopropyl)methyl]cyclopropyl]methyl] acid in human adipose tissue. We confirmed the presence of cyclopropaneoctanoic acid 2-hexyl by derivatization of FA extracted from human adipose tissue to picolinyl esters. Cyclopropaneoctanoic acid 2-hexyl was the main cyclopropane FA (approximately 0.4 % of total fatty acids in human adipose tissue, and about 0.2 % of total fatty acids in the serum). In adipose tissue cyclopropaneoctanoic acid 2-hexyl was found mainly in triacylglycerols, whereas in serum in phospholipids and triacylglycerols. The cyclopropaneoctanoic acid 2-hexyl has also been found in serum, and adipose tissue of rats in amounts comparable to humans. The content of cyclopropaneoctanoic acid 2-hexyl decreased in adipose tissue of rats maintained on a restricted diet for 1 month. In conclusion, we demonstrated that cyclopropaneoctanoic acid 2-hexyl is present in human adipose tissue and serum. Adipose tissue cyclopropaneoctanoic acid 2-hexyl is stored mainly in triacylglycerols and the storage of this cyclopropane FA is affected by food restriction.

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

    Science.gov (United States)

    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. Using competitive protein adsorption to measure fibrinogen in undiluted human serum

    Science.gov (United States)

    Choi, Seokheun; Wang, Ran; Lajevardi-Khosh, Arad; Chae, Junseok

    2010-12-01

    We report a unique sensing mechanism based on competitive protein adsorption to measure fibrinogen, a cardiovascular biomarker, in undiluted human serum. The method uses physical adsorption of proteins to a surface rather than complex and time-consuming immobilization procedures. Two fibrinogen concentrations were differentiated in spiked in human serum [3.0 mg/ml (normal concentration) versus 3.2 mg/ml (abnormal concentration with heart disease)]. Real-time surface plasmon resonance signals were monitored as fibrinogen displaced a preadsorbed protein, IgM, on a hydrophobic gold surface. The relatively strong-affinity protein, IgM, was displaced primarily by fibrinogen and much less by other proteins in human serum.

  5. Nanoparticles of Conjugated Methotrexate-Human Serum Albumin: Preparation and Cytotoxicity Evaluations

    Directory of Open Access Journals (Sweden)

    Azade Taheri

    2011-01-01

    Full Text Available Methotrexate-human serum albumin conjugates were developed by a simple carbodiimide reaction. Methotrexate-human serum albumin conjugates were then crosslinked with 1-ethyl-3-(3-dimethylaminopropyl carbodiimide HCl (EDC to form nanoparticles. The size of nanoparticles determined by laser light scattering and TEM was between 90–150 nm. Nanoparticles were very stable at physiologic conditions (PBS pH 7.4, 37∘C and after incubation with serum. The effect of amount of EDC used for crosslinking on the particle size and free amino groups of nanoparticles was examined. The amount of crosslinker showed no significant effect on the size of nanoparticles but free amino groups of nanoparticles were decreased by increasing the crosslinker. The physicochemical interactions between methotrexate and human serum albumin were investigated by differential scanning calorimetry (DSC. Nanoparticles were more cytotoxic on T47D cells compared to free methotrexate. Moreover, methotrexate-human serum albumin nanoparticles decreased the IC50 value of methotrexate on T47D cells in comparison with free methotrexate.

  6. Adsorption of human serum proteins onto TREN-agarose: purification of human IgG by negative chromatography.

    Science.gov (United States)

    Bresolin, Igor Tadeu Lazzarotto; Borsoi-Ribeiro, Mariana; Caro, Juliana Rodrigues; dos Santos, Francine Petit; de Castro, Marina Polesi; Bueno, Sonia Maria Alves

    2009-01-01

    Tris(2-aminoethyl)amine (TREN) - a chelating agent used in IMAC - immobilized onto agarose gel was evaluated for the purification of IgG from human serum by negative chromatography. A one-step purification process allowed the recovery of 73.3% of the loaded IgG in the nonretained fractions with purity of 90-95% (based on total protein concentration and nephelometric analysis of albumin, transferrin, and immunoglobulins A, G, and M). The binding capacity was relatively high (66.63 mg of human serum protein/mL). These results suggest that this negative chromatography is a potential technique for purification of IgG from human serum.

  7. The in vitro anti-HIV efficacy of negatively charged human serum albumin is antagonized by heparin

    NARCIS (Netherlands)

    Swart, P J; Sun, C S; Kuipers, M E; Asuncion, C; Josephs, S; Smit, C; Meijer, D K

    1997-01-01

    Succinylated human serum albumin (Suc-HSA) was synthesized by treating human serum albumin with succinic anhydride, Among similar proteins and neo(glyco)proteins tested, Suc-HSA exhibits a pronounced net negative charge, a feature that largely contributes to its efficacy against replication of human

  8. Influence of intralipid on free propofol fraction assayed in human serum albumin solutions and human plasma

    Institute of Scientific and Technical Information of China (English)

    Rafal KALITYNSKI; Andrzej L DAWIDOWICZ; Jacek POSZYTEK

    2006-01-01

    Aim: It is generally assumed that only unbound drugs can reach the site of action by diffusing across the membranes and exerting pharmacological effects by interacting with receptors. Recent research has shown that the percentage of free drugs may depend on the total drug concentration. The aim of the paper is to verify whether the mentioned dependence reported for propofol also takes place in plasma and human serum albumin samples in the presence of intralipid-the medium used as a vehicle for propofol infusions and a parenteral nutrition agent. Methods: Artificial plasma samples and human plasma were spiked with intralipid or ethanolic solutions of propofol. The samples were then assayed for free propofol concentration using ultrafiltration and high performance liquid chromatography with fluorimetric detection. Results: The decrease of the total drug concentration results in free propofol fraction increase, irrespectively of the used type of propofol solvent and sample type. The addition of intralipid causes the lowering of the overall free drug fraction with respect to the samples spiked with ethanolic solutions of the drug. Conclusion: The presence of intralipid does not influence the phenomenon of free propofol fraction rise at low total drug concentration. Such a rise cannot be ignored in clinical conditions when the drug is applied for sedative, antiemetic or other low-dosage purposes.

  9. Human serum antibodies to a major defined epitope of human herpesvirus 8 small viral capsid antigen.

    Science.gov (United States)

    Tedeschi, R; De Paoli, P; Schulz, T F; Dillner, J

    1999-04-01

    The major antibody-reactive epitope of the small viral capsid antigen (sVCA) of human herpesvirus 8 (HHV-8) was defined by use of overlapping peptides. Strong IgG reactivity was found among approximately 50% of 44 human immunodeficiency virus-positive or -negative patients with Kaposi's sarcoma and 13 subjects who were seropositive by immunofluorescence assay (IFA) for the latent HHV-8 nuclear antigen. Only 1 of 106 subjects seronegative for both lytic and latent HHV-8 antigens and 10 of 81 subjects IFA-seropositive only for the lytic HHV-8 antigen had strong IgG reactivity to this epitope. Among 534 healthy Swedish women, only 1.3% were strongly seropositive. Comparison of the peptide-based and purified sVCA protein-based ELISAs found 55% sensitivity and 98% specificity. However, only 1 of 452 serum samples from healthy women was positive in both tests. In conclusion, the defined sVCA epitope was a specific, but not very sensitive, serologic marker of active HHV-8 infection. Such infection appears to be rare among Swedish women, even with sexual risk-taking behavior.

  10. Impact of infusion method on amikacin serum levels in humans.

    Science.gov (United States)

    Simon, N; Décaudin, B; Lannoy, D; Odou, M F; De Broucker, M; Barthélémy, C; Poret, E; Dubreuil, L; Odou, P

    2010-08-01

    Aminoglycosides are broad-spectrum antibiotics with peak-dependent bactericidal activity, administered by gravity infusion or for more accuracy by electronic pump infusion. The aim of this study was to assess the difference between the two systems and its pharmacokinetic impact. Twenty-four patients hospitalised for community-acquired pulmonary infections received amikacin by IV route over 1 h with a targeted peak concentration of 35 mg/L. They were randomly distributed into two groups, one receiving infusion through a pump system, the other by gravity. Amikacin serum levels were determined at the end of infusion and 24 h later. C(max) values were significantly lower with gravity than pump (40.2 +/- 12.3 vs. 50.6 +/- 17.6 mg/L, respectively; p = 0.04). Elimination half-life time, volume of distribution and clearance did not differ significantly from one group to the other. The percentage of patients who failed to achieve the targeted peak concentration was significantly higher with gravity than pump (41.7% vs. 16.7%, respectively; p infusion flow-rate provides better control over amikacin C(max). This study underlines the fact that infusion device characteristics should be added to the physiopathological information of a patient if we are to make a better estimation of pharmacokinetic parameters.

  11. Serum cytokine levels in human ascariasis and toxocariasis.

    Science.gov (United States)

    Malla, Nancy; Fomda, Bashir Ahmad; Thokar, Manzoor Ahmad

    2006-03-01

    Cytokine-mediated regulation of chronic intestinal helminth infections is well documented. The present study reports the serum cytokine responses in 38 ascariasis (stool samples positive for Ascaris lumbricoides ova) and toxocariasis (seropositive) patients, 8 ascariasis-positive and toxocariasis-seronegative patients, 22 endemic, normal, healthy subjects residing in areas hyperendemic for ascariasis and 16 normal healthy subjects residing in a low-endemic area in India. The results indicated T-helper type-2-type cytokine responses in ascariasis and toxocariasis (seropositive) and ascariasis-positive and toxocariasis-seronegative patients. The important observation was that both patients and healthy individuals in ascariasis-hyperendemic areas had significantly higher interleukin-5 (IL-5) responses than non-endemic control subjects. The altered immune responses of patients in areas hyperendemic of ascariasis may have further implications. Earlier reports suggest that the geohelminth parasites in endemic areas may modulate the immune response to oral vaccines. A critical role for IL-5 in the immune response against challenge infection consistent with the association of type-2 cytokines with vaccine-mediated protection has been reported. Furthermore, co-infection by pathogens that elicit opposing immune responses, particularly helminths vs HIV and tuberculosis, could influence the infection dynamics, progression and immunoprophylaxis of the diseases they cause. Further studies are warranted to ascertain these findings.

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

    Science.gov (United States)

    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.

  13. Radioimmunoassay for zearalenone and zearalanol in human serum: production, properties, and use of porcine antibodies

    Energy Technology Data Exchange (ETDEWEB)

    Thouvenot, D.; Morfin, R.F.

    1983-01-01

    To produce antigens susceptible to raise antibodies for resorcylic acid lactones, the 6'-carboxymethyloxime derivatives of zearalenone and zearalanone were bound to bovine serum albumin. Pigs could be immunized by using these antigens, the best titer in antibodies being obtained with the zearalenone antigen. The procine antibodies were specific for the resorcylic acid lactones of structural resemblance with zearalenone. This specificity made the antibodies usable for a radioimmunoassay of zearalenone and zearalanol, which may be found in human and animal sera. The range of the assay was between 0.25 and 10 ng. The limit of detection was 5 ppb (5 ng/ml) in human serum.

  14. Analysis of PAHs in human serum using cloud point extraction and LC/MS

    Energy Technology Data Exchange (ETDEWEB)

    Sirimanne, S.R.; Ma, Li; McClure, C. [National Center for Health, Environmental Health and Lab. Sciences, Atlanta, GA (United States)

    1995-12-31

    In these laboratories, the authors assess human exposure to environmental toxicants by quantifying the levels of target toxicants in serum of exposed individuals. The levels of target analytes are often in trace levels and hence preconcentration and cleanup are prerequisites to the analysis of these trace toxicants. The authors have identified cloud point extraction as an impressive alternative to conventional solvent extraction due its beneficial properties and the greater extraction efficiencies. The authors report for the first time, extraction of PAHs from human serum using cloud point technology and analysis by HPLC.

  15. Detection and identification of individual antigen molecules in human serum with pulsed semiconductor lasers

    Science.gov (United States)

    Sauer, M.; Zander, C.; Müller, R.; Ullrich, B.; Drexhage, K. H.; Kaul, S.; Wolfrum, J.

    1997-09-01

    The fluorescence bursts of individual antibody molecules BM-7 (IgG1) labeled with single dye molecules were detected and identified by the characteristic fluorescence lifetimes of the dyes Cy5 (1.5 ns) and JA169 (2.7 ns) directly in neat human serum. Fluorescence excitation was performed by a short-pulse (FWHMmucine (MUC1) was detected in neat human serum by single-molecule events containing both fluorescence lifetimes indicating specific binding of both antibody molecules (Cy5-BM-7 and JA169-BM-7). The sensitivity achieved allows the detection of antigens at concentrations below 10-11 M without separation steps.

  16. Competitive Protein Adsorption of Albumin and Immunoglobulin G from Human Serum onto Polymer Surfaces

    DEFF Research Database (Denmark)

    Holmberg, Maria; Hou, Xiaolin

    2010-01-01

    Competitive protein adsorption from human serum onto unmodified polyethylene terephthalate (PET) surfaces and plasma-polymerized PET surfaces, using the monomer diethylene glycol vinyl ether (DEGVE), has been investigated using radioactive labeling. Albumin and immunoglobulin G (IgG) labeled...... with two different iodine isotopes have been added to human serum solutions of different concentrations, and adsorption has been performed using adsorption times from approximately 5 s to 24 h. DEGVE surfaces showed indications of being nonfouling regarding albumin and IgG adsorption during competitive...

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

    Science.gov (United States)

    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. Characterization of Acute Renal Allograft Rejection by Human Serum Proteomic Analysis

    Institute of Scientific and Technical Information of China (English)

    Ying GAO; Ke WU; Yi XU; Hongmin ZHOU; Wentao HE; Weina ZHANG; Lanjun CAI; Xingguang LIN; Zemin FANG; Zhenlong LUO; Hui GUO; Zhonghua CHEN

    2009-01-01

    To identify acute renal allograft rejection biomarkers in human serum, two-dimensional differential in-gel electrophoresis (2-D DIGE) and reversed phase high-performance liquid chromatog-raphy (RP-HPLC) followed by electrospray ionization mass spectrometry (ESI-MS) were used. Serum samples from renal allograft patients and normal volunteers were divided into three groups: acute rejec-tion (AR), stable renal function (SRF) and normal volunteer (N). Serum samples were firstly processed using Multiple Affinity Removal Column to selectively remove the highest abundance proteins. Differ-entially expressed proteins were analyzed using 2-D DIGE. These differential protein spots were ex-cised, digested by trypsin, and identified by RP-HPLC-ESI/MS. Twenty-two differentially expressed proteins were identified in serum from AR group. These proteins included complement C9 precursor,apolipoprotein A-Ⅳ precursor, vitamin D-binding protein precursor, beta-2-glycoprotein 1 precursor,etc. Vitamin D-binding protein, one of these proteins, was confirmed by ELISA in the independent set of serum samples. In conclusion, the differentially expressed proteins as serum biomarker candidates may provide the basis of acute rejection noninvasive diagnosis. Confirmed vitamin D-binding protein may be one of serum biomarkers of acute rejection. Furthermore, it may provide great insights into un-derstanding the mechanisms and potential treatment strategy of acute rejection.

  19. Effect of calcium phosphate crystals induced by uremic serum on calcification of human aortic smooth muscle cells

    Institute of Scientific and Technical Information of China (English)

    刘曜蓉

    2013-01-01

    Objective To investigate the impact of calcium phosphate crystals induced by uremic serum on calcification of human aortic smooth muscle cells (HASMCs) .Methods Uremic serum was incubated at 37℃for 3days.Calcium phosphate crystals and uremic supernatant were isolated from uremic serum by ultracentrifugation.

  20. SPE-HPLC purification of endocrine disrupting compounds from human serum for assessment of xenoestrogenic activity

    DEFF Research Database (Denmark)

    Hjelmborg, P.S.; Ghisari, Mandana; Bonefeld-Jørgensen, Eva

    2006-01-01

    Assessment of xenoestrogenic activity in human serum samples requires the removal of endogenous sex hormones to assure that the activity measured originates from xenobiotic compounds only. Serum samples representing high, medium and lower accumulation of persistent organic pollutants (POPs) were...... extracted using solid-phase extraction (SPE) followed by normal-phase high-performance liquid chromatography (NP-HPLC) for separation of POPs from endogenous hormones. The recovery of polychlorinated biphenyl (PCB) congeners in spiked serum samples was up to 86 %, making the extraction method suitable...... measured by ERE-CALUX was validated and considered to be a valuable tool to assess the combined ER effect of lipophilic serum POPs where additive/synergistic and agonistic/antagonistic effects are integrated giving an overall estimate of exposure and bioactivity....

  1. Are typical human serum BPA concentrations measurable and sufficient to be estrogenic in the general population?

    Science.gov (United States)

    Teeguarden, Justin; Hanson-Drury, Sesha; Fisher, Jeffrey W; Doerge, Daniel R

    2013-12-01

    Mammalian estrogen receptors modulate many physiological processes. Chemicals with structural features similar to estrogens can interact with estrogen receptors to produce biological effects similar to those caused by endogenous estrogens in the body. Bisphenol A (BPA) is a structural analogue of estrogen that binds to estrogen receptors. Exposure to BPA in humans is virtually ubiquitous in industrialized societies, but BPA is rapidly detoxified by metabolism and does not accumulate in the body. Whether or not serum concentrations of BPA in humans are sufficiently high to disrupt normal estrogen-related biology is the subject of intense political and scientific debate. Here we show a convergence of robust methods for measuring or calculating serum concentrations of BPA in humans from 93 published studies of more than 30,000 individuals in 19 countries across all life stages. Typical serum BPA concentrations are orders of magnitude lower than levels measurable by modern analytical methods and below concentrations required to occupy more than 0.0009% of Type II Estrogen Binding Sites, GPR30, ERα or ERβ receptors. Occupancies would be higher, but ≤0.04%, for the highest affinity receptor, ERRγ. Our results show limited or no potential for estrogenicity in humans, and question reports of measurable BPA in human serum.

  2. Biparametric multicommutated flow analysis system for determination of human serum phosphoesterase activity.

    Science.gov (United States)

    Tymecki, Łukasz; Strzelak, Kamil; Koncki, Robert

    2013-10-03

    A multicommutated flow analysis (MCFA) system constructed of microsolenoid valves and pumps offering simultaneous determination of activity of acid phosphatase (ACP) and alkaline phosphatase (ALP) in human serum samples has been developed. The MCFA system is based on optoelectronic flow-through detector made of two light emitting diodes and operating according to paired emitter detector diode (PEDD) principle. This photometric PEDD device has been dedicated for detection of p-nitrophenol (NP) generated in the course of enzymatic hydrolysis of p-nitrophenyl phosphate and optimized for the determination of NP in human serum samples. The developed PEDD-based MCFA system allows independent optimization of conditions for reaction and detection steps of photometric ACP and ALP bioassays. Moreover, it allows elimination of photometric interferences from serum matrix components according to two-points kinetic mode of measurement. The single measurement cycle takes 12 min, consists of four measurements (two for each phosphoesterase) and enables determination of serum ACP and ALP activities at physiological and pathological levels. The real analytical utility of the developed MCFA system has been confirmed by analysis of control sera as well as real human serum samples from healthy persons and oncological patients. Copyright © 2013 Elsevier B.V. All rights reserved.

  3. "Humanized" stem cell culture techniques: the animal serum controversy.

    Science.gov (United States)

    Tekkatte, Chandana; Gunasingh, Gency Ponrose; Cherian, K M; Sankaranarayanan, Kavitha

    2011-01-01

    Cellular therapy is reaching a pinnacle with an understanding of the potential of human mesenchymal stem cells (hMSCs) to regenerate damaged tissue in the body. The limited numbers of these hMSCs in currently identified sources, like bone marrow, adipose tissue, and so forth, bring forth the need for their in vitro culture/expansion. However, the extensive usage of supplements containing xenogeneic components in the expansion-media might pose a risk to the post-transplantation safety of patients. This warrants the necessity to identify and develop chemically defined or "humanized" supplements which would make in vitro cultured/processed cells relatively safer for transplantation in regenerative medicine. In this paper, we outline the various caveats associated with conventionally used supplements of xenogenic origin and also portray the possible alternatives/additives which could one day herald the dawn of a new era in the translation of in vitro cultured cells to therapeutic interventions.

  4. Chromatofocusing of apolipoproteins from human serum high density lipoprotein.

    Science.gov (United States)

    Knipping, G; Steyrer, E; Holasek, A

    1984-01-01

    Human HDL was delipidated and the apolipoproteins were fractionated by chromatofocusing. Chromatofocusing, which separates proteins due to their differing isoelectric points, resulted in 8 peaks with corresponding pI values of 7.40, 6.92, 6.64, 5.48, 5.30, 5.18, 4.92 and 4.63. By one single chromatofocusing run four apolipoproteins were obtained in pure form. Two additional polypeptides could be purified during the desalting step using phenyl-Sepharose.

  5. A Novel Immunological Assay for Hepcidin Quantification in Human Serum

    OpenAIRE

    Vasiliki Koliaraki; Martha Marinou; Vassilakopoulos, Theodoros P.; Eustathios Vavourakis; Emmanuel Tsochatzis; Pangalis, Gerassimos A.; George Papatheodoridis; Alexandra Stamoulakatou; Dorine W Swinkels; George Papanikolaou; Avgi Mamalaki

    2009-01-01

    BACKGROUND: Hepcidin is a 25-aminoacid cysteine-rich iron regulating peptide. Increased hepcidin concentrations lead to iron sequestration in macrophages, contributing to the pathogenesis of anaemia of chronic disease whereas decreased hepcidin is observed in iron deficiency and primary iron overload diseases such as hereditary hemochromatosis. Hepcidin quantification in human blood or urine may provide further insights for the pathogenesis of disorders of iron homeostasis and might prove a v...

  6. Crystallization And Preliminary Crystallographic Analysis of Recombinant Human Galectin-1

    Energy Technology Data Exchange (ETDEWEB)

    Scott, S.A.; Scott, K.; Blanchard, H.

    2009-06-04

    Human galectin-1 has been cloned, expressed in E. coli, purified and crystallized in the presence of both lactose (ligand) and {beta}-mercaptoethanol under six different conditions. The X-ray diffraction data obtained have enabled the assignment of unit-cell parameters for two novel crystal forms of human galectin-1.

  7. Human Serum Albumin (HSA) Suppresses the Effects of Glycerol Monolaurate (GML) on Human T Cell Activation and Function

    Science.gov (United States)

    Zhang, Michael S.; Houtman, Jon C. D.

    2016-01-01

    Glycerol monolaurate (GML) is a monoglyceride with well characterized anti-microbial properties. Because of these properties, GML is widely used in food, cosmetics, and personal care products and currently being tested as a therapeutic for menstrual associated toxic shock syndrome, superficial wound infections, and HIV transmission. Recently, we have described that GML potently suppresses select T cell receptor (TCR)-induced signaling events, leading to reduced human T cell effector functions. However, how soluble host factors present in the blood and at sites of infection affect GML-mediated human T cell suppression is unknown. In this study, we have characterized how human serum albumin (HSA) affects GML-induced inhibition of human T cells. We found that HSA and other serum albumins bind to 12 carbon acyl side chain of GML at low micromolar affinities and restores the TCR-induced formation of LAT, PLC-γ1, and AKT microclusters at the plasma membrane. Additionally, HSA reverses GML mediated inhibition of AKT phosphorylation and partially restores cytokine production in GML treated cells. Our data reveal that HSA, one of the most abundant proteins in the human serum and at sites of infections, potently reverses the suppression of human T cells by GML. This suggests that GML-driven human T cell suppression depends upon the local tissue environment, with albumin concentration being a major determinant of GML function. PMID:27764189

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

    Science.gov (United States)

    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...

  9. Evidence for the existence of mammalian acetoacetate decarboxylase: with special reference to human blood serum

    NARCIS (Netherlands)

    Stekelenburg, Gerard J. van; Koorevaar, Gerrit

    In this article evidence is presented for the existence of mammalian acetoacetate decarboxylase (acetoacetate carboxy-lyase: E.G. 4.1.1.4). From experiments with human blood serum the presence of a non-ultrafiltrable activator, accelerating the decomposition of acetoacetate into acetone and carbon

  10. A spectroscopic and molecular docking approach on the binding of tinzaparin sodium with human serum albumin

    Science.gov (United States)

    Abdullah, Saleh M. S.; Fatma, Sana; Rabbani, Gulam; Ashraf, Jalaluddin M.

    2017-01-01

    Protein bound toxins are poorly removed by conventional extracorporeal therapies. Venous thromboembolism (VTE) is a major cause of morbidity and mortality in patients with cancer. The interaction between tinzaparin, an inhibitor of angiotensin converting enzyme and human serum albumin, a principal plasma protein in the liver has been investigated in vitro under a simulated physiological condition by UV-vis spectrophotometry and fluorescence spectrometry. The intrinsic fluorescence intensity of human serum albumin was strongly quenched by tinzaparin (TP). The binding constants and binding stoichiometry can be calculated from the data obtained from fluorescence quenching experiments. The negative value of ΔG° reveals that the binding process is a spontaneous process. Thermodynamic analysis shows that the HSA-TP complex formation occurs via hydrogen bonds, hydrophobic interactions and undergoes slight structural changes as evident by far-UV CD. It indicated that the hydrophobic interactions play a main role in the binding of TP to human serum albumin. In addition, the distance between TP (acceptor) and tryptophan residues of human serum albumin (donor) was estimated to be 2.21 nm according to the Förster's resonance energy transfer theory. For the deeper understanding of the interaction, thermodynamic, and molecular docking studies were performed as well. Our docking results suggest that TP forms stable complex with HSA (Kb ∼ 104) and its primary binding site is located in subdomain IIA (Sudlow Site I). The results obtained herein will be of biological significance in pharmacology and clinical medicine.

  11. Covalent binding of nitrogen mustards to the cysteine-34 residue in human serum albumin

    NARCIS (Netherlands)

    Noort, D.; Hulst, A.G.; Jansen, R.

    2002-01-01

    Covalent binding of various clinically important nitrogen mustards to the cysteine-34 residue of human serum albumin, in vitro and in vivo, is demonstrated. A rapid method for detection of these adducts is presented, based on liquid chromatography-tandem mass spectrometry analysis of the adducted

  12. Platelet-rich plasma can replace fetal bovine serum in human meniscus cell cultures

    NARCIS (Netherlands)

    Gonzales, V.K.; Mulder, E.L.W. de; Boer, T. den; Hannink, G.; Tienen, T.G. van; Heerde, W.L. van; Buma, P.

    2013-01-01

    Concerns over fetal bovine serum (FBS) limit the clinical application of cultured tissue-engineered constructs. Therefore, we investigated if platelet-rich plasma (PRP) can fully replace FBS for meniscus tissue engineering purposes. Human PRP and platelet-poor plasma (PPP) were isolated from three h

  13. Linear regression of postevacuation serum human chorionic gonadotropin concentrations predicts postmolar gestational trophoblastic neoplasia

    NARCIS (Netherlands)

    Lybol, C.; Sweep, F.C.; Ottevanger, P.B.; Massuger, L.F.A.G.; Thomas, C.M.G.

    2013-01-01

    OBJECTIVE: Currently, human chorionic gonadotropin (hCG) follow-up after evacuation of hydatidiform moles is essential to identify patients requiring chemotherapeutic treatment for gestational trophoblastic neoplasia (GTN). We propose a model based on linear regression of postevacuation serum hCG co

  14. Strategy to tether organometallic ruthenium-arene anticancer compounds to recombinant human serum albumin.

    Science.gov (United States)

    Ang, Wee Han; Daldini, Elisa; Juillerat-Jeanneret, Lucienne; Dyson, Paul J

    2007-10-29

    In order to utilize macromolecules for drug targeting and delivery, a strategy to tether organometallic ruthenium-arene drugs to carrier protein molecules was developed. The approach involves the design of a drug fragment capable of conjugating to linker molecules on a modified carrier protein via hydrazone bond formation. The proof-of-concept using recombinant human serum albumin is described.

  15. Targeted non-covalent self-assembled nanoparticles based on human serum albumin

    NARCIS (Netherlands)

    Bunschoten, Anton; Buckle, Tessa; Kuil, Joeri; Luker, Gary D.; Luker, Kathryn E.; Nieweg, Omgo; van Leeuwen, Fijs W. B.

    2012-01-01

    Human serum albumin (HSA) is a biological nanocarrier that forms non-covalent complexes with a number of synthetic and biomolecules. Previously we demonstrated radiolabeled HSA-based nanoparticles can form non-covalent complexes with fluorescent cyanine dyes yielding imaging agents for surgical guid

  16. Assay of Cysteine in Human Serum with Quinine-Ce4+ Chemiluminescence System

    Institute of Scientific and Technical Information of China (English)

    2002-01-01

    A sensitive and selective chemiluminescence (CL) method was developed for the determination of cysteine. This method is based on that the weak CL of cysteine oxidized with cerium (IV) can be greatly enhanced by quinine, and the total cysteine in human serum can be detected through simply diluting with water, showing a simpler analytical characteristic.

  17. Human skin condition and its associations with nutrient concentrations in serum and diet

    NARCIS (Netherlands)

    Boelsma, E.; Vijver, L.P.L. van de; Goldbohm, R.A.; Klöpping-Ketelaars, I.A.A.; Hendriks, H.F.J.; Roza, L.

    2003-01-01

    Background: Nutritional factors exert promising actions on the skin, but only scant information is available on the modulating effects of physiologic concentrations of nutrients on the skin condition of humans. Objective: The objective was to evaluate whether nutrient concentrations in serum and die

  18. Evidence for the existence of mammalian acetoacetate decarboxylase: with special reference to human blood serum

    NARCIS (Netherlands)

    Stekelenburg, Gerard J. van; Koorevaar, Gerrit

    1972-01-01

    In this article evidence is presented for the existence of mammalian acetoacetate decarboxylase (acetoacetate carboxy-lyase: E.G. 4.1.1.4). From experiments with human blood serum the presence of a non-ultrafiltrable activator, accelerating the decomposition of acetoacetate into acetone and carbon d

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

    Science.gov (United States)

    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...

  20. Ionization of tyrosine residues in human serum albumin and in its complexes with bilirubin and laurate

    DEFF Research Database (Denmark)

    Honoré, B; Brodersen, R

    1992-01-01

    Spectrophotometric titration of human serum albumin indicates that ionization of the 18 tyrosine residues takes place between pH 9 and 12.7. A Hill plot indicates that protons dissociate co-operatively from tyrosine residues, in pure albumin between pH 11.0 and 11.4 with a Hill coefficient 1.7, a...

  1. Human skin condition and its associations with nutrient concentrations in serum and diet

    NARCIS (Netherlands)

    Boelsma, E.; Vijver, L.P.L. van de; Goldbohm, R.A.; Klöpping-Ketelaars, I.A.A.; Hendriks, H.F.J.; Roza, L.

    2003-01-01

    Background: Nutritional factors exert promising actions on the skin, but only scant information is available on the modulating effects of physiologic concentrations of nutrients on the skin condition of humans. Objective: The objective was to evaluate whether nutrient concentrations in serum and

  2. Fractionation of human serum lipoproteins and simultaneous enzymatic determination of cholesterol and triglycerides

    NARCIS (Netherlands)

    Qureshi, R.N.; Kok, W.T.; Schoenmakers, P.J.

    2009-01-01

    A method based on Asymmetric Flow Field-Flow Fractionation (AF4) was developed to separate different types of lipoproteins from human serum. The emphasis in the method optimization was on the possibilities to characterize the largest lipoprotein fractions (LDL and VLDL), which is usually not

  3. Urinary estrogen excretion and concentration of serum human placental lactogen in pregnancies following legally induced abortion

    DEFF Research Database (Denmark)

    Obel, E B; Madsen, Mette

    1980-01-01

    Feto-placental function was assessed by 24-hour excretion of estrogen in urine and by the concentration of human Placental Lactogen (hPL) in serum in pregnant women whose previous pregnancy was terminated by legally induced abortion. The mean 24-hour excretion of estrogens in urine and the mean c...

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

    Science.gov (United States)

    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.

  5. Effects of topical human amniotic fluid and human serum in a mouse model of keratoconjunctivitis sicca.

    Science.gov (United States)

    Quinto, Guilherme G; Camacho, Walter; Castro-Combs, Juan; Li, Li; Martins, Suy Anne R; Wittmann, Priscila; Campos, Mauro; Behrens, Ashley

    2012-04-01

    To compare the effects of topical human amniotic fluid (HAF), topical human serum (HS), and topical artificial tears in a mouse model of dry eye. Thirty C57BL/6 mice were divided into 3 treatment groups: HAF, HS, and preservative-free artificial tears. Dry eye was induced by an injection of botulinum toxin B (BTX-B) into the lacrimal gland. Tear production and ocular surface fluorescein staining were evaluated in each mouse at 6 time points during a 4-week period. Goblet cell density was assessed in stained histological sections. Apoptotic keratocytes were evaluated by terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling test assay. A significant decrease in tear production was observed 3 days after BTX-B injection in all groups. At week 1, the HAF and HS groups had improved tear production compared with the control group (P < 0.001 and P = 0.003, respectively). HAF had a significantly improved fluorescein staining score compared with the HS (P = 0.043) and control (P = 0.007) groups at week 2. Goblet cell density was significantly decreased in the control group compared with the HAF and HS groups (P < 0.001). No difference in the amount of terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling-positive keratocytes was observed among the groups. HAF was superior to HS and artificial tears for improving corneal staining within 2 weeks of therapy in this induced mouse model of keratoconjunctivitis sicca. Clinical studies are needed to ascertain the benefits of these therapies in patients with ocular surface disorders associated with dry eye.

  6. [Development and evaluation of a serological protocol of fluorescence polarization for the preliminary study of Brucella spp antibodies in humans].

    Science.gov (United States)

    Sánchez-Villalobos, Alfredo; Urdaneta-Fernández, Margelys; Rubio-Fuenmayor, Elí; Molero-Saras, Gladys; Luzardo-Charris, Carlos; Corona-Mengual, Carlos

    2011-03-01

    In order to show the development and scope of a serological analysis method based on fluorescence polarization assay (FPA) from a drop of blood obtained by the capillary technique, a Brucella antibody assay was performed on a group of 321 high-risk workers. The results were compared with data from the analysis of blood serum by FPA and a competitive enzyme immunoassay (ELISA-c). The number of concordance was 318 (99.06%), and discordant 3 (0.93%), which were negative in serum by fluorescence polarization (FPAs) and ELISA-c, but positive with capillary FPA (FPAc). The comparative results FPAc were: sensitivity 100%; specificity: 99.05%; positive predictive value 66.67%; negative predictive value 100.0%; false positive rate: 0.95%; false negative rate: 0%; accuracy: 98.0%; odds ratio: 203.00. The youden J for both FPA methods was 0.667. The identification was considered reliable and the correlation of both procedures, FPA and ELISA-c, was no statistically different (P > 0.05%), which allows to highly recommend the study implementation of human brucellosis with capillary blood as a preliminary method.

  7. HPLC separation of human serum albumin isoforms based on their isoelectric points

    OpenAIRE

    Turell, Lucía; Botti, Horacio; Bonilla, Lucía; Torres, María José; Schopfer, Francisco; Freeman, Bruce A.; Armas, Larissa; Ricciardi, Alejandro; Alvarez, Beatriz; Radi, Rafael

    2013-01-01

    Human serum albumin (HSA) is the most abundant protein in plasma. Cys34, the only free Cys residue, is the predominant plasma thiol and a relevant sacrificial antioxidant. Both in vivo circulating HSA and pharmaceutical preparations are heterogeneous with respect to the oxidation state of Cys34. In this work, we developed an external pH gradient chromatofocusing procedure that allows the analysis of the oxidation status of HSA in human plasma and biopharmaceutical products based on the differ...

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

    Science.gov (United States)

    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.

  9. Effects of consumption of probiotics and prebiotics on serum lipid levels in humans.

    Science.gov (United States)

    Pereira, Dora I A; Gibson, Glenn R

    2002-01-01

    The objective of this article is to review existing studies concerning the effects of probiotics and prebiotics on serum cholesterol concentrations, with particular attention on the possible mechanisms of their action. Although not without exception, results from animal and human studies suggest a moderate cholesterol-lowering action of dairy products fermented with appropriate strain(s) of lactic acid bacteria and bifidobacteria. Mechanistically, probiotic bacteria ferment food-derived indigestible carbohydrates to produce short-chain fatty acids in the gut, which can then cause a decrease in the systemic levels of blood lipids by inhibiting hepatic cholesterol synthesis and/or redistributing cholesterol from plasma to the liver. Furthermore, some bacteria may interfere with cholesterol absorption from the gut by deconjugating bile salts and therefore affecting the metabolism of cholesterol, or by directly assimilating cholesterol. For prebiotic substances, the majority of studies have been done with the fructooligosaccharides inulin and oligofructose, and although convincing lipid-lowering effects have been observed in animals, high dose levels had to be used. Reports in humans are few in number. In studies conducted in normal-lipidemic subjects, two reported no effect of inulin or oligofructose on serum lipids, whereas two others reported a significant reduction in serum triglycerides (19 and 27%, respectively) with more modest changes in serum total and LDL cholesterol. At present, data suggest that in hyperlipidemic subjects, any effects that do occur result primarily in reductions in cholesterol, whereas in normal lipidemic subjects, effects on serum triglycerides are the dominant feature.

  10. Extending the Serum Half-Life of G-CSF via Fusion with the Domain III of Human Serum Albumin

    Directory of Open Access Journals (Sweden)

    Shuqiang Zhao

    2013-01-01

    Full Text Available Protein fusion technology is one of the most commonly used methods to extend the half-life of therapeutic proteins. In this study, in order to prolong the half-life of Granulocyte colony stimulating factor (G-CSF, the domain III of human serum albumin (3DHSA was genetically fused to the N-terminal of G-CSF. The 3DHSA-G-CSF fusion gene was cloned into pPICZαA along with the open reading frame of the α-factor signal under the control of the AOX1 promoter. The recombinant expression vector was transformed into Pichia pastoris GS115, and the recombinant strains were screened by SDS-PAGE. As expected, the 3DHSA-G-CSF showed high binding affinity with HSA antibody and G-CSF antibody, and the natural N-terminal of 3DHSA was detected by N-terminal sequencing. The bioactivity and pharmacokinetic studies of 3DHSA-G-CSF were respectively determined using neutropenia model mice and human G-CSF ELISA kit. The results demonstrated that 3DHSA-G-CSF has the ability to increase the peripheral white blood cell (WBC counts of neutropenia model mice, and the half-life of 3DHSA-G-CSF is longer than that of native G-CSF. In conclusion, 3DHSA can be used to extend the half-life of G-CSF.

  11. Comparative evaluation of various solid phases for the development of coated tube assays for the estimation of progesterone in human serum, bovine serum and bovine milk

    Energy Technology Data Exchange (ETDEWEB)

    Karir, Tarveen [Board of Radiation and Isotope Technology (BRIT), BARC Vashi Complex, Department of Atomic Energy (DAE), Navi Mumbai 400 705 (India); Samuel, Grace [Board of Radiation and Isotope Technology (BRIT), BARC Vashi Complex, Department of Atomic Energy (DAE), Navi Mumbai 400 705 (India)], E-mail: grace_samuel1955@rediffmail.com; Sivaprasad, N.; Venkatesh, Meera [Board of Radiation and Isotope Technology (BRIT), BARC Vashi Complex, Department of Atomic Energy (DAE), Navi Mumbai 400 705 (India)

    2009-11-15

    Immobilization of progesterone antibody using three polystyrene surfaces and two progesterone radiotracers for use in the development of a coated tube assay for the evaluation of progesterone levels in human serum, bovine serum and bovine milk was studied. The selection of the solid phase and the tracers were based on the maximum binding, non-specific binding, sensitivity and percentage recovery. Amongst the polystyrene tubes studied, streptavidin coated tubes showed the acceptable assay features such as low non-specific binding (0.5-1.0%), adequate sensitivity (0.13-0.16 ng/ml) and recovery (85-115%) for all the three sample matrices, human serum, bovine serum and bovine milk.

  12. 1β,25-Dihydroxyvitamin D3: A new vitamin D metabolite in human serum.

    Science.gov (United States)

    Pauwels, Steven; Jans, Ivo; Billen, Jaak; Heijboer, Annemieke; Verstuyf, Annemieke; Carmeliet, Geert; Mathieu, Chantal; Maestro, Miguel; Waelkens, Etienne; Evenepoel, Pieter; Bouillon, Roger; Vanderschueren, Dirk; Vermeersch, Pieter

    2017-10-01

    The measurement of 1α,25(OH)2D3 in human serum poses a true challenge as concentrations are very low and structurally similar metabolites can interfere. During optimization of our in-house LC-MSMS method for serum 1α,25(OH)2D3 a previously co-eluting isobaric interference was separated. The isobar was identified as 1β,25(OH)2D3 by comparing retention time and fragmentation spectra to standards (other isobaric dihydroxylated vitamin D3 analogs). 1β,25(OH)2D3 showed specific cluster formation (water), not present in 1α,25(OH)2D3. 1β,25(OH)2D3 was measured in serum of apparently healthy human volunteers (n=20), patients with high serum 25-hydroxyvitamin D [25(OH)D] concentrations (>50ng/mL) (n=33 among which 4 with very high levels (>150ng/mL)) and patients with kidney failure (n=68; 39 stage 1-3, 29 stage 4-5). Pearson's r was calculated for correlations and Mann-Whitney statistic to compare group medians. Median serum 1β,25(OH)2D3 was 11pg/mL in apparently healthy volunteers and increased to 20pg/mL for serum 25(OH)D concentrations above 80ng/mL (n=22) (pD3 concentrations were significantly correlated to serum 25(OH)D concentrations (r=0.85) for the combined results from healthy volunteers and patient sera (n=53) (pD3 was 7pg/mL and not different from the median level in healthy volunteers (p=0.06). The median concentration did not vary with different stages. We present evidence for the widespread presence of 1β,25(OH)2D3, a new vitamin D metabolite, in human serum. The level increases with rising serum 25(OH)D concentrations and is particularly high in patients with very high 25(OH)D levels. We previously demonstrated that 1β,25(OH)2D3 is a poor genomic agonist but a potent non-genomic antagonist of 1α,25(OH)2D3. The clinical implications of the presence of this analog therefore require further exploration. Copyright © 2017 Elsevier Ltd. All rights reserved.

  13. Serum α-klotho concentrations during preimplantation can predict aging or quality of human oocytes and clinical pregnancy rates

    OpenAIRE

    Takemura, Takashi; Okabe, Midori

    2016-01-01

    Background To discover simple biomarkers to evaluate the aging or quality of human oocytes and clinical pregnancy rates is needed. However, the association among serum α-klotho concentrations during preimplantation, the aging or quality of human oocytes and clinical pregnancy rates has not been investigated. Findings The serum α-klotho concentrations during preimplantation decreased due to aging (p 

  14. Substantially improved pharmacokinetics of recombinant human butyrylcholinesterase by fusion to human serum albumin

    Directory of Open Access Journals (Sweden)

    Pierson Janice

    2008-05-01

    Full Text Available Abstract Background Human butyrylcholinesterase (huBChE has been shown to be an effective antidote against multiple LD50 of organophosphorus compounds. A prerequisite for such use of huBChE is a prolonged circulatory half-life. This study was undertaken to produce recombinant huBChE fused to human serum albumin (hSA and characterize the fusion protein. Results Secretion level of the fusion protein produced in vitro in BHK cells was ~30 mg/liter. Transgenic mice and goats generated with the fusion constructs expressed in their milk a bioactive protein at concentrations of 0.04–1.1 g/liter. BChE activity gel staining and a size exclusion chromatography (SEC-HPLC revealed that the fusion protein consisted of predominant dimers and some monomers. The protein was confirmed to have expected molecular mass of ~150 kDa by Western blot. The purified fusion protein produced in vitro was injected intravenously into juvenile pigs for pharmacokinetic study. Analysis of a series of blood samples using the Ellman assay revealed a substantial enhancement of the plasma half-life of the fusion protein (~32 h when compared with a transgenically produced huBChE preparation containing >70% tetramer (~3 h. In vitro nerve agent binding and inhibition experiments indicated that the fusion protein in the milk of transgenic mice had similar inhibition characteristics compared to human plasma BChE against the nerve agents tested. Conclusion Both the pharmacokinetic study and the in vitro nerve agent binding and inhibition assay suggested that a fusion protein retaining both properties of huBChE and hSA is produced in vitro and in vivo. The production of the fusion protein in the milk of transgenic goats provided further evidence that sufficient quantities of BChE/hSA can be produced to serve as a cost-effective and reliable source of BChE for prophylaxis and post-exposure treatment.

  15. Preliminary Findings of Serum Creatinine and Estimated Glomerular Filtration Rate (eGFR) in Adolescents with Intellectual Disabilities

    Science.gov (United States)

    Lin, Jin-Ding; Lin, Lan-Ping; Hsieh, Molly; Lin, Pei-Ying

    2010-01-01

    The present study aimed to describe the kidney function profile--serum creatinine and estimated glomerular filtration rate (eGFR), and to examine the relationships of predisposing factors to abnormal serum creatinine in people with intellectual disabilities (ID). Data were collected by a cross-sectional study of 827 aged 15-18 years adolescents…

  16. Sensitive Procedure for Rapid Detection of Human Brucellosis, Based on PCR Method in Contaminated Serum Samples

    Directory of Open Access Journals (Sweden)

    Eslam Ghezelsofla

    2013-07-01

    Full Text Available AbstractBackground and objective: Brucellosis is a zoonosis transmittable to humans poses a significant public health problem in many developing countries and requires rapid and accurate diagnostic methods. Here, our aim was to develop a diagnostic polymerase chain reaction (PCR assay in artificially contaminated serum samples as a model for rapid and accurate laboratory confirmation of human brucellosis. Material and methods: In this study, initially the standard Brucella abortus strain (2308 were cultured on Brucella agar medium and then colonies were inactivated by formalin 10 %. Genomic DNA was extracted from inactivated bacterial colonies. Serial dilutions of bacterial-DNA were prepared in fetal bovine serum (FBS and water and subsequently DNA extraction were repeated on these artificially contaminated samples. The two pairs of primers amplified two different fragments included in: a gene encoding an outer membrane protein (omp-2 (primers JPF/JPR and a sequence 16S rRNA of B. abortus (primers F4/R2. Results: The two primers assayed showed a difference in sensitivity for detecting Brucella DNA, ranging between 5 pg and 50 pg for artificially contaminated serum samples and 50Fg and 5 pg for contaminated control samples. Therefore, the sensitivity of PCR using F4/R2 primers was greater than the PCR using JPF/JPR primers.Conclusion: Although the sensitivity of PCR using these primers was affected by serum inhibitors, they are still the most sensitive and they could provide a useful tool for the diagnosis of human brucellosis.

  17. GC-FID determination and pharmacokinetic studies of oleanolic acid in human serum.

    Science.gov (United States)

    Rada, Mirela; Castellano, José María; Perona, Javier S; Guinda, Ángeles

    2015-11-01

    Analytical interest of OA determination in human serum has increased owing to the increasing interest in pharmaceutical research by pharmaceutical properties. A simple, specific, precise and accurate GC method with flame ionization detector (FID) developed and validated for the determination of oleanolic acid (OA) in human serum (HS). To an aliquot of HS, internal standard was added and a combination of liquid-liquid extraction with a mixture of diethyl ether-isopropyl alcohol, filtration and consecutive GC resulted in separation and quantification of OA. The organic phase was analyzed using a GC system equipped with a 30 × 0.25 mm i.d. Rtx-65TG capillary column and FID detection. Total chromatographic time was 10 min and no interfering peaks from endogenous components in blank serum were observed. The OA/internal standard peak area ratio was linearly fitted to the OA concentration (r = 0.992) over the range 10-1500 ng/mL. The mean serum extraction recovery of OA was 96.7 ± 1.0% and the lower limit of quantification based on 5 mL of serum was 10.7 ng/mL. The intra-day coefficient of variation ranged from 1.3 to 3.6% and inter-day varied from 1.4 to 4.5%. The developed method was used to study the pharmacokinetics of OA after oral administration in humans. The assay was simple, sensitive, precise and accurate for the use in the study of the mechanisms of absorption and distribution of OA in humans.

  18. Determination of trace cobalt concentrations in human serum by adsorptive stripping voltammetry.

    Science.gov (United States)

    Kajic, Petra; Milosev, Ingrid; Pihlar, Boris; Pisot, Venceslav

    2003-01-01

    The goal of our study was to develop an accurate and reliable method for determining trace cobalt concentrations in human serum. The method was used to determine cobalt in the sera of healthy persons and patients with orthopaedic implants containing cobalt - a possible source of systemic release of cobalt into the human body. This goal is of vital interest since cobalt and its compounds are classified by IARC as potentially carcinogenic to humans. We used an electrochemical method, adsorptive stripping voltammetry (AdSV), which made possible the low detection limit and high sensitivity needed for measurements in human serum. The serum was acid digested by a combination of H2SO4, HNO3 and H2O2 in a 10 mL Kjeldhal flask. The digested sample was then dissolved in 0.1 mol/L ammonia buffer, pH 9.0 +/- 0.2. The determination is based on the adsorptive collection of the complex of cobalt (II) with dimethylglyoxime on a hanging mercury drop electrode (HMDE). The optimum values of adsorption potential and time were determined to be -0.8 V and 60 s. The optimisation of the sample digestion protocol and measurement procedures ensured the reliable assessment of low cobalt concentrations, down to 0.03 microg/L. The mean concentration of serum cobalt in four healthy persons was 0.11 +/- 0.06 microg/L, and in four patients with total hip replacements 0.34 +/- 0.07 microg/L. This method will be used routinely for measuring serum cobalt levels in patients with total hip replacements.

  19. Human-Robot Emergency Response - Experimental Platform and Preliminary Dataset

    Science.gov (United States)

    2014-07-28

    Proceedings of the IEEE International Conference on Robotics and Automation, Leuven, Belgium, May 16–21 1998, pp. 3715–3720. [13] itseez, “ Opencv ,” http...function and camshift function in OpenCV [13]. In each image obtained form cameras, we first calculate back projection of a histogram model of a human. In

  20. Biocides Steering Group on human exposure assessment: A preliminary report

    NARCIS (Netherlands)

    Hemmen, J.J. van

    1999-01-01

    In a project granted by DG XI of the European Commission, it is attempted to collate experimental and theoretical data on human (workers and consumers) exposure assessment to biocidal products, and to outline the methodology for sampling and measurement. On the basis of the available evidence, appro

  1. 人血清CYFRA21-1化学发光免疫分析方法的初步建立及应用%The Preliminary Establishment and Application of Chemiluminescence Immunoassay for Detection of Human CYFRA21 -1 in Serum

    Institute of Scientific and Technical Information of China (English)

    潘慧杰; 刘红; 唐磊; 李嘉; 杨萌萌; 杨铁生; 樊春红; 杨晓林; 孙旭东

    2012-01-01

    Objective To estimate an enhanced chemiluminescence immunoassay for detection CYFRA21-1 in serum.Methods CYFRA21-1 chemiluminescence quantitative immunoassay kit was used.308 clinical samples were detected, compared with ECLA CY-FRA21-1 immunoassay.Results The sensitivity of the assay was 0.04μg/L.The linear range was (1.5 ~ ICO) μg/L, and there was no significant cross-reaction with NSE or CEA.The recovery rate of addition and dilution were both below 10% .Besides, the intra- and inter-coefficients of variations were from 90% to 110% .The 0.95 confidence interval for normal samples was 0 ~3.4μg/L (n =231) .Comparing to ECLA, the clinical accordance rate was 96.65% .Conclusion The sensitivity, specificity, stability and detection range of this method are all good enough to meet the clinical requirement, furthermore, to take the place of the foreign like products.%目的:建立人血清CYFRA21-1化学发光免疫分析方法并在临床检测中的应用.方法:使用CYFRA21-1定量测定试剂盒(化学发光法)检测308例临床血清标本,并与CYFRA21-1电化学发光全自动免疫分析方法进行对比.结果:灵敏度0.04μg/L,线性范围(1.5~100)μg/L,与NSE及CEA无交叉反应,样本中抗凝剂量的柠檬酸钠、肝素、EDTA-Na2对测定结果没有影响.添加回收率和稀释回收率为90%~110%,分析内和分析间变异系数均<10.0%.该方法正常参考值为(0~3.4)μg/L(95%可信限).ECLA测定结果与本法相比,临床总符合率为96.65%.结论:该方法灵敏度高、特异性好、稳定性强,检测范围宽,有良好的准确性和重复性,完全可替代进口化学发光试剂用于临床样本的检测.

  2. Fractionation of cystine aminopeptidases ('oxytocinase') from term human placenta and maternal serum.

    Science.gov (United States)

    Gopalaswamy, G; Balasubramaniam, N; Kanagasabapathy, A S

    1984-12-15

    Cystine aminopeptidase (EC 3.4.11.3) enzymes, present in term human placenta and maternal serum, were compared with respect to their behaviour on ion-exchange columns, Km and pH optima, chelator, metal ion and L-methionine effects, Sepharose 6B elution profiles and molecular weights. From placental extracts two activity peaks (CAS I and II) hydrolysing S-benzyl L-cysteine paranitroanilide were separated on DEAE Sephacel. Differences in properties between the two forms were evident. Maternal serum enzyme eluted from the DEAE Sephacel column in a position similar to that of placental CAS I. In addition, the maternal serum enzyme was similar in properties to placental CAS I. It is possible that of all the different cystyl aminopeptidase enzyme systems present in placental tissue, only one appears in maternal blood.

  3. Determination of barnidipine in human serum and dog plasma by HPLC with electrochemical detection.

    Science.gov (United States)

    Takamura, K; Abdel-Wadood, H M; Kusu, F; Rafaat, I H; Saleh, G A; el-Rabbat, N A; Otagiri, M

    1995-10-01

    Barnidipine is a 1,4-dihydropyridine calcium antagonist. HPLC was conducted on a polybutadiene coated alumina column using an alkaline mobile phase and an electrochemical detector to determine the content of this drug in serum and plasma. A good linear relationship between barnidipine concentration and peak height was found in 5-500 ng/ml with a correlation coefficient of 0.998. The detection limit was 1 ng/ml. The within-day and day-to-day variations were examined for control human serum. Relative standard deviation of within-day assay for serum spiked with 10 ng/ml barnidipine.HCl was 6.9% and the recovery was 104%. A pharmacokinetic study was made in which the time course of barnidipine in dog plasma was followed.

  4. Headspace Solid Phase Micro Extraction Gas Chromatographic Determination of Fenthion in Human Serum

    Directory of Open Access Journals (Sweden)

    Kyriaki Machera

    2008-05-01

    Full Text Available A simple and effective analytical procedure was developed for the determination of fenthion residues in human serum samples. The sample treatment was performed using the headspace solid-phase micro extraction with polyacrylate fiber, which has the advantage to require low amount of serum (1 mL without tedious pre-treatment. The quantification of fenthion was carried out by gas chromatography-mass spectrometry and the recoveries ranged from 79 to 104% at two spiking levels for 6 replicates. Detection and quantification limits were calculated as 1.51 and 4.54 ng/mL of serum respectively. Two fenthion metabolites − fenoxon and fenthion–sulfoxide − were also identified.

  5. Chronic hepatitis B serum promotes apoptotic damage in human renal tubular cells

    Institute of Scientific and Technical Information of China (English)

    Cun-Liang Deng; Xin-Wen Song; Hai-Jun Liang; Chen Feng; Yun-Jian Sheng; Ming-Yong Wang

    2006-01-01

    AIM: To investigate the effect of the serum of patients with chronic hepatitis B (CHB) on apoptosis of renal tubular epithelial cells in vitro and to study the role of hepatitis B virus (HBV) and transforming growth factor-β1 (TGF-β1) in the pathogenesis of hepatitis B virus associated glomerulonephritis (HBV-GN).METHODS: The levels of serum TGF-β1 were measured by specific enzyme linked immunosorbent assay (ELISA) and HBV DNA was tested by polymerase chain reaction (PCR) in 44 patients with CHB ,and 20 healthy persons as the control. The normal human kidney proximal tubular cell (HK-2) was cultured together with the sera of healthy persons, CHB patients with HBV-DNA negative(20 cases) and HBV-DNA positive (24 cases) for up to 72 h. Apoptosis and Fas expression of the HK-2 were detected by flow cytometer.RESULTS: The apoptosis rate and Fas expression of HK-2 cells were significantly higher in HBV DNA positive serum group 19.01±5.85% and 17.58±8.35%, HBV DNA negative serum group 8.12±2.80% and 6.96 ± 2.76% than those in control group 4.25±0.65% and 2.33 ± 1.09%, respectively (P < 0.01). The apoptosis rate and Fas expression of HK-2 in HBV DNA positive serum group was significantly higher than those in HBV DNA negative serum (P < 0.01). Apoptosis rate of HK-2 cells in HBV DNA positive serum group was positively correlated with the level of HBV-DNA (r = 0.657). The level of serum TGF-β1 in CHB group was 163.05 ± 91.35 μg/L, significantly higher as compared with 81.40 ± 40.75 μg/L in the control group (P < 0.01).CONCLUSION: The serum of patients with chronic hepatitis B promotes apoptotic damage in human renal tubular cells by triggering a pathway of Fas up-regulation. HBV and TGF-β1 may play important roles in the mechanism of hepatitis B virus associated glomerulonephritis.

  6. Detection of novel sequences related to african Swine Fever virus in human serum and sewage.

    Science.gov (United States)

    Loh, Joy; Zhao, Guoyan; Presti, Rachel M; Holtz, Lori R; Finkbeiner, Stacy R; Droit, Lindsay; Villasana, Zoilmar; Todd, Collin; Pipas, James M; Calgua, Byron; Girones, Rosina; Wang, David; Virgin, Herbert W

    2009-12-01

    The family Asfarviridae contains only a single virus species, African swine fever virus (ASFV). ASFV is a viral agent with significant economic impact due to its devastating effects on populations of domesticated pigs during outbreaks but has not been reported to infect humans. We report here the discovery of novel viral sequences in human serum and sewage which are clearly related to the asfarvirus family but highly divergent from ASFV. Detection of these sequences suggests that greater genetic diversity may exist among asfarviruses than previously thought and raises the possibility that human infection by asfarviruses may occur.

  7. Association of a Human FABP1 Gene Promoter Region Polymorphism with Altered Serum Triglyceride Levels.

    Directory of Open Access Journals (Sweden)

    Xian-E Peng

    Full Text Available Liver fatty acid-binding protein (L-FABP, also known as fatty acid-binding protein 1 (FABP1, is a key regulator of hepatic lipid metabolism. Elevated FABP1 levels are associated with an increased risk of cardiovascular disease (CVD and metabolic syndromes. In this study, we examine the association of FABP1 gene promoter variants with serum FABP1 and lipid levels in a Chinese population. Four promoter single-nucleotide polymorphisms (SNPs of FABP1 gene were genotyped in a cross-sectional survey of healthy volunteers (n = 1,182 from Fuzhou city of China. Results showed that only the rs2919872 G>A variant was significantly associated with serum TG concentration(P = 0.032.Compared with the rs2919872 G allele, rs2919872 A allele contributed significantly to reduced serum TG concentration, and this allele dramatically decreased the FABP1 promoter activity(P < 0.05. The rs2919872 A allele carriers had considerably lower serum FABP1 levels than G allele carriers (P < 0.01. In the multivariable linear regression analysis, the rs2919872 A allele was negatively associated with serum FABP1 levels (β = -0.320, P = 0.003, while serum TG levels were positively associated with serum FABP1 levels (β = 0.487, P = 0.014. Our data suggest that compared with the rs2919872 G allele, the rs2919872 A allele reduces the transcriptional activity of FABP1 promoter, and thereby may link FABP1 gene variation to TG level in humans.

  8. Localized-statistical quantification of human serum proteome associated with type 2 diabetes.

    Directory of Open Access Journals (Sweden)

    Rong-Xia Li

    Full Text Available BACKGROUND: Recent advances in proteomics have shed light to discover serum proteins or peptides as biomarkers for tracking the progression of diabetes as well as understanding molecular mechanisms of the disease. RESULTS: In this work, human serum of non-diabetic and diabetic cohorts was analyzed by proteomic approach. To analyze total 1377 high-confident serum-proteins, we developed a computing strategy called localized statistics of protein abundance distribution (LSPAD to calculate a significant bias of a particular protein-abundance between these two cohorts. As a result, 68 proteins were found significantly over-represented in the diabetic serum (p<0.01. In addition, a pathway-associated analysis was developed to obtain the overall pathway bias associated with type 2 diabetes, from which the significant over-representation of complement system associated with type 2 diabetes was uncovered. Moreover, an up-stream activator of complement pathway, ficolin-3, was observed over-represented in the serum of type 2 diabetic patients, which was further validated with statistic significance (p = 0.012 with more clinical samples. CONCLUSIONS: The developed LSPAD approach is well fit for analyzing proteomic data derived from biological complex systems such as plasma proteome. With LSPAD, we disclosed the comprehensive distribution of the proteins associated with diabetes in different abundance levels and the involvement of ficolin-related complement activation in diabetes.

  9. Study of OH● Radicals in Human Serum Blood of Healthy Individuals and Those with Pathological Schizophrenia

    Directory of Open Access Journals (Sweden)

    Wolfgang Linert

    2011-01-01

    Full Text Available The human body is constantly under attack from free radicals that occur as part of normal cell metabolism, and by exposure to environmental factors such as UV light, cigarette smoke, environmental pollutants and gamma radiation. The resulting “Reactive Oxygen Species” (ROS circulate freely in the body with access to all organs and tissues, which can have serious repercussions throughout the body. The body possesses a number of mechanisms both to control the production of ROS and to cope with free radicals in order to limit or repair damage to tissues. Overproduction of ROS or insufficient defense mechanisms leads to a dangerous disbalance in the organism. Thereby several pathomechanisms implicated in over 100 human diseases, e.g., cardiovascular disease, cancer, diabetes mellitus, physiological disease, aging, etc., can be induced. Thus, a detailed investigation on the quantity of oxygen radicals, such as hydroxyl radicals (OH● in human serum blood, and its possible correlation with antioxidant therapy effects, is highly topical. The subject of this study was the influence of schizophrenia on the amount of OH● in human serum blood. The radicals were detected by fluorimetry, using terephthalic acid as a chemical trap. For all experiments the serum blood of healthy people was used as a control group.

  10. Preliminary molecular characterization of the human pathogen Angiostrongylus cantonensis

    Directory of Open Access Journals (Sweden)

    He Ai

    2009-10-01

    Full Text Available Abstract Background Human angiostrongyliasis is an emerging food-borne public health problem, with the number of cases increasing worldwide, especially in mainland China. Angiostrongylus cantonensis is the causative agent of this severe disease. However, little is known about the genetics and basic biology of A. cantonensis. Results A cDNA library of A. cantonensis fourth-stage larvae was constructed, and ~1,200 clones were sequenced. Bioinformatic analyses revealed 378 cDNA clusters, 54.2% of which matched known genes at a cutoff expectation value of 10-20. Of these 378 unique cDNAs, 168 contained open reading frames encoding proteins containing an average of 238 amino acids. Characterization of the functions of these encoded proteins by Gene Ontology analysis showed enrichment in proteins with binding and catalytic activity. The observed pattern of enzymes involved in protein metabolism, lipid metabolism and glycolysis may reflect the central nervous system habitat of this pathogen. Four proteins were tested for their immunogenicity using enzyme-linked immunosorbent assays and histopathological examinations. The specificity of each of the four proteins was superior to that of crude somatic and excretory/secretory antigens of larvae, although their sensitivity was relatively low. We further showed that mice immunized with recombinant cystatin, a product of one of the four cDNA candidate genes, were partially protected from A. cantonensis infection. Conclusion The data presented here substantially expand the available genetic information about the human pathogen A. cantonensis, and should be a significant resource for angiostrongyliasis researchers. As such, this work serves as a starting point for molecular approaches for diagnosing and controlling human angiostrongyliasis.

  11. No donor age effect of human serum on collagen synthesis signaling and cell proliferation of human tendon fibroblasts

    DEFF Research Database (Denmark)

    Bayer, Monika L; Schjerling, Peter; Biskup, Edyta

    2012-01-01

    The aging process of tendon tissue is associated with decreased collagen content and increased risk for injuries. An essential factor in tendon physiology is transforming growth factor-ß1 (TGF-ß1), which is presumed to be reduced systemically with advanced age. The aim of this study was to invest......The aging process of tendon tissue is associated with decreased collagen content and increased risk for injuries. An essential factor in tendon physiology is transforming growth factor-ß1 (TGF-ß1), which is presumed to be reduced systemically with advanced age. The aim of this study...... was to investigate whether human serum from elderly donors would have an inhibiting effect on the expression of collagen and collagen-related genes as well as on cell proliferative capacity in tendon cells from young individuals. There was no difference in systemic TGF-ß1 levels in serum obtained from young...... and elderly donors, and we found no difference in collagen expression when cells were subjected to human serum from elderly versus young donors. In addition, tendon cell proliferation was similar when culture medium was supplemented with serum of different donor age. These findings suggest that factors...

  12. Crystallization and preliminary crystallographic analysis of human glycosylated haemoglobin

    Energy Technology Data Exchange (ETDEWEB)

    Syakhovich, Vitaly E. [Department of Biochemistry and Biophysics, International Sakharov Environmental University, Dolgobrodskaya St 23, 220009 Minsk (Belarus); Saraswathi, N. T.; Ruff, Marc, E-mail: ruff@igbmc.u-strasbg.fr [Département de Biologie et Génomique Structurales, Institut de Génétique et de Biologie Moléculaire et Cellulaire, 1 Rue Laurent Fries, BP 10142, 67404 Illkirch (France); Bokut, Sergey B. [Department of Biochemistry and Biophysics, International Sakharov Environmental University, Dolgobrodskaya St 23, 220009 Minsk (Belarus); Moras, Dino [Département de Biologie et Génomique Structurales, Institut de Génétique et de Biologie Moléculaire et Cellulaire, 1 Rue Laurent Fries, BP 10142, 67404 Illkirch (France); Department of Biochemistry and Biophysics, International Sakharov Environmental University, Dolgobrodskaya St 23, 220009 Minsk (Belarus)

    2006-02-01

    Non enzymatic modification of haemoglobin by glucose plays an important role in diabetes pathogenesis. Here the purification, characterization and crystallization of human glycosylated haemoglobin are reported. Human glycosylated haemoglobin A{sub 1C} is a stable minor variant formed in vivo by post-translational modification of the main form of haemoglobin by glucose. Crystals of oxyHbA{sub 1C} were obtained using the hanging-drop vapour-diffusion method and PEG as precipitant. The diffraction pattern of the crystal extends to a resolution of 2.3 Å at 120 K. The crystals belong to space group C2, with unit-cell parameters a = 237.98, b = 59.27, c = 137.02 Å, α = 90.00, β = 125.40, γ = 90.00°. The presence of two and a half molecules per asymmetric unit gives a crystal volume per protein weight (V{sub M}) of 9.70 Å{sup 3} Da{sup −1} and a solvent content of 49%.

  13. [Prokaryotic expression for fusion protein of human metapneumovirus and its preliminary application as an antigen for antibody detection].

    Science.gov (United States)

    Zhu, Ru-nan; Qian, Yuan; Zhao, Lin-qing; Sun, Yu; Deng, Jie; Wang, Fang

    2011-03-01

    To understand the effectiveness of prokaryotic expression of fusion protein (F) of human metapneumovirus (hMPV) and its application as antigen, F proteins from different genotypes of hMPV were expressed in prokaryotic expression system and purified by Ni-NTA affinity chromatography column. According to the hydrophobicity, antigen index and surface probability of F protein, the subunit 1 (F1) region of F protein was generated and expressed in E. Coil. BL21(DE3). The 6-His-F1 proteins with molecular weight of approximately 37 kD generated from hMPV of two genotypes were expressed efficiently mainly in inclusion body. The antigenicity and specificity of the expressed proteins were tested and confirmed by Western Blot using polyclonal antibody against hMPV and one serum specimen from a patient with confirmed hMPV acute infection,and polyclonal antibodies against human respiratory syncytial virus and parainfluenza virus 2 and 3. The results of preliminary use of the expressed proteins for detecting antibodies against hMPV in 457 serum specimens collected from different age groups in Beijing indicated that 66%-67% of sera in all age groups were positive. The positive rate of antibodies declined in children in age groups from birth to 2-year-old and then rose along with the increase in age, in which the lowest was in age group from 1 to 2-year-old and the highest in newborn and people older than 60 years. The data indicated the existence of maternal transferred antibodies against hMPV in infants and the risk of hMPV infections in children younger than 2 years old.

  14. Effect of transdermal magnesium cream on serum and urinary magnesium levels in humans: A pilot study.

    Science.gov (United States)

    Kass, Lindsy; Rosanoff, Andrea; Tanner, Amy; Sullivan, Keith; McAuley, William; Plesset, Michael

    2017-01-01

    Oral magnesium supplementation is commonly used to support a low magnesium diet. This investigation set out to determine whether magnesium in a cream could be absorbed transdermally in humans to improve magnesium status. In this single blind, parallel designed pilot study, n = 25 participants (aged 34.3+/-14.8y, height 171.5+/-11cm, weight 75.9 +/-14 Kg) were randomly assigned to either a 56mg/day magnesium cream or placebo cream group for two weeks. Magnesium serum and 24hour urinary excretion were measured at baseline and at 14 days intervention. Food diaries were recorded for 8 days during this period. Mg test and placebo groups' serum and urinary Mg did not differ at baseline. After the Mg2+ cream intervention there was a clinically relevant increase in serum magnesium (0.82 to 0.89 mmol/l,p = 0.29) that was not seen in the placebo group (0.77 to 0.79 mmol/L), but was only statistically significant (p = 0.02)) in a subgroup of non-athletes. Magnesium urinary excretion increased from baseline slightly in the Mg2+ group but with no statistical significance (p = 0.48). The Mg2+ group showed an 8.54% increase in serum Mg2+ and a 9.1% increase in urinary Mg2+ while these figures for the placebo group were smaller, i.e. +2.6% for serum Mg2+ and -32% for urinary Mg2+. In the placebo group, both serum and urine concentrations showed no statistically significant change after the application of the placebo cream. No previous studies have looked at transdermal absorbency of Mg2+ in human subjects. In this pilot study, transdermal delivery of 56 mg Mg/day (a low dose compared with commercial transdermal Mg2+ products available) showed a larger percentage rise in both serum and urinary markers from pre to post intervention compared with subjects using the placebo cream, but statistical significance was achieved only for serum Mg2+ in a subgroup of non-athletes. Future studies should look at higher dosage of magnesium cream for longer durations. ISRCTN registry ID No. ISRTN

  15. A direct electron transfer-based glucose/oxygen biofuel cell operating in human serum

    Energy Technology Data Exchange (ETDEWEB)

    Coman, V.; Gorton, L. [Department of Analytical Chemistry/Biochemistry, Lund University, 22100 Lund (Sweden); Ludwig, R. [Research Centre Applied Biocatalysis, 8010 Graz (Austria); Department of Food Sciences and Technology, BOKU-University of Natural Resources and Applied Life Sciences, 1190 Wien (Austria); Harreither, W.; Haltrich, D. [Department of Food Sciences and Technology, BOKU-University of Natural Resources and Applied Life Sciences, 1190 Wien (Austria); Ruzgas, T. [Biomedical Laboratory Science, Health and Society, Malmoe University, 20506 Malmoe (Sweden); Laboratory of Chemical Enzymology, A.N. Bach Institute of Biochemistry, 119071 Moscow (Russian Federation); Shleev, S.

    2010-02-15

    We report on the fabrication and characterisation of the very first direct electron transfer-based glucose/oxygen biofuel cell (BFC) operating in neutral glucose-containing buffer and human serum. Corynascus thermophilus cellobiose dehydrogenase and Myrothecium verrucaria bilirubin oxidase were used as anodic and cathodic bioelements, respectively. The following characteristics of the mediator-, separator- and membrane-less, a priori, non-toxic and simple miniature BFC, was obtained: an open-circuit voltage of 0.62 and 0.58 V, a maximum power density of ca. 3 and 4 {mu}W cm{sup -2} at 0.37 and 0.19 V of cell voltage, in phosphate buffer and human serum, respectively. (Abstract Copyright [2010], Wiley Periodicals, Inc.)

  16. Power Harvesting from Human Serum in Buckypaper-Based Enzymatic Biofuel Cell

    Directory of Open Access Journals (Sweden)

    Güray eGüven

    2016-02-01

    Full Text Available The requirement for a miniature, high density, long life, rechargeable power source is common to a vast majority of microsystems, including the implantable devices for medical applications. A model biofuel cell system operating in human serum has been studied for future applications of biomedical and implantable medical devices. Anodic and cathodic electrodes were made of carbon nanotube –buckypaper modified with PQQ-dependent glucose dehydrogenase and laccase, respectively. Modified electrodes were characterized electrochemically and assembled in a biofuel cell set-up. Power density of 16.12 μW/cm2 was achieved in human serum for lower than physiological glucose concentrations. Increasing the glucose concentration and biofuel cell temperature caused an increase on power output leading up to 49.16 μW/cm2.

  17. Analysis of organochlorine pesticides in human milk: preliminary results.

    Science.gov (United States)

    Campoy, C; Jiménez, M; Olea-Serrano, M F; Moreno-Frías, M; Cañabate, F; Olea, N; Bayés, R; Molina-Font, J A

    2001-11-01

    In the face of evidence of human milk contamination by organochlorine pesticides, an analysis was performed on samples of milk obtained from healthy lactating women in the provinces of Granada and Almeria in Southern Spain. The samples were obtained by the Neonate Section of the Department of Pediatrics of Granada University Hospital (Neonatology Division) and by the Neonatal Service of Poniente Hospital in El Ejido, Almería. A liquid-liquid extraction procedure was performed. The cleaning of the sample before gas chromatography-mass spectrometry (GC-MS) used silica Sep-Pak. Among other pesticides, aldrin, dieldrin, DDT and its metabolites, lindane, methoxychlor and endosulfan were identified. The presence of these products was confirmed by mass spectrometry. The identification and quantification of these organochlorine molecules is important because they have estrogenic effects.

  18. Functional characterization of the lectin pathway of complement in human serum.

    Science.gov (United States)

    Roos, Anja; Bouwman, Lee H; Munoz, Jeric; Zuiverloon, Tahlita; Faber-Krol, Maria C; Fallaux-van den Houten, Francien C; Klar-Mohamad, Ngaisah; Hack, C Erik; Tilanus, Marcel G; Daha, Mohamed R

    2003-01-01

    Mannan-binding lectin (MBL) is a major initiator of the lectin pathway (LP) of complement. Polymorphisms in exon 1 of the MBL gene are associated with impaired MBL function and infections. Functional assays to assess the activity of the classical pathway (CP) and the alternative pathway (AP) of complement in serum are broadly used in patient diagnostics. We have now developed a functional LP assay that enables the specific quantification of autologous MBL-dependent complement activation in human serum. Complement activation was assessed by ELISA using coated mannan to assess the LP and coated IgM to assess the CP. Normal human serum (NHS) contains IgG, IgA and IgM antibodies against mannan, as shown by ELISA. These antibodies are likely to induce CP activation. Using C1q-blocking and MBL-blocking mAb, it was confirmed that both the LP and the CP contribute to complement activation by mannan. In order to quantify LP activity without interference of the CP, LP activity was measured in serum in the presence of C1q-blocking Ab. Activation of serum on coated IgM via the CP resulted in a dose-dependent deposition of C1q, C4, C3, and C5b-9. This activation and subsequent complement deposition was completely inhibited by the C1q-blocking mAb 2204 and by polyclonal Fab anti-C1q Ab. Evaluation of the LP in the presence of mAb 2204 showed a strong dose-dependent deposition of C4, C3, and C5b-9 using serum from MBL-wildtype (AA) but not MBL-mutant donors (AB or BB genotype), indicating that complement activation under these conditions is MBL-dependent and C1q-independent. Donors with different MBL genotypes were identified using a newly developed oligonucleotide ligation assay (OLA) for detection of MBL exon 1 polymorphisms. We describe a novel functional assay that enables quantification of autologous complement activation via the LP in full human serum up to the formation of the membrane attack complex. This assay offers novel possibilities for patient diagnostics as well as

  19. Preliminary Study on Biological Properties of Adult Human Bone Marrow-derived Mesenchymal Stem Cells

    Institute of Scientific and Technical Information of China (English)

    WU Tao; BAI Hai; WANG Jingchang; SHI Jingyun; WANG Cunbang; LU Jihong; OU Jianfeng; WANG Qian

    2006-01-01

    Objective: To establish a method of culture and expansion of adult human bone marrow-derived MSCs in vitro and to explore their biological properties. Methods: Mononuclear cells were obtained from 5 mL adult human bone marrow by density gradient centrifugation with Percoll solution. Adult human MSCs were cultured in Dulbecco's Modified Eagle's Medium with low glucose (LG-DMEM) containing 10% fetal calf serum at a density of 2× 105 cell/cm2. The morphocytology was observed under phase-contrast microscope. The cell growth was measured by MTT method. The flow cytometer was performed to examine the expression of cell surface molecules and cell cycle. The ultrastructure of MSCs was observed under transmission electron microscope. The immunomodulatory functions of MSCs were measured by MTT method. The effects of MSCs on the growth of K562 cells and the dynamic change of HA, Ⅳ-C, LN concentration in the culture supernatant of MSCs was also observed. Results: The MSCs harvested in this study were homogenous population and exhibited a spindle-shaped fibroblastic morphology. The cell growth curve showed that MSCs had a strong ability of proliferation. The cells were positive for CD44,while negative for hematopoietic cell surface marker such as CD3, CD4, CD7, CD13, CD14, CD15, CD19,CD22, CD33, CD34, CD45 and HLA-DR, which was closely related to graft versus host disease. Above 90% cells of MSCs were found at G0/G1 phase. The ultrastructure of MSCs indicated that there were plenty of cytoplasmic organelles. Allogeneic peripheral blood lymphocytes proliferation was suppressed by MSCs and the inhibition ratio was 60.68% (P<0.01). The suppressive effect was also existed in the culture supernatant of MSCs and the inhibition ratio was 9.00% (P<0.05). When lymphocytes were stimulated by PHA, the suppression effects of the culture supernatant were even stronger and the inhibition ratio was 20.91%(P<0.01). Compared with the cell growth curve of the K562 cells alone, the K562

  20. Rapid Detection of Trypanosoma cruzi in Human Serum by Use of an Immunochromatographic Dipstick Test ▿

    OpenAIRE

    Reithinger, Richard; Mario J Grijalva; Chiriboga, Rosa F.; Alarcón de Noya, Belkisyolé; Jaime R. Torres; Pavia-Ruz, Norma; MANRIQUE-SAIDE, Pablo; Cardinal, Marta V.; Ricardo E. Gürtler

    2010-01-01

    We evaluated a commercially available immunochromatographic dipstick test to detect Trypanosoma cruzi infection in 366 human serum samples with known serological results from Argentina, Ecuador, Mexico, and Venezuela. One hundred forty-nine of 366 (40.7%) and 171/366 (46.7%) samples tested positive by dipstick and serology, respectively. Dipstick sensitivity was calculated to be 84.8% (range between countries, 77.5 to 95%), and specificity was 97.9% (95.9 to 100%).

  1. Thermodynamic study on the interaction between anti-tumor drug tegafur and human serum albumin

    Institute of Scientific and Technical Information of China (English)

    2007-01-01

    The changes of thermodynamic properties of the system on interaction between tegafur and human serum albumin (HSA) and the changes of secondary structure units of HSA in the system at 298.15 K have been investigated by the Nano-Watt-Scale isothermal titration calorimetry (ITC), the Langmuirs binding model and the circular dichroism (CD) spectrometry.(C) 2007 Lin Wei Li. Published by Elsevier B.V. on behalf of Chinese Chemical Society. All rights reserved.

  2. Removal of Endotoxin from Human Serum Albumin Solutions by Hydrophobic and Cationic Charged Membrane

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

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

  3. Impact of Polychlorinated Biphenyls Contamination on Estrogenic Activity in Human Male Serum

    OpenAIRE

    Plíšková, Martina; Vondráček, Jan; Canton, Rocio Fernandez; Nera, Jiřií; Kočan, Anton; Petrík, Ján; Trnovec, Tomáš; Sanderson, Thomas; van den Berg, Martin; Machala, Miroslav

    2005-01-01

    Polychlorinated biphenyls (PCBs) are thought to cause numerous adverse health effects, but their impact on estrogen signaling is still not fully understood. In the present study, we used the ER-CALUX bioassay to determine estrogenic/antiestrogenic activities of the prevalent PCB congeners and PCB mixtures isolated from human male serum. The samples were collected from residents of an area with an extensive environmental contamination from a former PCB production site as well as from a neighbo...

  4. Human CRISP-3 binds serum alpha(1)B-glycoprotein across species

    DEFF Research Database (Denmark)

    Udby, Lene; Johnsen, Anders H; Borregaard, Niels

    2010-01-01

    CRISP-3 was previously shown to be bound to alpha(1)B-glycoprotein (A1BG) in human serum/plasma. All mammalian sera are supposed to contain A1BG, although its presence in rodent sera is not well-documented. Since animal sera are often used to supplement buffers in experiments, in particular such ...... such that involve cell cultures, binding proteins present in sera might interfere in the experiments....

  5. Molecular interaction of PCB153 to human serum albumin: Insights from spectroscopic and molecular modeling studies

    Energy Technology Data Exchange (ETDEWEB)

    Han, Chao; Fang, Senbiao; Cao, Huiming; Lu, Yan; Ma, Yaqiong [School of Pharmacy, Lanzhou University, Lanzhou 730000 (China); Wei, Dongfeng [Institute of Basic Research in Clinical Medicine, China Academy of Chinese Medical Sciences, Beijing 100700 (China); Xie, Xiaoyun [College of Earth and Environmental Science, Lanzhou University, Lanzhou 730000 (China); Liu, Xiaohua [School of Pharmacy, Lanzhou University, Lanzhou 730000 (China); Li, Xin [College of Food and Bioengineering, Henan University of Science and Technology, Luoyang 471003 (China); Fei, Dongqing [School of Pharmacy, Lanzhou University, Lanzhou 730000 (China); Zhao, Chunyan, E-mail: zhaochy07@lzu.edu.cn [School of Pharmacy, Lanzhou University, Lanzhou 730000 (China)

    2013-03-15

    Highlights: ► We identify the binding mode of PCB153 to human serum albumin (HSA). ► Spectroscopic and molecular modeling results reveal that PCB153 binds at the site II. ► The interaction is mainly governed by hydrophobic and hydrogen bond forces. ► The work helps to probe transporting, distribution and toxicity effect of PCBs. -- Abstract: Polychlorinated biphenyls (PCBs) possessed much potential hazard to environment because of its chemical stability and biological toxicity. Here, we identified the binding mode of a representative compound, PCB153, to human serum albumin (HSA) using fluorescence and molecular dynamics simulation methods. The fluorescence study showed that the intrinsic fluorescence of HSA was quenched by addition of PCB153 through a static quenching mechanism. The thermodynamic analysis proved the binding behavior was mainly governed by hydrophobic force. Furthermore, as evidenced by site marker displacement experiments using two probe compounds, it revealed that PCB153 acted exactly on subdomain IIIA (site II) of HSA. On the other hand, the molecular dynamics studies as well as free energy calculations made another important contribution to understand the conformational changes of HSA and the stability of HSA-PCB153 system. Molecular docking revealed PCB153 can bind in a large hydrophobic activity of subdomain IIIA by the hydrophobic interaction and hydrogen bond interactions between chlorine atoms and residue ASN391. The present work provided reasonable models helping us further understand the transporting, distribution and toxicity effect of PCBs when it spread into human blood serum.

  6. Investigation of the interaction of deltamethrin (DM) with human serum albumin by multi-spectroscopic method

    Science.gov (United States)

    Wang, Jiaman; Ma, Liang; Zhang, Yuhao; Jiang, Tao

    2017-02-01

    The interaction of Deltamethrin (DM) with human serum albumin (HSA) under the condition of simulating human blood pH environment (pH = 7.4) was investigated by fluorescence, UV-Vis absorbance and circular dichroism (CD) spectroscopy. It was shown that DM was a static quencher of HSA. The binding constants (Ka) are 3.598 × 104 L mol-1 (25 °C); the thermodynamic parameters (ΔH = -3.269 × 104 kJ mol-1, ΔS = -22.81 kJ mol-1 k-1, ΔG = -25889.8 kJ mol-1) obtained with the thermodynamic equation. The hydrogen bond and Vander Waals were the main driving force. The effect of DM on the conformation of HSA was observed by three-dimensional (3D) fluorescence and circular dichroism spectra, indicating that the interaction between DM and HSA was achieved through the binding of DM with the tryptophan and tyrosine residues of HSA. The study on the interaction of DM and Bovine Serum Albumin (BSA) was researched and compared. Difference exists in the interactions of with each of the serum albumins. We will verify and supplement that DM residue in animals and human metabolism, toxicology and other mechanisms are different.

  7. Quantifying glucose and lipid components in human serum by Raman spectroscopy and multivariate statistics.

    Science.gov (United States)

    Silveira, Landulfo; Borges, Rita de Cássia Fernandes; Navarro, Ricardo Scarparo; Giana, Hector Enrique; Zângaro, Renato Amaro; Pacheco, Marcos Tadeu Tavares; Fernandes, Adriana Barrinha

    2017-05-01

    Raman spectroscopy has been employed in the quantitative analysis of biochemical components in human serum. This study aimed to develop a spectral model to estimate the concentration of glucose and lipid fractions in human serum, thus evaluating the feasibility of Raman spectroscopy technique for diagnostic purposes. A total of 44 samples of blood serum were collected from volunteers submitted to routine blood biochemical assay analysis. The biochemical concentrations of glucose, triglycerides, cholesterol, and high-density and low-density lipoproteins (HDL and LDL) were obtained by colorimetric method. Serum samples (200 μL) were submitted to Raman spectroscopy (830 nm, 250 mW, 50-s accumulation). The spectra of sera present peaks related to the main constituents, particularly proteins and lipids. A quantitative model based on partial least squares (PLS) regression has been developed to estimate the concentration of these compounds, taking the biochemical concentrations assayed by the colorimetric method as sample's actual concentrations. The PLS model based on leave-one-out cross-validation approach estimated the concentration of triglycerides and cholesterol with r = 0.98 and 0.96, and root mean square error of 35.4 and 15.9 mg/dL, respectively. For the other biochemicals, the r was ranging from 0.75 to 0.86. These results evidenced the possibility of performing biochemical assay in blood serum samples by Raman spectroscopy and PLS regression and may be employed as a means of diagnosis in routine clinical analysis.

  8. Expression, purification and preliminary crystallographic studies of human ketohexokinase.

    Science.gov (United States)

    Kozak, M; Hayward, B; Borek, D; Bonthron, D T; Jaskólski, M

    2001-04-01

    Ketohexokinase (KHK; E.C. 2.7.1.3) catalyses the (reversible) phosphorylation of fructose to fructose-1-phosphate. KHK is the first enzyme in a specialized catabolic pathway metabolizing dietary fructose to the glycolytic intermediate glyceraldehyde-3-phosphate. Mutations inactivating KHK underlie the metabolic disorder essential fructosuria. The primary structure of KHK shows no significant homology to other mammalian hexokinases. It is most similar to prokaryotic ribokinases, but catalyses a distinct phosphorylation reaction. Recombinant human KHK has been crystallized in the orthorhombic form (space group P2(1)2(1)2 or P2(1)2(1)2(1)). Single crystals of this polymorph suitable for X-ray diffraction have been obtained by vapour diffusion using 2-propanol and MPD as precipitants (pH 7.5). The crystals have unit-cell parameters a = 93.4, b = 121.5, c = 108.4 A. Diffraction data were collected to 4.3 A resolution. The asymmetric unit contains four protein molecules.

  9. Loxoprofen sodium induces the production of complement C5a in human serum.

    Science.gov (United States)

    Kumagai, Tomoaki; Yamaguchi, Nozomi; Hirai, Hiroyuki; Kojima, Shigeyuki; Kodani, Yoshiko; Hashiguchi, Akihiko; Haida, Michiko; Nakamura, Masataka

    2016-04-01

    Basophil activation test (BAT) is an in vitro allergy test that is useful to identify allergens that cause IgE-dependent allergies. The test has been used to detect not only food allergies and allergies caused by environmental factors but also to detect drug hypersensitivity, which has been known to include IgE-independent reactions. In our preliminary studies in which BAT was applied to detect hypersensitivity of loxoprofen, a non-steroidal anti-inflammatory drug (NSAID), conventional BAT with incubation for 30min did not show basophil activation by means of increased CD203c expression. In this study, we extended the incubation time to 24h on the basis of the hypothesis that loxoprofen indirectly activates basophils. Basophils from healthy control donors as well as allergic patients showed up-regulation of CD203c after incubation with loxoprofen for 24h. Activation was induced using loxoprofen-treated serum. Proteomic and pharmacologic analyses revealed that serum incubation with loxoprofen generated an active complement component C5a, which induced CD203c expression via binding to the C5a receptor on basophils. Because C3a production was also detected after incubation for 24h, loxoprofen is likely to stimulate the complement classical pathway. Our findings suggest that the complement activation is involved in drug hypersensitivity and the suppression of this activation may contribute to the elimination of false positive of BAT for drug allergies.

  10. Measurements of vitamin B12 in human blood serum using resonance Raman spectroscopy

    Science.gov (United States)

    Tsiminis, G.; Schartner, E. P.; Brooks, J. L.; Hutchinson, M. R.

    2016-12-01

    Vitamin B12 (cobalamin and its derivatives) deficiency has been identified as a potential modifiable risk factor for dementia and Alzheimer's disease. Chronic deficiency of vitamin B12 has been significantly associated with an increased risk of cognitive decline. An effective and efficient method for measuring vitamin B12 concentration in human blood would enable ongoing tracking and assessment of this potential modifiable risk factor. In this work we present an optical sensor based on resonance Raman spectroscopy for rapid measurements of vitamin B12 in human blood serum. The measurement takes less than a minute and requires minimum preparation (centrifuging) of the collected blood samples.

  11. Investigation into the interaction of losartan with human serum albumin and glycated human serum albumin by spectroscopic and molecular dynamics simulation techniques: A comparison study.

    Science.gov (United States)

    Moeinpour, Farid; Mohseni-Shahri, Fatemeh S; Malaekeh-Nikouei, Bizhan; Nassirli, Hooriyeh

    2016-09-25

    The interaction between losartan and human serum albumin (HSA), as well as its glycated form (gHSA) was studied by multiple spectroscopic techniques and molecular dynamics simulation under physiological conditions. The binding information, including the binding constants, effective quenching constant and number of binding sites showed that the binding partiality of losartan to HSA was higher than to gHSA. The findings of three-dimensional fluorescence spectra demonstrated that the binding of losartan to HSA and gHSA would alter the protein conformation. The distances between Trp residue and the binding sites of the drug were evaluated on the basis of the Förster theory, and it was indicated that non-radiative energy transfer from HSA and gHSA to the losartan happened with a high possibility. According to molecular dynamics simulation, the protein secondary and tertiary structure changes were compared in HSA and gHSA for clarifying the obtained results.

  12. An approach to remove albumin for the proteomic analysis of low abundance biomarkers in human serum.

    Science.gov (United States)

    Ahmed, Nuzhat; Barker, Gillian; Oliva, Karen; Garfin, David; Talmadge, Kenneth; Georgiou, Harry; Quinn, Michael; Rice, Greg

    2003-10-01

    Proteomic technologies are being used to discover and identify disease-associated biomarkers. The application of these technologies in the search for potential diagnostic/prognostic biomarkers in the serum of patients has been limited by the presence of highly abundant albumin and immunoglobulins that constitute approximately 60-97% of the total serum proteins. The purpose of the study was to evaluate whether treatment of human serum with Affi-Gel Blue alone or in combination with Protein A (Aurum serum protein mini kit, Bio-Rad) before two-dimensional gel electrophoresis (2-DE) analysis removed high abundance proteins to allow the visualization of low abundant proteins. Serum samples were treated with either Affi-Gel Blue or Aurum kit and then subjected to 2-DE using 11 cm, pH 4-7 isoelectric focussing strips for the first dimension and 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis for second dimension. Protein spots were visualized using a fluorescent protein dye (SYPRO Ruby, Bio-Rad). Comparison between treatment methods showed significant removal of albumin by both Affi-Gel Blue and Aurum kit and considerable differences in the protein profile of the gels after each treatment. Direct comparison between treatments revealed twenty-eight protein spots unique to Affi-Gel Blue while only two spots were unique after Aurum kit treatment. Unique spots in Affi-Gel Blue and Aurum kit treated serum were not visualized in untreated serum. Sixteen hours of Affi-Gel Blue treatment resulted in enhanced visualization of fifty-three protein spots by two-fold, thirty-one by five-fold, twelve by ten-fold and six by twenty-fold. In parallel after Aurum kit treatment two-, five-, ten- and twenty-fold enhancements of thirty, thirteen, eight and five protein spots, respectively, were observed. The pattern of increased visualization of protein spots with both treatment methods was similar. In conclusion, treatment of serum samples with Affi-Gel Blue or Aurum kit before

  13. Gene expression signature of fibroblast serum response predicts human cancer progression: similarities between tumors and wounds.

    Directory of Open Access Journals (Sweden)

    Howard Y Chang

    2004-02-01

    Full Text Available Cancer invasion and metastasis have been likened to wound healing gone awry. Despite parallels in cellular behavior between cancer progression and wound healing, the molecular relationships between these two processes and their prognostic implications are unclear. In this study, based on gene expression profiles of fibroblasts from ten anatomic sites, we identify a stereotyped gene expression program in response to serum exposure that appears to reflect the multifaceted role of fibroblasts in wound healing. The genes comprising this fibroblast common serum response are coordinately regulated in many human tumors, allowing us to identify tumors with gene expression signatures suggestive of active wounds. Genes induced in the fibroblast serum-response program are expressed in tumors by the tumor cells themselves, by tumor-associated fibroblasts, or both. The molecular features that define this wound-like phenotype are evident at an early clinical stage, persist during treatment, and predict increased risk of metastasis and death in breast, lung, and gastric carcinomas. Thus, the transcriptional signature of the response of fibroblasts to serum provides a possible link between cancer progression and wound healing, as well as a powerful predictor of the clinical course in several common carcinomas.

  14. The effect of the binge drinking session on the activity of salivary, serum and urinary beta-hexosaminidase: preliminary data.

    Science.gov (United States)

    Waszkiewicz, Napoleon; Szajda, Slawomir Dariusz; Jankowska, Anna; Kepka, Alina; Dobryniewski, Jacek; Szulc, Agata; Zwierz, Krzysztof

    2008-01-01

    Our report is the first to show that an acute ingestion (6 h) of a relatively large, yet tolerable dose of alcohol (120-160 g), significantly increases activity of total serum beta-hexosaminidase (total beta-HEX), beta-HEX A and beta-HEX B isoenzymes, as well as salivary total beta-HEX and urinary beta-HEX A, in eight infrequent binge drinkers. An increase in the activity of serum and urinary total HEX is mainly due to its secretory isoenzyme beta-HEX A.

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

    Science.gov (United States)

    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. Copyright © 2014 International Society for Cellular Therapy. Published by Elsevier Inc. All rights reserved.

  16. Highly efficient human serum filtration with water-soluble nanoporous nanoparticles

    Directory of Open Access Journals (Sweden)

    Antonella Pujia

    2010-11-01

    Full Text Available Antonella Pujia1, Francesco De Angelis1,2, Domenica Scumaci3, Marco Gaspari3, Carlo Liberale1,2, Patrizio Candeloro1, Giovanni Cuda3, Enzo Di Fabrizio1,21BIONEM Laboratory, Department of Experimental and Clinical Medicine, University of Catanzaro “Magna Graecia”, Germaneto (CZ, Italy; 2IIT, Italian Institute of Technology, Genova, Italy; 3Proteomics and Mass Spectrometry Laboratory, Department of Experimental and Clinical Medicine, University of Catanzaro “Magna Graecia”, Germaneto (CZ, ItalyBackground: Human serum has the potential to become the most informative source of novel biomarkers, but its study is very difficult due to the incredible complexity of its molecular composition. We describe a novel tool based on biodegradable nanoporous nanoparticles (NPNPs that allows the harvesting of low-molecular-weight fractions of crude human serum or other biofluids. NPNPs with a diameter of 200 nm and pore size of a few nm were obtained by ultrasonication of nanoporous silicon. When incubated with a solution, the NPNPs harvest only the molecules small enough to be absorbed into the nanopores. Then they can be recovered by centrifugation and dissolved in water, making the harvested molecules available for further analyses.Results: Fluorescence microscopy, gel electrophoresis, and mass spectrometry were used to show the enrichment of low-molecular-weight fraction of serum under physiological conditions, with a cut-off of 13 kDa and an enrichment factor >50.Conclusion: From these findings, we conclude that ability to tune pore size, combined with the availability of hundreds of biomolecule cross-linkers, opens up new perspectives on complex biofluid analysis, discovery of biomarkers, and in situ drug delivery.Keywords: nanoporous silicon, nanoparticle, biomarker discovery, human serum proteomics, harvesting

  17. First-trimester maternal serum human thyroid-stimulating hormone in chromosomally normal and Down syndrome pregnancies

    NARCIS (Netherlands)

    Pratt, JJ; de Wolf, BTHM; Mantingh, A

    2001-01-01

    Maternal serum human thyroid-stimulating hormone (TSH) levels were investigated in chromosomally normal and Down syndrome pregnancies to determine whether TSH can be used as a marker for Down syndrome in the first trimester. Measurements were conducted on stored serum samples collected from 23 Down

  18. Detection and preliminary characterization of antibacterial protein(s in the serum of mud crab, Scylla serrata

    Directory of Open Access Journals (Sweden)

    V Meiyalagan

    2015-11-01

    Full Text Available Serum of mud crab, Scylla serrata has been found to possess significant antibacterial activity against some of the resident specific bacteria including Bacillus sp. N1, Bacillus flexus N3, Escherichia coli as well as crustacean pathogenic bacteria viz., Vibrio harveyi, V. alginolyticus and V. vulnificus. The physico-chemical characterization reveals the molecule responsible for antibacterial activity in the serum over 14 kDa, stable in the pH range of 6 to 8 and between the temperatures 20 to 40 ºC. Precipitation of respective molecule(s with 75 % ammonium sulphate or the supernatant obtained after precipitating the protein with 10 % TCA indicated that the molecule(s responsible for serum antibacterial activity appear to be proteinaceous in nature. Further studies demonstrated that antibacterial molecule(s against E. coli and V. harveyi appeared to be trypsin and pronase resistant and the molecule(s or domain responsible for antibacterial activity against Bacillus sp. N1 and B. flexus N3 appeared to be protease sensitive, thereby implicating possible involvement of multiple antibacterial factors in the serum of mud crab, S. serrata.

  19. DEAE-Affi-Gel Blue chromatography of human serum: use for purification of native transferrin.

    Science.gov (United States)

    Werner, P A; Galbraith, R M; Arnaud, P

    1983-10-01

    Human serum was subjected to chromatography on DEAE-Affi-Gel Blue which combines ion-exchange and pseudo-ligand-affinity chromatography in a 0.02 M phosphate buffer, pH 7.0. All serum proteins were bound with the exception of transferrin, IgG (immunoglobulin G) and trace amounts of IgA. After a second step of Sephadex G-100 gel chromatography, or affinity chromatography against goat anti-human IgG F(ab')2 coupled to AH-Sepharose 4B, IgG and IgA were removed. The transferrin obtained was homogeneous and of high yield (greater than 80%), and was unaltered as judged by analyses of molecular weight, isoelectric point, iron-binding capacity, antigenicity, and ability to bind to high-affinity specific cellular receptors. Thus, DEAE-Affi-Gel Blue chromatography may be used as the basis for a simple, rapid, two-step method for the purification of large amounts of native transferrin from serum.

  20. Carbofuran poisoning detected by mass spectrometry of butyrylcholinesterase adduct in human serum.

    Science.gov (United States)

    Li, He; Ricordel, Ivan; Tong, Larry; Schopfer, Lawrence M; Baud, Frédéric; Mégarbane, Bruno; Maury, Eric; Masson, Patrick; Lockridge, Oksana

    2009-03-01

    Carbofuran is a pesticide whose acute toxicity is due to inhibition of acetylcholinesterase. Butyrylcholinesterase (BChE) in plasma is inhibited by carbofuran and serves as a biomarker of poisoning by carbofuran. The goal was to develop a method to positively identify poisoning by carbofuran. Sera from an attempted murder and an attempted suicide were analyzed for the presence of carbofuran adducts on BChE. The BChE from 1 ml of serum was rapidly purified on a 0.2 ml procainamide-Sepharose column. Speed was essential because the carbofuran-BChE adduct decarbamylates with a half-life of about 2 h. The partially purified BChE was boiled to denature the protein, thus stopping decarbamylation and making the protein vulnerable to digestion with trypsin. The labeled peptide was partially purified by HPLC before analysis by LC/MS/MS in the multiple reaction monitoring mode on the QTRAP 2000 mass spectrometer. Carbofuran was found to be covalently bound to Ser 198 of human BChE in serum samples from two poisoning cases. Multiple reaction monitoring triggered MS/MS spectra positively identified the carbofuran-BChE adduct. In conclusion a mass spectrometry method to identify carbofuran poisoning in humans has been developed. The method uses 1 ml of serum and detects low-level exposure associated with as little as 20% inhibition of plasma butyrylcholinesterase.

  1. Detection of human liver-specific F antigen in serum and its preliminary application

    Institute of Scientific and Technical Information of China (English)

    1999-01-01

    @@ INTRODUCTION Aspartate aminotransferase (AST) and alanine aminotransferase (ALT) activity are usually used as biochemical criteria for the evaluation of liver lesion. These enzymes will release into the blood when the integrity of hepatocyte is compromised. Although the aminotransferase has been used as a criterion of liver function for decades, it can not show the accurate hepatohistological characteristics when it is abnormal in patients with chronic liver disease[1].

  2. Electrochemical biosensor for quantitation of anti-DNA autoantibodies in human serum.

    Science.gov (United States)

    Rubin, Robert L; Wall, David; Konstantinov, Konstantin N

    2014-01-15

    Measurement of serum autoantibody is a critical tool in the diagnosis and management of autoimmune diseases. However, rapid and convenient methods at the point-of care have not been achieved in large part because any one antibody species is a heterogeneous and miniscule fraction of the total serum immunoglobulin displaying identical properties other than its antigen-binding specificity. The present system addresses these challenges by vacuum-mediated transport of diluted serum through an antigen-coated porous membrane. To measure anti-DNA autoantibodies, native DNA was immobilized into a poly(vinylidene fluoride) membrane pre-coated with a synthetic phenylalanine/lysine co-polymer. Flow-through of primary and peroxidase-conjugated secondary antibodies over the course of 3 min enhanced productive antibody-antigen interactions by bringing the reactants into close mutual proximity. Signal was quantified electrochemically during the enzymatic conversion of the tetramethylbenzidine substrate to a charge-transfer complex. The electrochemical signals generated by sera from patients with systemic lupus erythematosus using this device showed good quantitative correlation with a standard enzyme-linked immunosorbent assay and displayed similar detection limits. Inter- and intra-assay variability and electrode uniformity were favorable as was a two-month test of the stability of the DNA-coated membrane. While refining the fluidics requirements of this biosensor will be needed, its capacity to quantify over the course of 30 min anti-DNA antibodies in fresh human serum without background reactivity of normal serum makes this a promising technology as a point-of care device of clinical utility.

  3. Direct detection of antibody concentration and affinity in human serum using microscale thermophoresis.

    Science.gov (United States)

    Lippok, Svenja; Seidel, Susanne A I; Duhr, Stefan; Uhland, Kerstin; Holthoff, Hans-Peter; Jenne, Dieter; Braun, Dieter

    2012-04-17

    The direct quantification of both the binding affinity and absolute concentration of disease-related biomarkers in biological fluids is particularly beneficial for differential diagnosis and therapy monitoring. Here, we extend microscale thermophoresis to target immunological questions. Optically generated thermal gradients were used to deplete fluorescently marked antigens in 2- and 10-fold-diluted human serum. We devised and validated an autocompetitive strategy to independently fit the concentration and dissociation constant of autoimmune antibodies against the cardiac β1-adrenergic receptor related to dilated cardiomyopathy. As an artificial antigen, the peptide COR1 was designed to mimic the second extracellular receptor loop. Thermophoresis resolved antibody concentrations from 2 to 200 nM and measured the dissociation constant as 75 nM. The approach quantifies antibody binding in its native serum environment within microliter volumes and without any surface attachments. The simplicity of the mix and probe protocol minimizes systematic errors, making thermophoresis a promising detection method for personalized medicine.

  4. Serum profiling of healthy aging identifies phospho- and sphingolipid species as markers of human longevity.

    Science.gov (United States)

    Montoliu, Ivan; Scherer, Max; Beguelin, Fiona; DaSilva, Laeticia; Mari, Daniela; Salvioli, Stefano; Martin, Francois-Pierre J; Capri, Miriam; Bucci, Laura; Ostan, Rita; Garagnani, Paolo; Monti, Daniela; Biagi, Elena; Brigidi, Patrizia; Kussmann, Martin; Rezzi, Serge; Franceschi, Claudio; Collino, Sebastiano

    2014-01-01

    As centenarians well represent the model of healthy aging, there are many important implications in revealing the underlying molecular mechanisms behind such successful aging. By combining NMR metabonomics and shot-gun lipidomics in serum we analyzed metabolome and lipidome composition of a group of centenarians with respect to elderly individuals. Specifically, NMR metabonomics profiling of serum revealed that centenarians are characterized by a metabolic phenotype distinct from that of elderly subjects, in particular regarding amino acids and lipid species. Shot- gun lipidomics approach displays unique changes in lipids biosynthesis in centenarians, with 41 differently abundant lipid species with respect to elderly subjects. These findings reveal phospho/sphingolipids as putative markers and biological modulators of healthy aging, in humans. Considering the particular actions of these metabolites, these data are suggestive of a better counteractive antioxidant capacity and a well-developed membrane lipid remodelling process in the healthy aging phenotype.

  5. Zinc phthalocyanine-conjugated with bovine serum albumin mediated photodynamic therapy of human larynx carcinoma

    Science.gov (United States)

    Silva, E. P. O.; Santos, E. D.; Gonçalves, C. S.; Cardoso, M. A. G.; Soares, C. P.; Beltrame, M., Jr.

    2016-10-01

    Phthalocyanines, which are classified as second-generation photosensitizers, have advantageous photophysical properties, and extensive studies have demonstrated their potential applications in photodynamic therapy. The present work describes the preparation of a new zinc phthalocyanine conjugated to bovine serum albumin (compound 4a) and its photodynamic efficiency in human larynx-carcinoma cells (HEp-2 cells). The unconjugated precursor (compound 4) was also studied. Compounds 4 and 4a penetrated efficiently into the cell, exhibiting cytoplasmic localization, and showed no cytotoxicity in the dark. However, high photodynamic activities were observed in HEp-2 cells after treatments with 5 µM photosensitizers and 4.5 J cm-2 light. These conditions were sufficient to decrease the cell viability to 57.93% and 32.75% for compounds 4 and 4a, respectively. The present results demonstrated high photodynamic efficiency of zinc phthalocyanine conjugated with bovine serum albumin in destroying the larynx-carcinoma cells.

  6. Stereospecific antibodies to methadone. I. Radioimmunoassay of d,l-methadone in human serum.

    Science.gov (United States)

    Bartos, F; Olsen, G D; Leger, R N; Bartos, D

    1977-01-01

    Anti-d,l-methadone antibodies were produced in rabbits immunized with d,l-methadol-hemisuccinate thyroglobulin conjugate. Using the antiserum, a radioimmunoasay (RIA) for determination of d,l-methadone in human serum has been developed and is described. Concentration of d,l-methadone of 1.4 pmol in a native serum sample (volume 0.1 ml or less) could be measured directly by RIA. The antibodies crossreact 100% with d,l-methadone, 50% with d-methadone, 50% with l-methadone and 100% with alpha-d-methadol. No crossreactivity was found with alpha 1-methadol, morphine, meperidine, dextropropoxyphene, 2-ethyl-5-methyl-3,3-diphenyl-l-pyrroline and 2-ethylidene-l, 5-dimethyl-3,3-diphenylpyrrolidene. High sensitivity and small sample requirements make this method suitable for future monitoring of patients on methadone maintenance and for studies where other procedures have lack of sensitivity.

  7. Human serum teratogenicity studies using in vitro cultures of rat embryos

    Energy Technology Data Exchange (ETDEWEB)

    Klein, N.W.; Chatot, C.L.; Plenefisch, J.D.; Carey, S.W.

    1982-01-01

    Those conditions that constitute reproductive risks to man are being analyzed. Particular concern is with those conditions that cannot be or have not been identified by present methodologies. These conditions constitute the majority of factors causing fetal wastages and birth defects. The test system uses intact rat embryos that are cultured in vitro for 2 days. Findings to date suggest that this system may have a number of distinct advantages: (1) whole-embryo culture provides the test with the entire repertoire of processes involved in embryonic development; (2) whole-rat embryos can be cultured on high levels of blood serum; and (3) they can be cultured on serum from human subjects, which provides a direct and unique evaluation of the principal organism of concern. In regard to this last point, it is important to recognize that there is a large range of teratogenic responses and sensitivities to teratogens dependent upon both individual and species differences. (ERB)

  8. Elevated serum progesterone/ MII oocyte ratio on the day of human chorionic gonadotropin administration can predict impaired endometrial receptivity

    OpenAIRE

    2015-01-01

    Background: Increased serum progesterone on the day of human chorionic gonadotropin administration may affect in vitro fertilization (IVF) outcome. Objective: The aim of this study was to evaluate whether progesterone elevation on the day of human chorionic gonadotropin administration is associated with poor IVF outcome. Materials and Methods: To determine the relationship between serum progesterone on the day of HCG and the outcome of IVF-embryo transfer treatment, 378 infertile patients und...

  9. Human Serum is as Efficient as Fetal Bovine Serum in Supporting Proliferation and Differentiation of Human Multipotent Stromal (Mesenchymal) Stem Cells In Vitro and In Vivo

    DEFF Research Database (Denmark)

    Aldahmash, Abdullah; Haack-Sørensen, Mandana; Al-Nbaheen, May

    2011-01-01

    BACKGROUND: Human multipotent stromal (skeletal, mesenchymal) stem cells (hMSC) are employed in an increasing number of clinical trials for tissue regeneration of age-related degenerative diseases. However, routine use of fetal bovine sera (FBS) for their in vitro expansion is not optimal and may...... pose a health risk for patients. METHODS: We carried out a side-by-side comparison of the effects of allogenic pooled human serum (HuS) versus FBS on hMSC proliferation and differentiation in vitro and in vivo. As a model for hMSC, we employed telomerase-immortalized hMSC; hMSC-TERT cell line. RESULTS......) or adipocytic markers (PPAR-gamma2, lipoprotein lipase (LPL), aP2), respectively. In order to test for the functional capacity of hMSC-TERT that have been maintained in long-term cultures in the presence of HuS vs. FBS, the cells were mixed with hydroxyapatite/tricalcium phosphate (HA/TCP) and implanted...

  10. In vitro biotransformation of tris(2-butoxyethyl) phosphate (TBOEP) in human liver and serum

    Energy Technology Data Exchange (ETDEWEB)

    Van den Eede, Nele, E-mail: nele.vandeneede@uantwerpen.be [Toxicological Center, Department of Pharmaceutical Sciences, University of Antwerp, Universiteitsplein 1, 2610 Wilrijk, Antwerp (Belgium); Erratico, Claudio [Toxicological Center, Department of Pharmaceutical Sciences, University of Antwerp, Universiteitsplein 1, 2610 Wilrijk, Antwerp (Belgium); Exarchou, Vassiliki [Natural Products & Food Research and Analysis (NatuRA), Department of Pharmaceutical Sciences, University of Antwerp, Universiteitsplein 1, 2610 Wilrijk, Antwerp (Belgium); Maho, Walid; Neels, Hugo [Toxicological Center, Department of Pharmaceutical Sciences, University of Antwerp, Universiteitsplein 1, 2610 Wilrijk, Antwerp (Belgium); Covaci, Adrian, E-mail: adrian.covaci@uantwerpen.be [Toxicological Center, Department of Pharmaceutical Sciences, University of Antwerp, Universiteitsplein 1, 2610 Wilrijk, Antwerp (Belgium)

    2015-04-15

    Tris(2-butoxyethyl) phosphate (TBOEP) is a plasticizer present in indoor dust, reaching levels of several micrograms per gram. Such levels could lead to significant daily exposure of adults and children. Currently, no toxicokinetic data are available to estimate TBOEP clearance in humans after uptake and therefore, one objective of this study was to investigate intrinsic clearance of TBOEP by human liver microsome (HLM) and serum enzymes. Another objective was to generate information to identify and prioritize several metabolites of TBOEP for investigation of human exposure by biomonitoring. 1D and 2D-NMR methodologies were successfully applied on a mixture of the metabolites to confirm the structure of 3-HO-TBOEP (bis(2-butoxyethyl) 3-hydroxyl-2-butoxyethyl phosphate) and to tentatively assign structures to 1-HO-TBOEP and 2-HO-TBOEP. HO-TBOEP isomers and bis(2-butoxyethyl) phosphate (BBOEP), bis(2-butoxyethyl) hydroxyethyl phosphate (BBOEHEP) were further monitored by liquid chromatography–tandem mass spectrometry. Rates of formation of BBOEHEP and HO-TBOEP metabolites by liver enzymes were best described by the Michaelis–Menten model. Apparent K{sub m} values for BBOEHEP, 3-HO-TBOEP, and sum of 1- and 2-HO-TBOEP isomer formation were 152, 197 and 148 μM, respectively. Apparent V{sub max} values for the formation of BBOEHEP, 3-HO-TBOEP, and the sum of 1- and 2-HO-TBOEP isomers were 2560, 643, and 254 pmol/min/mg protein, respectively. No detectable formation of BBOEP occurred with liver or serum enzymes. Our findings indicate that intrinsic clearance of TBOEP is mainly catalyzed by oxidative enzymes in the liver and that its major in vitro metabolite is BBOEHEP. These findings can be applied in human biomonitoring studies and risk assessment. - Highlights: • First steps in the elucidation of TBOEP toxicokinetics • Quantification of TBOEP metabolites in human serum and liver microsomes • No detectable formation of BBOEP occurred with liver or serum

  11. Maintenance of human embryonic stem cells in animal serum- and feeder layer-free culture conditions.

    Science.gov (United States)

    Amit, Michal; Itskovitz-Eldor, Joseph

    2006-01-01

    The availability of human embryonic stem cells (hESCs) reflects their outstanding potential for research areas such as human developmental biology, teratology, and cell-based therapies. To allow their continuous growth as undifferentiated cells, isolation and culturing were traditionally conducted on mouse embryonic fibroblast feeder layers, using medium supplemented with fetal bovine serum. However, these conditions allow possible exposure of the cells to animal pathogens. Because both research and future clinical application require an animal-free and well-defined culture system for hESCs, these conventional conditions would prevent the use of hESCs in human therapy. This chapter describes optional culture conditions based on either animal-free or feeder-free culture methods for hESCs.

  12. PRM-151 (recombinant human serum amyloid P/pentraxin 2) for the treatment of fibrosis.

    Science.gov (United States)

    Duffield, Jeremy S; Lupher, Mark L

    2010-06-01

    Serum amyloid P or pentraxin 2 (PTX2) is a highly phylogenetically conserved, naturally circulating plasma protein and a soluble pattern recognition receptor of the innate immune system. The unique binding activities of PTX2 suggest that it may localize specifically to sites of injury and function to aid in the removal of damaged tissue. The recent discovery of its ability to regulate certain monocyte differentiation states has identified PTX2 as a novel and potentially powerful antifibrotic agent. A fully recombinant form of the human PTX2 protein, designated PRM-151, has recently initiated human clinical trials. Here we review the molecular, cellular and structural biology of PRM-151/PTX2 in vitro and in several in vivo preclinical models of fibrotic disease that demonstrate its potential as a first-in-class natural modulator of fibrotic pathology with significant potential to treat a wide variety of human diseases.

  13. Effects of Fenton Reaction on Human Serum Albumin: An In Vitro Study

    Science.gov (United States)

    Khosravifarsani, Meysam; Monfared, Ali Shabestani; Pouramir, Mahdi; Zabihi, Ebrahim

    2016-01-01

    Introduction Human serum albumin (HSA) is a critical protein in human blood plasma, which can be highly damaged by oxidative stress. The aim of this study was to analyze modifications of this protein after oxidation using a Fenton system. Methods In this 2015 experiment, different ratios of Fenton reagent (Fe2+/H2O2) was incubated with one concentration of human serum albumin (1mg/ml). Hence, HSA was incubated 30 min with various combinations of a Fenton system and quantified oxidation products such as carbonyl groups, fragmentations, degradations, and oxidized free thiol group using reliable techniques. Image and data analysis were carried out using ImageJ software and Excel (version 2007), respectively. Results An SDS-PAGE profile showed no cross link and aggregation. However, protein band intensity has decreased to 50% in the highest ratio of H2O2/Fe. Carbonylation assay indicated carbonyl/protein (molc/molp) ratio increased linearly in lower ratios and the values plateau at higher levels of H2O2/Fe 2+. The only free sulfhydryl group on HSA was oxidized in all ratios of the Fenton system. Conclusion To sum, the structure of HSA has been changed following treatment with Hydroxyl Radical as the main product of Fenton reaction. These data confirm the antioxidant activity of HSA.

  14. Improvement in skin wrinkles using a preparation containing human growth factors and hyaluronic acid serum.

    Science.gov (United States)

    Lee, Do Hyun; Oh, In Young; Koo, Kyo Tan; Suk, Jang Mi; Jung, Sang Wook; Park, Jin Oh; Kim, Beom Joon; Choi, Yoo Mi

    2015-02-01

    Skin aging is accompanied by wrinkle formation. At some sites, such as the periorbital skin, this is a relatively early phenomenon. We evaluated the anti-wrinkle effect of a preparation containing human growth factor and hyaluronic acid serum on periorbital wrinkles (crow's feet). In total, 23 Korean women (age range: 39-59 years), who were not pregnant, nursing, or undergoing any concurrent therapy, were enrolled in this study. All the patients completed an 8-week trial of twice-daily application of human growth factor and hyaluronic acid serum on the entire face. Efficacy was based on a global photodamage score, photographs, and image analysis using replicas and visiometer analysis every 4 weeks. The standard wrinkle and roughness parameters used in assessing skin by visiometer were calculated and statistically analyzed. Periorbital wrinkles were significantly improved after treatment, with improvements noted both by physician's assessment and visiometer analysis. Topical application of human growth factor and hyaluronic acid was beneficial in reducing periorbital wrinkles.

  15. Serial color Doppler flow of uterine vasculature combined with serum beta-hCG measurements for improved monitoring of patients with gestational trophoblastic disease. A preliminary report.

    Science.gov (United States)

    Maymon, R; Schneider, D; Shulman, A; Bukowsky, I; Weinraub, Z

    1996-01-01

    Weekly serum beta-hCG measurements and transvaginal ultrasound scans coupled with color Doppler flow were performed on 8 patients with hydatidiform mole. Two patients later developed persistent trophoblastic disease, necessitating chemotherapy. The correlation coefficients between Doppler flow indices, systolic-diastolic (S/D) ratio and pulsatility index (PI) with log beta-hCG were -0.96 and -0.97, respectively. The weekly S/D and PI indices were plotted on an individual curve. Only the 2 patients who developed persistent gestational trophoblastic disease had PI index levels of < or = 1.5 as early as 2 weeks after molar evacuation. At that stage their serum beta-hCG levels were not different from some of the other patients. In this preliminary report, the regression of the disease could be reliably assessed by observing the changes in low resistance flow which paralleled the gradual decrements in serial beta-hCG levels. Thus, the contribution of this noninvasive imaging technique encourages the authors to further investigate Doppler flow monitoring among a larger sample of patients suffering from various gestational trophoblastic diseases.

  16. Human Umbilical Cord Blood-Derived Serum for Culturing the Supportive Feeder Cells of Human Pluripotent Stem Cell Lines

    Directory of Open Access Journals (Sweden)

    Ruttachuk Rungsiwiwut

    2016-01-01

    Full Text Available Although human pluripotent stem cells (hPSCs can proliferate robustly on the feeder-free culture system, genetic instability of hPSCs has been reported in such environment. Alternatively, feeder cells enable hPSCs to maintain their pluripotency. The feeder cells are usually grown in a culture medium containing fetal bovine serum (FBS prior to coculture with hPSCs. The use of FBS might limit the clinical application of hPSCs. Recently, human cord blood-derived serum (hUCS showed a positive effect on culture of mesenchymal stem cells. It is interesting to test whether hUCS can be used for culture of feeder cells of hPSCs. This study was aimed to replace FBS with hUCS for culturing the human foreskin fibroblasts (HFFs prior to feeder cell preparation. The results showed that HFFs cultured in hUCS-containing medium (HFF-hUCS displayed fibroblastic features, high proliferation rates, short population doubling times, and normal karyotypes after prolonged culture. Inactivated HFF-hUCS expressed important genes, including Activin A, FGF2, and TGFβ1, which have been implicated in the maintenance of hPSC pluripotency. Moreover, hPSC lines maintained pluripotency, differentiation capacities, and karyotypic stability after being cocultured for extended period with inactivated HFF-hUCS. Therefore, the results demonstrated the benefit of hUCS for hPSCs culture system.

  17. Thermodynamic Study of Human Serum Albumin upon Interaction with Ytterbium (III

    Directory of Open Access Journals (Sweden)

    G. Rezaei Behbehani

    2013-01-01

    Full Text Available Complexation reaction between Yb3+ and human serum albumin is examined using isothermal titration calorimetry (ITC. The extension solvation theory was used to reproduce the enthalpies of HAS + Yb3+ interactions over the whole range of Yb3+ concentrations. The binding parameters recovered from this model were attributed to the structural change of HSA. The results show that Yb3+ ions bind to HSA with three equivalent affinity sites. It was found that in the high concentrations of the ytterbium ions, the HSA structure was destabilized.

  18. Thermal investigation of Human Serum Albumin upon Interaction with Ytterbium (III

    Directory of Open Access Journals (Sweden)

    Gholamreza Rezaei Behbehani

    2012-01-01

    Full Text Available In this paper complexation reaction between Yb3+ and Human serum albumin is examined using isothermal titration calorimetry (ITC. The extended solvation model was used to reproduce the enthalpies of HAS+Yb3+ interactions over the whole range of Yb3+ concentrations. The binding parameters recovered from this model were attributed to the structural change of HSA. The results show that Yb3+ ions bind to HSA with three equivalent affinity sites. It was found that in the high concentrations of the ytterbium ions, the HSA structure was destabilized.

  19. Congener Specific Analysis of Polychlorinated Biphenyls (PCBs) in Human Blood Serum from Croatia

    OpenAIRE

    Krauthacker, Blanka; Reiner, Elsa

    2000-01-01

    A gas-chromatographic method on capillary columns is described for measuring concentrations of total PCBs and of six PCB congeners, PCB-28, PCB-52, PCB-101, PCB-138, PCB-153 and PCB-180, in human blood serum. Recovery of compounds was evaluated, and the repeatability and reproducibility of the results tested on samples analysed on the same day and over a period of two years. The method was verified in an international AQA Study in three rounds of measurements. The method was applied for the a...

  20. Investigation of the Interaction between Adenosine and Human Serum Albumin by Fluorescent Spectroscopy and Molecular Modeling

    Institute of Scientific and Technical Information of China (English)

    CUI Feng-Ling; WANG Jun-Li; LI Fang; FAN Jing; QU Gui-Rong; YAO Xiao-Jun; LEI Bei-Lei

    2008-01-01

    The binding interaction of adenosine with human serum albumin (HSA) was investigated under simulative physiological conditions by fluorescence spectroscopy in combination with a molecular modeling method. A strong fluorescence quenching reaction of adenosine to HSA was observed and the quenching mechanism was suggested as static quenching according to the Stern-Volmer equation. The binding constants (K) at different temperatures as well as thermodynamic parameters, enthalpy change (ΔH) and entropy change (ΔS), were calculated according to relevant fluorescent data and Vant'Hoff equation. The hydrophobic interaction was a predominant intermolecular force in order to stabilize the complex, which was in agreement with the results of molecular modeling study.

  1. 6-Nitro-L-tryptophan: a novel spectroscopic probe of trp aporepressor and human serum albumin.

    Science.gov (United States)

    Phillips, R S; Marmorstein, R Q

    1988-04-01

    The binding of 6-nitro-L-tryptophan to trp aporepressor and human serum albumin has been examined by visible difference spectroscopy and circular dichroism. 6-Nitro-L-tryptophan, prepared by nitration of L-tryptophan with nitric acid in glacial acetic acid, exhibits a visible and near-uv absorption spectrum with lambda max at about 330 nm (epsilon = 7 X 10(3) M-1 cm-1) and a shoulder near 380 nm in H2O. In the presence of trp aporepressor, the visible absorption intensity is sharply diminished. Visible difference spectral titration data give KD = 1.27 X 10(-4) M and n = 0.95 per subunit at 25 degrees C. While 6-nitro-L-tryptophan exhibits no significant circular dichroism between 300 and 500 nm, the complex with trp aporepressor exhibits strong circular dichroism signals, with a negative maximum at 386 nm (delta epsilon = -7.5 M-1 cm-1) and a positive maximum at 310 nm (delta epsilon = +6 M-1 cm-1). Circular dichroism titration data give KD = 1.69 X 10(-4) M and n = 0.90 per subunit at 25 degrees C. The KD values determined spectroscopically are in excellent agreement with that determined by equilibrium dialysis, KD = 1.5 X 10(-4) M at 25 degrees C. In the presence of human serum albumin, the spectrum of 6-nitro-L-tryptophan exhibits a blue shift and an increase in absorption intensity; similar changes are observed in solvents of low dielectric contrast such as 80% aqueous dioxane. Visible difference spectral titration data give KD = 8.0 X 10(-5) M and n = 0.95 for human serum albumin. The complex of 6-nitro-L-tryptophan with human serum albumin exhibits a strong positive circular dichroism maximum at 380 nm (delta epsilon = +9.8 M-1 cm-1) with a shoulder at 310-320 nm. Circular dichroism titration data give KD = 6.4 X 10(-5) M and n = 0.83, in good agreement with the visible difference spectral results. Taken together, our results demonstrate the utility of 6-nitro-L-tryptophan as a spectroscopic probe for tryptophan-binding proteins.

  2. VX Hydrolysis by Human Serum Paraoxonase 1: A Comparison of Experimental and Computational Results

    OpenAIRE

    Matthew W Peterson; Steven Z Fairchild; Otto, Tamara C.; Mojdeh Mohtashemi; Cerasoli, Douglas M.; Wenling E Chang

    2011-01-01

    Human Serum paraoxonase 1 (HuPON1) is an enzyme that has been shown to hydrolyze a variety of chemicals including the nerve agent VX. While wildtype HuPON1 does not exhibit sufficient activity against VX to be used as an in vivo countermeasure, it has been suggested that increasing HuPON1's organophosphorous hydrolase activity by one or two orders of magnitude would make the enzyme suitable for this purpose. The binding interaction between HuPON1 and VX has recently been modeled, but the mech...

  3. Effects of Krill Oil on serum lipids of hyperlipidemic rats and human SW480 cells

    Directory of Open Access Journals (Sweden)

    Qian Wen-Bin

    2008-08-01

    Full Text Available Abstract Background Cardiovascular disease (CVD and colon cancer incidence are known to be closely related to dietary factors. This article evaluated effects of krill oil (KO on serum lipids of hyperlipidemia rats and human colon cancer cells (SW480. Serum lipids of rats fed with high fat diet (HFD and different doses of KO were measured by automatic analyzer. Effect of KO on viability of cells was determined by methyl thiazolyl tetrazolium (MTT assay. Results Except for higher dose group, body weights decreased significantly. Total cholesterol (TC, LDL-cholesterol (LDL-C of all dose groups, Triglycerides (TG of low and mid dose groups descended significantly, while there were no significant differences of HDL-cholesterol (HDL-C, compared with control group. Treatment of colon cancer cells with KO also resulted in time-dependent inhibition of cell growth. Conclusion Our findings indicated that the consumption of KO may provide benefits to control serum lipid levels in certain diseases and inhibit growth of colon cancer cells. Therefore, KO may be a good candidate for development as a functional food and nutraceutical.

  4. Derivation and characterization of human embryonic germ cells: serum-free culture and differentiation potential.

    Science.gov (United States)

    Hua, Jinlian; Yu, Haisheng; Liu, Sheng; Dou, Zhongying; Sun, Yadong; Jing, Xiaoqi; Yang, Chunrong; Lei, Anmin; Wang, Huayan; Gao, Zhimin

    2009-08-01

    This study examined the effects of a chemically defined culture medium supplement, knock-out serum replacement (KSR), on the growth and differentiation of human embryonic germ cells (hEgc) and found that the efficiency of the initial establishment of hEGC lines in KSR medium was significantly higher than in fetal calf serum (FCS) medium. The percentage of undifferentiated hEGC colonies growing in KSR medium was significantly higher than in FCS-based medium (P embryonic germ cell-like morphology. They showed normal and stable diploid karyotype and expressed alkaline phosphatase (AP), stage-specific embryonic antigens (SSEA) and other specific markers of pluripotent cells. In addition, hEGC could form simple and cystic embryoid bodies (EB) that consisted of various cell types including neural, epithelial and rhythmically beating cardiac cells, even sperm-like and oocyte-like cells. Tumour-like outgrowths were formed in nude mice and found to contain a variety of cell types, including uterine epithelium, adipocytes, squamous tissue and skin structures. In conclusion, an appropriate serum-free culture system has been developed for the establishment of hEGC lines. This may provide an in-vitro model to study differentiation and can be used as a potential source of therapy for infertility and regenerative medicine.

  5. Effects of a honeybee sting on the serum free amino acid profile in humans.

    Directory of Open Access Journals (Sweden)

    Jan Matysiak

    Full Text Available The aim of this study was to assess the response to a honeybee venom by analyzing serum levels of 34 free amino acids. Another goal of this study was to apply complex analytic-bioinformatic-clinical strategy based on up-to-date achievements of mass spectrometry in metabolomic profiling. The amino acid profiles were determined using hybrid triple quadrupole/linear ion trap mass spectrometer coupled with a liquid chromatography instrument. Serum samples were collected from 27 beekeepers within 3 hours after they were stung and after a minimum of 6 weeks following the last sting. The differences in amino acid profiles were evaluated using MetaboAnalyst and ROCCET web portals. Chemometric tests showed statistically significant differences in the levels of L-glutamine (Gln, L-glutamic acid (Glu, L-methionine (Met and 3-methyl-L-histidine (3MHis between the two analyzed groups of serum samples. Gln and Glu appeared to be the most important metabolites for distinguishing the beekeepers tested shortly after a bee sting from those tested at least 6 weeks later. The role of some amino acids in the response of an organism to the honeybee sting was also discussed. This study indicated that proposed methodology may allow to identify the individuals just after the sting and those who were stung at least 6 weeks earlier. The results we obtained will contribute to better understanding of the human body response to the honeybee sting.

  6. Determination of guaifenesin in human serum by capillary gas chromatography and electron capture detection.

    Science.gov (United States)

    Sharaf, Maged H M; Stiff, Dwight D

    2004-06-29

    A method for the quantitation of guaifenesin in human serum has been developed and validated. The procedure involves liquid-liquid extraction of the serum sample in the presence of mephenesin as an internal standard, followed by derivatization and analysis using capillary gas chromatography (GC) and electron capture detection (ECD). Different solvents were tested for extraction of guaifenesin from serum. n-Hexane/dichloromethane (1:1, v/v) gave the highest recovery and the lowest background and was chosen as the extraction solvent. After extraction, the residue of guaifenesin was derivatized at 60 degrees C for 30 min, with trifluoroacetic acid anhydride (TFAA) in toluene in the presence of pyridine. Excess trifluoroacetic acid anhydride was removed using dilute solution of ammonium hydroxide. The method proved to be linear over the range of 25.0-1000 ng/ml. Recovery of guaifenesin from spiked samples was consistent, averaging 75.5% at 50.0 ng/ml with a range of 72.0-80.0% (N = 8 determinations) and averaging 78% at 800 ng/ml with a range of 76.0-81.0% (N = 8 determinations). The internal standard recovery was also consistent averaging 72.8% with a range of 67.0-76.0% (N = 16 determinations). Copyright 2004 Elsevier B.V.

  7. The proteomic analysis of human neonatal umbilical cord serum by mass spectrometry

    Institute of Scientific and Technical Information of China (English)

    Hong-juan SONG; Ping ZHANG; Xue-jiang GUO; Lian-ming LIAO; Zuo-min ZHOU; Jia-hao SHA; Yu-gui CUI; Hui JI; Jia-yin LIU

    2009-01-01

    Aim: To investigate the proteome composition and function of human neonatal arterial umbilical cord.Methods: Serum proteomic analyses were performed on samples from both males and females by using a combination of techniques: (1) removal of six high-abundance proteins, (2) tryptic digestion of low-abundance proteins, (3) separation of peptide mixtures by reverse-phase high-performance liquid chromatography (RP-HPLC), and (4) peptide identification using electrospray ionization tandem mass spectrometry (ESI-MS/MS).Results: A total of 837 non-redundant proteins were identified, with 213 male-specific and 239 female-specific proteins. Among them, 319 proteins were identified by at least 2 distinct peptides. The subcellular localization, function, and pathway involvement for each of the identified proteins were analyzed. A comparison of this neonatal proteome to that of adult serum proteome revealed novel bioma-rkers, such as alpha-fetoprotein and periostin that were specific to newborn infants.Conclusion: These data will contribute to a better understanding of the composition of umbilical cord serum and aid the discovery of novel biomarkers for the prenatal diagnosis of fetal abnormalities.

  8. Determination of meropenem levels in human serum by high-performance liquid chromatography with ultraviolet detection.

    Science.gov (United States)

    Roth, Thomas; Fiedler, Stella; Mihai, Sidonia; Parsch, Hans

    2017-05-01

    Meropenem is a β-lactam broad-spectrum antibiotic and belongs to the subgroup of carbapenems. It is primarily used in intensive care units for intravenous treatment of severe infections. To avoid bacterial resistance or toxic side effects, the determination of serum meropenem concentration is highly advisable. A simple and fast method for the quantitative determination of meropenem in human serum using high-performance liquid chromatography with ultraviolet detection (HPLC/UV) was developed and validated. Meropenem was determined by an isocratic HPLC using a tris(hydroxymethyl)aminomethane buffer (pH 8.5; 15% methanol) as a mobile phase and UV detection at 300 nm, with a flow rate of 1.0 mL/min and an analysis time of 10 min. Chromatographic separation was performed on a Kinetex C18 column (5 μm, 150 × 4.6 mm). In order to remove undesired serum components, solid-phase extraction was used for sample preparation. Since meropenem is not stable in solution, sample and stock solution were stored at -80°C. After preparation, samples were stable at room temperature for at least 6 h. The calibration curve was linear from 3.5 to 200 mg/L with a correlation coefficient r(2) of 0.999. The method is accurate with an intra- and inter-assay precision <18.5%. Copyright © 2016 John Wiley & Sons, Ltd.

  9. Detection of platelet deposition in cases of arteriosclerosis obliterans (ASO) using indium-111 platelets and Tc-99m human serum albumin

    Energy Technology Data Exchange (ETDEWEB)

    Kitagawa, Kazuo; Miyai, Motonobu; Etani, Hideki

    1987-02-01

    We evaluated platelet deposition in vivo in 10 patients with arteriosclerosis obliterans (ASO) of the lower limbs and 8 normal subjects with a dual-tracer technique using indium-111 platelets and Tc-99m human serum albumin. Each patient with ASO showed intermittent claudication and angiographically occlusive vascular lesions in either the aorta, common iliac artery, external iliac artery, internal iliac artery or femoral artery. Of the 8 patients who were not under antiplatelet medication, 5 showed positive platelet deposition at angiographically occlusive vascular sites, whereas none of the normal subjects showed in vivo platelet deposition. In three patients who showed platelet deposition without antiplatelet medication and thereafter received aspirin therapy (650 mg/day), platelet deposition at all occlusive vascular sites was resolved after aspirin therapy. This preliminary study indicated that platelet scintigraphy might be useful in evaluating thrombogenicity and the effect of antiplatelet medication in vivo, in patients with ASO.

  10. [Influence of the human blood serum on contractility and beta-adrenoreactyvity of the isolated human myocardium].

    Science.gov (United States)

    Korotaeva, K N; Viaznikov, V A; Tsirkin, V I; Kostiaev, A A

    2011-01-01

    On strips of the isolated myocardium of right hearts auriculum of the 43 patients with ischemic illness of heart and 9 patients with heart diseases of various ethyology at statement venous canule during aorto-coronary shunting, estimated influence of adrenaline (10(-9)-10(-4) g/ml) on amplitude caused by electrostimulus (1H, 5ms, 25-30 V) contractions, and also inotropic and adrenomodulation activity of serum blood (in dilution 1 : 10000, 1: 1000, 1 : 500, 1: 100, 1 : 50, 1: 10 and 1 : 5) nonpregnant women. Direct dependence of amplitude of contraction on size of fraction of of blood emission on Teyholts is revealed. It means, that strips of right auriculum myocardium reflect contractility of a left ventriculum myocardium. Adrenaline in concentration 10(-7)-10(-6) g/ml dependent of dose raised amplitude of the caused contraction not influencing it in concentration of 10(-9) and 10(-8) g/ml (the constant of dissotiation has 2 x 10(-7) g/ml), that as a whole, speaks about decrease in efficiency of activation beta-AP. Blood Serum in dissolutions 1 : 10000-1 : 50 did not influence on amplitude of contraction, and in dissolutions 1 : 10 and 1 : 5 strengthened it, that speaks presence in blood the endogenous activator of myocyte contractility (EAMC). Serum showed beta-adrenomodulation activity that speaks presence in it endogenous sensitizer of beta-adrenoreceptors (ESBAR) and endogenous blocker of beta-adrenoreceptors (EBBAR). In particular, in experiences with adrenaline in subthreshold concentration (10(-8) g/ml) serum showed ESBAR-activity (in dissolutions 1 : 1000, 1 : 500, 1 : 100 and 1 : 50), and in experiences with adrenaline in as much as possible effective concentration (10(-6) g/ml) serum showed ESBAR-activity (in dissolutions 1 : 50 and 1 : 10) and EBBAR-activity (in dissolutions 1:500) Hence, containing in blood serum endogenous modulators of beta-adrenoreactivity - ESBAR and EBBAR can modulate efficiency of beta-adrenoreceptors activation of human

  11. Autologous serum skin test reactivity and basophil histamine release test in patients with nasal polyposis: preliminary results.

    Science.gov (United States)

    Zambetti, G; Ciofalo, A; Soldo, P; Fusconi, M; Romeo, R; Greco, A; Altissimi, G; Macri, G F; Marinelli, C; Pagliuca, G; De Vincentiis, M

    2010-01-01

    An eosinophilic inflammatory process is generally observed in patients suffering from nasal polyposis (NP), however its onset has not yet been defined. It has been suggested that immune activation of inflammatory cells may be the cause. The aim of this study is to verify whether autoantibodies and/or histamine-releasing factors are present in the serum of patients suffering from NP. In fact, we assume that autoantibodies and/or histamine-releasing factors, as already demonstrated in chronic idiopathic urticaria and asthma, may be involved in the pathogenesis of NP. In this case-control analytical study 40 patients with NP and 27 control subjects underwent the in vivo autologous serum skin test (ASST). The sera from 6 patients suffering from NP and 9 control group subjects, who had all been previously studied and randomly selected, underwent basophil histamine release assay from normal donor as a pilot study. The ASST showed positive results in 55% of patients suffering from NP versus 8% of the control group (p= .00006), the basophil histamine release test (BHRT) turned out positive in all patients tested and in 11% of the control group. We found a weak positive correlation between the percentage of histamine release and the wheal diameter. ASST reactivity is very frequent in patients suffering from NP, thus suggesting the presence of histamine-releasing factors in the blood stream. The BHRT was positive in the serum of all patients, thus suggesting the presence of anti-FcepsilonRI, anti-IgE autoantibodies and/or other histamine-releasing factors, the presence of which can play a role in triggering and maintaining the eosinophilic inflammatory process in NP.

  12. Kinetics of tris (1-chloro-2-propyl) phosphate (TCIPP) metabolism in human liver microsomes and serum.

    Science.gov (United States)

    Van den Eede, Nele; Tomy, Gregg; Tao, Fang; Halldorson, Thor; Harrad, Stuart; Neels, Hugo; Covaci, Adrian

    2016-02-01

    Tris(1-chloro-2-propyl) phosphate (TCIPP) is an emerging contaminant which is ubiquitous in the indoor and outdoor environment. Moreover, its presence in human body fluids and biota has been evidenced. Since no quantitative data exist on the biotransformation or stability of TCIPP in the human body, we performed an in vitro incubation of TCIPP with human liver microsomes (HLM) and human serum (HS). Two metabolites, namely bis(2-chloro-isopropyl) phosphate (BCIPP) and bis(1-chloro-2-propyl) 1-hydroxy-2-propyl phosphate (BCIPHIPP), were quantified in a kinetic study using HLM or HS (only BCIPP, the hydrolysis product) and LC-MS. The Michaelis-Menten model fitted best the NADPH-dependent formation of BCIPHIPP and BCIPP in HLM, with respective V(MAX) of 154 ± 4 and 1470 ± 110 pmol/min/mg protein and respective apparent K(m) of 80.2 ± 4.4 and 96.1 ± 14.5 μM. Hydrolases, which are naturally present in HLM, were also involved in the production of BCIPP. A HS paraoxonase assay could not detect any BCIPP formation above 38.6 ± 10.8 pmol/min/μL serum. Our data indicate that BCIPP is the major metabolite of TCIPP formed in the liver. To our knowledge, this is the first quantitative assessment of the stability of TCIPP in tissues of humans or any other species. Further research is needed to confirm whether these biotransformation reactions are associated with a decrease or increase in toxicity.

  13. Column-switching LC-MS/MS analysis for quantitative determination of testosterone in human serum.

    Science.gov (United States)

    Borrey, Daniëlle; Moerman, Elke; Cockx, An; Engelrelst, Veronique; Langlois, Michel R

    2007-07-01

    An accurate measurement of testosterone is needed in many clinical applications for correct diagnosis and appropriate treatment. Our aim was to develop a fast and robust high-throughput LC-MSMS method for quantification of serum testosterone in women. Testosterone was derivatized by oximation and extracted with methyl tert-butyl ether from 200 microL of serum. Further matrix elimination was achieved on-line using a column-switching LC-method. The instrumental analysis was performed on an API4000 tandem mass spectrometer equipped with an Agilent series 1312A binary pump and an Agilent series 1311A quaternary pump. The MRM transitions were 304-->124 and 304-->112 for testosterone and 307-->124 and 307-->112 for d(3)-testosterone. The total analysis time of the column-switching method was 3 min. Linear calibration curves were obtained in the concentration range from 0.035 nmol/L (0.01 microg/L) to 6.92 nmol/L (2 microg/L). Within-day and between-day precision, expressed as the relative standard deviation at four different concentrations ranged from 4.70% to 9.35%. Correlation with the in-house method (solvent-extraction RIA) showed r(2)=0.920. The presented column-switching method offers a simple, fast and economical analysis of testosterone in human serum. The procedure requires only small sample volumes and is well suited for quantification of testosterone in serum from women and children.

  14. Metallomics studies of human blood serum from treated bipolar disorder patients.

    Science.gov (United States)

    Sussulini, Alessandra; Kratzin, Hartmut; Jahn, Olaf; Banzato, Claudio E Muller; Arruda, Marco A Zezzi; Becker, Johanna Sabine

    2010-07-01

    In the present work, metallomics studies using biomolecular (matrix-assisted laser desorption ionization time-of-flight tandem mass spectrometry, MALDI-TOF MS/MS) and elemental mass spectrometry (laser ablation inductively coupled plasma mass spectrometry, LA-ICPMS) of human blood serum samples from bipolar disorder (BD) patients compared to controls were performed. The serum samples from three different groups: control (n = 25), BD patients treated with Li (n = 15), and BD patients not treated with Li (n = 10), were pooled according to their groups and separated by two-dimensional polyacrylamide gel electrophoresis (2-D PAGE). Then, in order to determine the metals bound to the protein spots and search for differences among the studied groups, the 2-D gels were analyzed by LA-ICPMS in three distinct modes: bioimaging of metals in gel sections, line scan through the protein spots, and microlocal analysis of selected protein spots. MALDI-TOF MS/MS characterized 32 serum proteins, and they were associated with the metals previously detected. When comparing control and treated BD patient groups, a differentiation in terms of metals bound to proteins was possible to observe. The main metals bound to proteins found in all groups were Na, Mg, Zn, Ca, and Fe. Mn was only detected in the control group; Co was only observed in the control and BD patients treated with Li group. K and Ti were only found in the BD patient groups, and P was only observed in control and BD patients not treated with Li drugs. This exploratory work shows that the association of LA-ICPMS with MALDI-TOF MS/MS is a powerful strategy in metallomics studies applied to determine differences in metal-containing proteins, being able to play an important role on the discovery of potential markers for BD and its treatment with Li in serum samples.

  15. Radioimmunoassay of a human serum growth factor for Balb/c-3T3 cells: derivation from platelets.

    Science.gov (United States)

    Antoniades, H N; Scher, C D

    1977-05-01

    A radioimmunoassay has been developed for the detection and quantification of a human serum polypeptide that has growth-promoting activity for confluent Balb/c-3T3 cells. Antiserum to this growth factor does not recognize antigens in mouse, guinea pig, or bovine serum but does detect some crossreacting antigen in the serum of the New World monkey Cebus albifrons and more in the serum of the Old World rhesus monkeys Macaca mulatta and M. fascicularis, demonstrating that the antigenic determinants of the growth factor have a degree of species specificity. Serum derived from whole human blood contains approximately 770 pg of the growth factor per mg of protein; serum derived from platelet-poor blood contains about 112 pg of the growth factor per mg of protein. As much as 1 microng of the growth factor per mg of protein has been recovered from human platelets by heating them at 100 degrees for 2 min. Approximately 1-2 ng of the growth factor, in either whole serum or platelets, stimulates 5 to 10 X 10(3) confluent Balb/c-3T3 cells to replicate. The heat treatment of platelets allows the purification and quantitative recovery of the growth factor from blood.

  16. Preliminary study on human fibroblasts as feeder layer for human embryonic stem cells culture in vitro

    Institute of Scientific and Technical Information of China (English)

    2003-01-01

    To avoid the direct contact with mouse cells and possible heterogeneous pathogen in future application, we need to replace mouse embryonic fibroblastswith human fibroblasts as the feeder layer to maintain human embryonic stem cells growth in the undifferentiated state. We successfully use human fibroblasts derived from aborted fetus and adult prepuce as feeder layer to maintain human embryonic stem cells growth. During the passage and growth on this feeder layer, the human embryonic stem cells can keep their undifferentiated state.

  17. Cabazitaxel-loaded human serum albumin nanoparticles as a therapeutic agent against prostate cancer

    Directory of Open Access Journals (Sweden)

    Qu N

    2016-07-01

    Full Text Available Na Qu,1 Robert J Lee,1,2 Yating Sun,1 Guangsheng Cai,1 Junyang Wang,1 Mengqiao Wang,1 Jiahui Lu,1 Qingfan Meng,1 Lirong Teng,1 Di Wang,1 Lesheng Teng1,3 1School of Life Sciences, Jilin University, Changchun, People’s Republic of China; 2Division of Pharmaceutics, College of Pharmacy, The Ohio State University, Columbus, OH, USA; 3State Key Laboratory of Long-acting and Targeting Drug Delivery System, Yantai, People’s Republic of China Abstract: Cabazitaxel-loaded human serum albumin nanoparticles (Cbz-NPs were synthesized to overcome vehicle-related toxicity of current clinical formulation of the drug based on Tween-80 (Cbz-Tween. A salting-out method was used for NP synthesis that avoids the use of chlorinated organic solvent and is simpler compared to the methods based on emulsion-solvent evaporation. Cbz-NPs had a narrow particle size distribution, suitable drug loading content (4.9%, and superior blood biocompatibility based on in vitro hemolysis assay. Blood circulation, tumor uptake, and antitumor activity of Cbz-NPs were assessed in prostatic cancer xenograft-bearing nude mice. Cbz-NPs exhibited prolonged blood circulation and greater accumulation of Cbz in tumors along with reduced toxicity compared to Cbz-Tween. Moreover, hematoxylin and eosin histopathological staining of organs revealed consistent results. The levels of blood urea nitrogen and serum creatinine in drug-treated mice showed that Cbz-NPs were less toxic than Cbz-Tween to the kidneys. In conclusion, Cbz-NPs provide a promising therapeutic for prostate cancer. Keywords: cabazitaxel, human serum albumin, nanoparticle, drug delivery, toxicity, pros­tate cancer

  18. Quantification of a cardiac biomarker in human serum using extraordinary optical transmission (EOT.

    Directory of Open Access Journals (Sweden)

    Tao Ding

    Full Text Available Nanoimprinting lithography (NIL is a manufacturing process that can produce macroscale surface areas with nanoscale features. In this paper, this technique is used to solve three fundamental issues for the application of localized surface plasmonic resonance (LSPR in practical clinical measurements: assay sensitivity, chip-to-chip variance, and the ability to perform assays in human serum. Using NIL, arrays of 140 nm square features were fabricated on a sensing area of 1.5 mm x 1.5 mm with low cost. The high reproducibility of NIL allowed for the use of a one-chip, one-measurement approach with 12 individually manufactured surfaces with minimal chip-to-chip variations. To better approximate a real world setting, all chips were modified with a biocompatible, multi-component monolayer and inter-chip variability was assessed by measuring a bioanalyte standard (2.5-75 ng/ml in the presence of a complex biofluid, human serum. In this setting, nanoimprinted LSPR chips were able to provide sufficient characteristics for a 'low-tech' approach to laboratory-based bioanalyte measurement, including: 1 sufficient size to interface with a common laboratory light source and detector without the need for a microscope, 2 high sensitivity in serum with a cardiac troponin limit of detection of 0.55 ng/ml, and 3 very low variability in chip manufacturing to produce a figure of merit (FOM of 10.5. These findings drive LSPR closer to technical comparability with ELISA-based assays while preserving the unique particularities of a LSPR based sensor, suitability for multiplexing and miniaturization, and point-of-care detections.

  19. Quantification of a cardiac biomarker in human serum using extraordinary optical transmission (EOT).

    Science.gov (United States)

    Ding, Tao; Hong, Minghui; Richards, A Mark; Wong, Ten It; Zhou, Xiaodong; Drum, Chester Lee

    2015-01-01

    Nanoimprinting lithography (NIL) is a manufacturing process that can produce macroscale surface areas with nanoscale features. In this paper, this technique is used to solve three fundamental issues for the application of localized surface plasmonic resonance (LSPR) in practical clinical measurements: assay sensitivity, chip-to-chip variance, and the ability to perform assays in human serum. Using NIL, arrays of 140 nm square features were fabricated on a sensing area of 1.5 mm x 1.5 mm with low cost. The high reproducibility of NIL allowed for the use of a one-chip, one-measurement approach with 12 individually manufactured surfaces with minimal chip-to-chip variations. To better approximate a real world setting, all chips were modified with a biocompatible, multi-component monolayer and inter-chip variability was assessed by measuring a bioanalyte standard (2.5-75 ng/ml) in the presence of a complex biofluid, human serum. In this setting, nanoimprinted LSPR chips were able to provide sufficient characteristics for a 'low-tech' approach to laboratory-based bioanalyte measurement, including: 1) sufficient size to interface with a common laboratory light source and detector without the need for a microscope, 2) high sensitivity in serum with a cardiac troponin limit of detection of 0.55 ng/ml, and 3) very low variability in chip manufacturing to produce a figure of merit (FOM) of 10.5. These findings drive LSPR closer to technical comparability with ELISA-based assays while preserving the unique particularities of a LSPR based sensor, suitability for multiplexing and miniaturization, and point-of-care detections.

  20. Impact of polychlorinated biphenyls contamination on estrogenic activity in human male serum.

    Science.gov (United States)

    Plísková, Martina; Vondrácek, Jan; Canton, Rocio Fernandez; Nera, Jirí; Kocan, Anton; Petrík, Ján; Trnovec, Tomás; Sanderson, Thomas; van den Berg, Martin; Machala, Miroslav

    2005-10-01

    Polychlorinated biphenyls (PCBs) are thought to cause numerous adverse health effects, but their impact on estrogen signaling is still not fully understood. In the present study, we used the ER-CALUX bioassay to determine estrogenic/antiestrogenic activities of the prevalent PCB congeners and PCB mixtures isolated from human male serum. The samples were collected from residents of an area with an extensive environmental contamination from a former PCB production site as well as from a neighboring background region in eastern Slovakia. We found that the lower-chlorinated PCBs were estrogenic, whereas the prevalent higher-chlorinated PCB congeners 138, 153, 170, 180, 187, 194, 199, and 203, as well as major PCB metabolites, behaved as antiestrogens. Coplanar PCBs had no direct effect on estrogen receptor (ER) activation in this in vitro model. In human male serum samples, high levels of PCBs were associated with a decreased ER-mediated activity and an increased dioxin-like activity, as determined by the DR-CALUX assay. 17beta-Estradiol (E2) was responsible for a major part of estrogenic activity identified in total serum extracts. Significant negative correlations were found between dioxin-like activity, as well as mRNA levels of cytochromes P450 1A1 and 1B1 in lymphocytes, and total estrogenic activity. For sample fractions containing only persistent organic pollutants (POPs), the increased frequency of antiestrogenic samples was associated with a higher sum of PCBs. This suggests that the prevalent non-dioxin-like PCBs were responsible for the weak antiestrogenic activity of some POPs fractions. Our data also suggest that it might be important to pay attention to direct effects of PCBs on steroid hormone levels in heavily exposed subjects.

  1. p53 Response to Ultrasound: Preliminary Observations in MCF7 Human Breast Cancer Cells

    Science.gov (United States)

    Burns, Janis M.; Campbell, Paul A.

    2011-09-01

    Mutated p53 can be found in approximately half of all human cancers. Strategies which seek to restore, or at least exercise a level of external control over, p53 functionality are thus potentially useful as adjuncts to therapy. Here, we report our preliminary measurements in this area, and demonstrate that short-burst pulsed ultrasound can indeed affect p53 activity. Specifically, we have observed that expression of the p53 protein can be regulated in the period immediately following low intensity short pulse (millisecond) ultrasound exposure, and that altered activity levels return to basal levels over a 24 hour period post-insonation.

  2. Targeted pharmacological depletion of serum amyloid P component for treatment of human amyloidosis.

    Science.gov (United States)

    Pepys, M B; Herbert, J; Hutchinson, W L; Tennent, G A; Lachmann, H J; Gallimore, J R; Lovat, L B; Bartfai, T; Alanine, A; Hertel, C; Hoffmann, T; Jakob-Roetne, R; Norcross, R D; Kemp, J A; Yamamura, K; Suzuki, M; Taylor, G W; Murray, S; Thompson, D; Purvis, A; Kolstoe, S; Wood, S P; Hawkins, P N

    2002-05-16

    The normal plasma protein serum amyloid P component (SAP) binds to fibrils in all types of amyloid deposits, and contributes to the pathogenesis of amyloidosis. In order to intervene in this process we have developed a drug, R-1-[6-[R-2-carboxy-pyrrolidin-1-yl]-6-oxo-hexanoyl]pyrrolidine-2-carboxylic acid, that is a competitive inhibitor of SAP binding to amyloid fibrils. This palindromic compound also crosslinks and dimerizes SAP molecules, leading to their very rapid clearance by the liver, and thus produces a marked depletion of circulating human SAP. This mechanism of drug action potently removes SAP from human amyloid deposits in the tissues and may provide a new therapeutic approach to both systemic amyloidosis and diseases associated with local amyloid, including Alzheimer's disease and type 2 diabetes.

  3. Chronocoulometric determination of urea in human serum using an inkjet printed biosensor.

    Science.gov (United States)

    Suman; O'Reilly, Emmet; Kelly, Michele; Morrin, Aoife; Smyth, Malcolm R; Killard, Anthony J

    2011-07-04

    A biosensor for the determination of urea in human serum was fabricated using a combination of inkjet printed polyaniline nanoparticles and inkjet printed urease enzyme deposited sequentially onto screen-printed carbon paste electrodes. Chronocoulometry was used to measure the decomposition of urea via the doping of ammonium at the polyaniline-modified electrode surface at -0.3 V vs. Ag/AgCl. Ammonium could be measured in the range from 0.1 to 100 mM. Urea could be measured by the sensor in the range of 2-12 mM (r(2)=0.98). The enzyme biosensor was correlated against a spectrophotometric assay for urea in 15 normal human serum samples which yielded a correlation coefficient of 0.85. Bland-Altman plots showed that in the range of 5.8-6.6 mM urea, the developed sensor had an average positive experimental bias of 0.12 mM (<2% RSD) over the reference method.

  4. Effects of fluorinated and hydrogenated surfactants on human serum albumin at different pHs.

    Science.gov (United States)

    Sabín, Juan; Prieto, Gerardo; González-Pérez, Alfredo; Ruso, Juan M; Sarmiento, Félix

    2006-01-01

    Complexation between human serum albumin (HSA) and two different surfactants, one fully fluorinated (sodium perfluorooctanoate, SPFO) and one fully hydrogenated (sodium caprylate, SO), was studied using zeta-potential measurements and difference spectroscopy. The study was carried out at three different pHs, 3.2, 6.7, and 10.0. The spectroscopy study was performed at pHs 6.7 and 10.0, given that at pH 3.2 high turbidity was observed in the wide range of surfactant concentrations. The results were interpreted in terms of the electrostatic and hydrophobic contributions to the stability of the different phases formed in the water-surfactant-HSA system. Solutions and precipitates were observed in the concentration range investigated in more detail. Using Pace methods, the thermodynamic values of the surfactant-induced conformational changes in HSA were determined for sodium perfluorooctanoate in the concentration range 2-12 mmol dm(-3) at pH 6.7 and 5-22 mmol dm(-3) at pH 10.0. Electrophoretic measurements were used to characterize surfactant adsorption by determining the number of molecules adsorbed on the surface of HSA and the Gibbs energy of adsorption. Finally, the interactions between human serum albumin and other anionic surfactants studied by other authors were compared with those observed in the present work.

  5. Molecular displacement of warfarin from human serum albumin by flavonoid aglycones

    Energy Technology Data Exchange (ETDEWEB)

    Poór, Miklós [Institute of Laboratory Medicine, University of Pécs, Pécs H-7624 (Hungary); Li, Yin; Kunsági-Máté, Sándor [Department of General and Physical Chemistry, University of Pécs, Pécs H-7624 (Hungary); János Szentágothai Research Center, H-7624 Pécs (Hungary); Petrik, József [Department of Medical Biochemistry and Hematology, Faculty of Pharmacy and Biochemistry, University of Zagreb, HR-10000 Zagreb (Croatia); Vladimir-Knežević, Sanda [Department of Pharmacognosy, Faculty of Pharmacy and Biochemistry, University of Zagreb, HR-10000 Zagreb (Croatia); Kőszegi, Tamás, E-mail: koszegit@freemail.hu [Institute of Laboratory Medicine, University of Pécs, Pécs H-7624 (Hungary)

    2013-10-15

    The well-known 4-hydroxycoumarin derivative warfarin is a widespread anticoagulant drug. Besides its strong albumin binding property warfarin has a narrow therapeutic window. Therefore, a few percent of displacement from albumin can result in serious biological consequences. The flavonoid molecular group also shows very strong plasma albumin binding characteristics occupying the same binding site. It is plausible to hypothesize that flavonoid aglycones may be able to displace warfarin from human serum albumin (HSA). In our study the competing activities of different flavone (acacetin, apigenin, chrysin, luteolin), flavonol (galangin, quercetin) and flavanone (hesperetin, naringenin) aglycones were investigated using fluorescence spectroscopy. Our results represent that flavonoids are able to displace warfarin from the surface of HSA. On the other hand, when comparing flavone or flavonol groups to flavanones the latter group seems to be much weaker competitor. These observations were also supported by calculation of stability constants. Our investigations strongly suggest that we should reckon with the described molecular displacement. However, further in vivo studies are needed to support the findings of our model system. -- Highlights: • Various flavonoids are able to displace warfarin from human serum albumin. • Flavones and flavonols are much more effective competitors than flavanones. • Even 300 nM aglycone concentrations show the interaction with 3 μM warfarin. • Flavonoid pairs show quasi-additive desorbing property. • Flavones and flavonols are much stronger competitors than the examined drugs.

  6. Interactions of hybrid gold-tannic acid nanoparticles with human serum albumin.

    Science.gov (United States)

    Sekowski, Szymon; Tomaszewska, Emilia; Soliwoda, Katarzyna; Celichowski, Grzegorz; Grobelny, Jaroslaw

    2017-01-01

    Nanoparticles present a wide spectrum of chemical, biological, and physical properties which result in their usage in many branches of science. We present an investigation of the interaction between human serum albumin and hybrid gold-tannic acid nanoparticles synthesized via a chemical reduction method. The results obtained demonstrate that tannic acid can be a very effective reducing and stabilizing agent and allows monodisperse hybrid gold nanomaterial to be obtained. The synthesized hybrid gold-tannic acid nanoparticles strongly interact with human serum albumin by formation of protein-corona complexes. The strength of the interaction with albumin depends on the number of tannic acid molecules on the surface of the nanoparticles and the presence of citric acid. Nanoparticles of large size and rich in tannic acid react more strongly with the protein [K SV = (8.00 ± 0.2) × 10(5) M(-1)] compared with smaller ones [K SV = (6.83 ± 0.5) × 10(4) M(-1)] containing citric acid and low concentration of tannic acid.

  7. Structural consistency analysis of recombinant and wild-type human serum albumin

    Science.gov (United States)

    Cao, Hui-Ling; Sun, Li-Hua; Liu, Li; Li, Jian; Tang, Lin; Guo, Yun-Zhu; Mei, Qi-Bing; He, Jian-Hua; Yin, Da-Chuan

    2017-01-01

    Recombinant human serum albumin (rHSA) is potential alternatives for human serum albumin (HSA) which may ease severe shortage of HSA worldwide. In theory, rHSA and HSA are the same. Structure decides function. Therefore, the 3D structural consistency analysis of rHSA and HSA is outmost importance, which is the base of their function consistency. In this paper, the crystal structures of rHSA at resolution limit of 2.22 Å and HSA at 2.30 Å were determined by X-ray diffraction (XRD), which were deposited in the Protein Data Bank (PDB) with accession codes 4G03 (rHSA) and 4G04 (HSA). The differences between rHSA and HSA were systematically analyzed from the crystallization behavior, diffraction data and three-dimensional (3D) structure. The superimposed contrasted analysis indicated that rHSA and HSA achieved a structural similarity of 99% with an r.m.s. deviation of 0.397 Å for the corresponding overall Cα atoms. In addition, the number of α-helices in the rHSA or HSA molecule was verified to be 30. As a result, rHSA can potentially replace HSA. The study provides a theoretical and experimental basis for the clinical and additional applications of rHSA. Meanwhile, it is also a good example for applications of genetic engineering.

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

    Energy Technology Data Exchange (ETDEWEB)

    Kielmas, Martyna; Szewczuk, Zbigniew; Stefanowicz, Piotr, E-mail: Piotr.stefanowicz@chem.uni.wroc.pl

    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.

  9. Serum or breast milk immunoglobulins mask the self-reactivity of human natural IgG antibodies.

    Science.gov (United States)

    Djoumerska-Alexieva, Iglika; Manoylov, Iliyan; Dimitrov, Jordan D; Tchorbanov, Andrey

    2014-04-01

    B cells producing IgG antibodies specific to a variety of self- or foreign antigens are a normal constituent of the immune system of all healthy individuals. These naturally occurring IgG antibodies are found in the serum, external secretions, and pooled human immunoglobulin preparations. They bind with low affinity to antigens, which can also be targets for pathologic autoantibodies. An enhancement of naturally occurring IgG autoantibody activity was observed after treatment of human IgG molecules with protein-destabilizing agents. We have investigated the interactions of human immunoglobulins that were obtained from serum or from breast milk of healthy individuals or IVIg with human liver antigens. Proteins from an individual serum or milk were isolated by two methods, one of which included exposure to low pH and the other did not. Purified serum, mucosal IgM, IgA, and the fraction containing immunoglobulin G F(ab')2 fragments each inhibited the binding of a single donor or pooled IgG to human liver antigens. Our study presents findings regarding the role of the breast milk or serum antibodies in blocking the self-reactivity of IgG antibodies. It supports the suggestion that not IVIg only, but also the pooled human IgM and IgA might possess a potent beneficial immunomodulatory activity in autoimmune patients.

  10. In vitro biotransformation of tris(2-butoxyethyl) phosphate (TBOEP) in human liver and serum.

    Science.gov (United States)

    Van den Eede, Nele; Erratico, Claudio; Exarchou, Vassiliki; Maho, Walid; Neels, Hugo; Covaci, Adrian

    2015-04-15

    Tris(2-butoxyethyl) phosphate (TBOEP) is a plasticizer present in indoor dust, reaching levels of several micrograms per gram. Such levels could lead to significant daily exposure of adults and children. Currently, no toxicokinetic data are available to estimate TBOEP clearance in humans after uptake and therefore, one objective of this study was to investigate intrinsic clearance of TBOEP by human liver microsome (HLM) and serum enzymes. Another objective was to generate information to identify and prioritize several metabolites of TBOEP for investigation of human exposure by biomonitoring. 1D and 2D-NMR methodologies were successfully applied on a mixture of the metabolites to confirm the structure of 3-HO-TBOEP (bis(2-butoxyethyl) 3-hydroxyl-2-butoxyethyl phosphate) and to tentatively assign structures to 1-HO-TBOEP and 2-HO-TBOEP. HO-TBOEP isomers and bis(2-butoxyethyl) phosphate (BBOEP), bis(2-butoxyethyl) hydroxyethyl phosphate (BBOEHEP) were further monitored by liquid chromatography-tandem mass spectrometry. Rates of formation of BBOEHEP and HO-TBOEP metabolites by liver enzymes were best described by the Michaelis-Menten model. Apparent Km values for BBOEHEP, 3-HO-TBOEP, and sum of 1- and 2-HO-TBOEP isomer formation were 152, 197 and 148μM, respectively. Apparent Vmax values for the formation of BBOEHEP, 3-HO-TBOEP, and the sum of 1- and 2-HO-TBOEP isomers were 2560, 643, and 254pmol/min/mg protein, respectively. No detectable formation of BBOEP occurred with liver or serum enzymes. Our findings indicate that intrinsic clearance of TBOEP is mainly catalyzed by oxidative enzymes in the liver and that its major in vitro metabolite is BBOEHEP. These findings can be applied in human biomonitoring studies and risk assessment.

  11. 1H NMR analysis of GHB and GBL: further findings on the interconversion and a preliminary report on the analysis of GHB in serum and urine.

    Science.gov (United States)

    Del Signore, Anthony G; McGregor, Michael; Cho, Bongsup P

    2005-01-01

    A 1H nuclear magnetic resonance (1H NMR) method for the determination of gamma-hydroxybutyric acid (GHB) and gamma-hydroxybutyrolactone (GBL) in human serum and urine using spiked samples has been developed. The method gives linear responses (correlation coefficients of 0.99 or greater) over the concentration range 0.01 mg/mL to 4.0 mg/mL in urine and 0.3 mg/mL to 2.0 mg/mL in serum. No sample pretreatment is required. Studies of the chemical interconversion of GBL and GHB showed hydrolysis of GBL to be rapid at pH 11.54, slower and less complete (30% hydrolysis) at pH 2.54 and slowest at pH 7.0, reaching 30% hydrolysis in about 40 days. No esterification of GHB was observed at any pH.

  12. Cupric ion reducing antioxidant capacity assay for antioxidants in human serum and for hydroxyl radical scavengers.

    Science.gov (United States)

    Apak, Reşat; Güçlü, Kubilay; Ozyürek, Mustafa; Bektaşoğlu, Burcu; Bener, Mustafa

    2010-01-01

    Tests measuring the combined antioxidant effect of the nonenzymatic defenses in biological fluids may be useful in providing an index of the organism's capability to counteract reactive species known as pro-oxidants, resist oxidative damage, and combat oxidative stress-related diseases. The selected chromogenic redox reagent for the assay of human serum should be easily accessible, stable, selective, and respond to all types of biologically important antioxidants such as ascorbic acid, alpha-tocopherol, beta-carotene, reduced glutathione (GSH), uric acid, and bilirubin, regardless of chemical type or hydrophilicity. Our recently developed cupric reducing antioxidant capacity (CUPRAC) spectrophotometric method for a number of polyphenols and flavonoids using the copper(II)-neocuproine reagent in ammonium acetate buffer is now applied to a complete series of plasma antioxidants for the assay of total antioxidant capacity of serum, and the resulting absorbance at 450 nm is recorded either directly (e.g., for ascorbic acid, alpha-tocopherol, and glutathione) or after incubation at 50 degrees C for 20 min (e.g., for uric acid, bilirubin, and albumin), quantitation being made by means of a calibration curve. The lipophilic antioxidants, alpha-tocopherol and beta-carotene, are assayed in dichloromethane. Lipophilic antioxidants of serum are extracted with n-hexane from an ethanolic solution of serum subjected to centrifugation. Hydrophilic antioxidants of serum are assayed in the centrifugate after perchloric acid precipitation of proteins. The CUPRAC molar absorptivities, linear ranges, and TEAC (trolox equivalent antioxidant capacity) coefficients of the serum antioxidants are established, and the results are evaluated in comparison with the findings of the ABTS/TEAC reference method. The intra- and inter-assay coefficients of variation (CVs) are 0.7 and 1.5%, respectively, for serum. The CUPRAC assay proved to be efficient for glutathione and thiol-type antioxidants

  13. Serum-free human MSC medium supports consistency in human but not in equine adipose-derived multipotent mesenchymal stromal cell culture.

    Science.gov (United States)

    Schubert, Susanna; Brehm, Walter; Hillmann, Aline; Burk, Janina

    2017-09-19

    For clinical applications of multipotent mesenchymal stromal cells (MSCs), serum-free culture is preferable to standardize cell products and prevent contamination with pathogens. In contrast to human MSCs, knowledge on serum-free culture of large animal MSCs is limited, despite its relevance for preclinical studies and development of veterinary cellular therapeutics. This study aimed to evaluate the suitability of a commercially available serum-free human MSC medium for culturing equine adipose-derived MSCs in comparison with human adipose MSCs. Enzyme-free isolation by explant technique and expansion of equine and human cells in the serum-free medium were feasible. However, serum-free culture altered the morphology and complicated handling of equine MSCs, with cell aggregation and spontaneous detachment of multilayers, compared to culture in standard medium supplemented with fetal bovine serum. Furthermore, proliferation and the surface immunophenotype of equine cells were more variable compared to the controls and appeared to depend on the lot of the serum-free medium. Particularly the expression of CD90 was different between experimental groups (P cells found in equine MSC samples cultured in serum-free medium (5.21-83.40%) compared to standard medium (86.20-99.50%). Additionally, small subpopulations expressing MSC exclusion markers such as CD14 (0.28-11.60%), CD34 (0.00-9.87%), CD45 (0.35-10.50%), or MHCII (0.00-3.67%) were found in equine samples after serum-free culture. In contrast, human samples displayed a more consistent morphology and a consistent CD29(+) (98.60-99.90%), CD73(+) (94.60-98.40%), CD90(+) (99.60-99.90%), and CD105(+) (97.40-99.80%) immunophenotype after culture in serum-free medium. The obtained data demonstrate that the serum-free medium was suitable for human MSC culture but did not lead to entirely satisfactory results in equine MSCs. This underlines that requirements regarding serum-free culture conditions are species

  14. Apolipoprotein modulation of streptococcal serum opacity factor activity against human plasma high-density lipoproteins.

    Science.gov (United States)

    Rosales, Corina; Gillard, Baiba K; Courtney, Harry S; Blanco-Vaca, Francisco; Pownall, Henry J

    2009-08-25

    Human plasma HDL are the target of streptococcal serum opacity factor (SOF), a virulence factor that clouds human plasma. Recombinant (r) SOF transfers cholesteryl esters (CE) from approximately 400,000 HDL particles to a CE-rich microemulsion (CERM), forms a cholesterol-poor HDL-like particle (neo HDL), and releases lipid-free (LF) apo A-I. Whereas the rSOF reaction requires labile apo A-I, the modulation effects of other apos are not known. We compared the products and rates of the rSOF reaction against human HDL and HDL from mice overexpressing apos A-I and A-II. Kinetic studies showed that the reactivity of various HDL species is apo-specific. LpA-I reacts faster than LpA-I/A-II. Adding apos A-I and A-II inhibited the SOF reaction, an effect that was more profound for apo A-II. The rate of SOF-mediated CERM formation was slower against HDL from mice expressing human apos A-I and A-II than against WT mice HDL and slowest against HDL from apo A-II overexpressing mice. The lower reactivity of SOF against HDL containing human apos is due to the higher hydropathy of human apo A-I, particularly its C-terminus relative to mouse apo A-I, and the higher lipophilicity of human apo A-II. The SOF-catalyzed reaction is the first to target HDL rather than its transporters and receptors in a way that enhances reverse cholesterol transport (RCT). Thus, effects of apos on the SOF reaction are highly relevant. Our studies show that the "humanized" apo A-I-expressing mouse is a good animal model for studies of rSOF effects on RCT in vivo.

  15. Predicted serum folate concentrations based on in vitro studies and kinetic modeling are consistent with measured folate concentrations in humans

    NARCIS (Netherlands)

    Verwei, M.; Freidig, A.P.; Havenaar, R.; Groten, J.P.

    2006-01-01

    The nutritional quality of new functional or fortified food products depends on the bioavailability of the nutrient(s) in the human body. Bioavailability is often determined in human intervention studies by measurements of plasma or serum profiles over a certain time period. These studies are time a

  16. Potent neutralizing serum immunoglobulin A (IgA) in human immunodeficiency virus type 2-exposed IgG-seronegative individuals

    DEFF Research Database (Denmark)

    Lizeng, Q; Nilsson, C; Sourial, S

    2004-01-01

    Links Potent neutralizing serum immunoglobulin A (IgA) in human immunodeficiency virus type 2-exposed IgG-seronegative individuals.Lizeng Q, Nilsson C, Sourial S, Andersson S, Larsen O, Aaby P, Ehnlund M, Bjorling E. Research Center, South Hospital, Stockholm, Sweden. The mechanisms behind...... the resistance to human immunodeficiency virus type 2 (HIV-2) infection are still not fully understood. In the present study, we explored the HIV-2-specific humoral serum immunoglobulin A (IgA) immune response in HIV-2-exposed IgG-seronegative (EGSN) individuals. Serum samples from heterosexual EGSN individuals...... and their known HIV-2-infected partners, as well as controls originating from Guinea-Bissau in Africa, were studied. Antibody reactivity to native and recombinant envelope glycoproteins was investigated, and the capacity of purified serum IgA to neutralize HIV-2(SBL6669) was tested. Our results showed that 16...

  17. The development of a radioimmunoassay for reverse triiodothyronine sulfate in human serum and amniotic fluid

    Energy Technology Data Exchange (ETDEWEB)

    Wu, Sing-Yung (Veterans Administration Medical Center, Long Beach, CA (United States)); Huang, Wen-Sheng; Chen, Wei-Lian (Tri-Service General Hospital, Taipei (Taiwan, Province of China)); Polk, D.; Reviczky, A.; Williams, J. III; Chopra, I.J.; Fisher, D.A. (Univ. of California, Los Angeles (United States))

    1993-06-01

    Sulfated iodothyronines including T[sub 4]-sulfate (T[sub 4]S) and T[sub 3]-sulfate (T[sub 3]S) have been identified in human serum and amniotic fluid. Little is know, however, about the existence of sulfate conjugation of reverse T[sub 3] (rT[sub 3]S) in man. In this report, the authors employed a novel, sensitive, and specific rT[sub 3]S RIA to address this question. The rabbit antiserum to rT[sub 3]S was highly specific; T[sub 4], T[sub 3], rT[sub 3], and 3,3'-T[sub 2] showed less than 0.002% cross-reaction with the antiserum. Only T[sub 4]S and T[sub 3]S cross-reacted significantly (0.3% and 0.01%, respectively); other analogs cross-reacted less than 0.0001%. The detection threshold of the RIA was 14 pmol/L (1.0 ng/dL). The mean serum rT[sub 3]S concentration (pmol/L) was 40 in euthyroid subjects. Values were similar in hypothyroid patients (38) and pregnant women (52) but significantly (P < 0.01) elevated to 176 in hyperthyroid patient, 74 in patients with nonthyroid illnesses, and 684 in cord sera of newborns. Serum rT[sub 3]S increased significantly in hyperthyroid patients 1 day after administration of 1 g sodium ipodate orally. Reverse T[sub 3]S was detected consistently in amniotic fluid at 14 to 22 weeks of gestation and showed a marked rise 1-3 weeks after intraamniotic administration of 500-1000 [mu]g T[sub 4]. The various data suggest that : (1) rT[sub 3]S is a normal component of human serum and amniotic fluid; (2) it is derived from metabolism of T[sub 4] or rT[sub 3]; (3) circulating rT[sub 3]S increases in hyperthyroidism and in circumstances where type I 5'-monodeiodinating activity is low, e.g. nonthyroid illnesses, fetal life, and after administration of ipodate. 20 refs., 4 figs.

  18. Quantitative GSL-glycome analysis of human whole serum based on an EGCase digestion and glycoblotting method[S

    Science.gov (United States)

    Furukawa, Jun-ichi; Sakai, Shota; Yokota, Ikuko; Okada, Kazue; Hanamatsu, Hisatoshi; Kobayashi, Takashi; Yoshida, Yasunobu; Higashino, Kenichi; Tamura, Tomohiro; Igarashi, Yasuyuki; Shinohara, Yasuro

    2015-01-01

    Glycosphingolipids (GSLs) are lipid molecules linked to carbohydrate units that form the plasma membrane lipid raft, which is clustered with sphingolipids, sterols, and specific proteins, and thereby contributes to membrane physical properties and specific recognition sites for various biological events. These bioactive GSL molecules consequently affect the pathophysiology and pathogenesis of various diseases. Thus, altered expression of GSLs in various diseases may be of importance for disease-related biomarker discovery. However, analysis of GSLs in blood is particularly challenging because GSLs are present at extremely low concentrations in serum/plasma. In this study, we established absolute GSL-glycan analysis of human serum based on endoglycoceramidase digestion and glycoblotting purification. We established two sample preparation protocols, one with and the other without GSL extraction using chloroform/methanol. Similar amounts of GSL-glycans were recovered with the two protocols. Both protocols permitted absolute quantitation of GSL-glycans using as little as 20 μl of serum. Using 10 healthy human serum samples, up to 42 signals corresponding to GSL-glycan compositions could be quantitatively detected, and the total serum GSL-glycan concentration was calculated to be 12.1–21.4 μM. We further applied this method to TLC-prefractionated serum samples. These findings will assist the discovery of disease-related biomarkers by serum GSL-glycomics. PMID:26420879

  19. Dimensions of Human-Work Domain Interaction: A Preliminary Analysis for the Design of a Corporate Digital Library.

    Science.gov (United States)

    Xie, Hong

    2003-01-01

    Applies the cognitive system engineering approach to investigate human-work interaction at a corporate setting. Reports preliminary analysis of data collected from diary analysis and interview of 20 subjects. Results identify three dimensions for each of four interactive activities involved in human-work interaction and their relationships.…

  20. Study on Interaction of Ginsenosides with Bovine or Human Serum Albumin Using Wavelength Modulation Surface Plasmon Resonance Biosensor

    Institute of Scientific and Technical Information of China (English)

    LIU Xia; SUN Ying; SONG Da-Qian; LI Xu-Wen; ZHANG Qing-Lin; TIAN Yuan; LIU Zhong-Ying; ZHANG Han-Qi

    2006-01-01

    To use a newly developed wavelength modulation surface plasmon resonance (SPR) biosensor, an experimental protocol was developed to investigate the interaction of ginsenosides with serum albumin. With a known concentration of the ginsenosides, bound percentages of the ginsenosides with human serum albumin (HSA) or bovine serum albumin (BSA) were obtained. SPR technique could require no labeling and this method provided the detailed information on association and disassociation of molecules in real time. The results indicate that the sensitivity of wavelength modulation SPR biosensor is sufficient for detection and characterization of binding events involving low-molecular weight compounds and their immobilized protein targets.

  1. High-performance liquid chromatographic determination of free and total polyamines in human serum as fluorescamine derivatives.

    Science.gov (United States)

    Kai, M; Ogata, T; Haraguchi, K; Ohkura, Y

    1979-06-11

    A highly sensitive and simple fluorimetric method for the determination of free and total polyamines, spermidine, spermine, putrescine and cadaverine, in human serum by high-performance liquid chromatography is described. The polyamines, obtained after clean-up of deproteinized serum by Cellex P column chromatography, are converted to their fluorescamine derivatives in the presence of nickel ion which inhibits the reaction of interfering amines with fluorescamine, and the derivatives are separated simultaneously by reversed-phase chromatography (LiChrosorb RP-18) with a linear gradient elution. The lower limits of detection are 10 and 15 pmole for spermine and the others in 0.5 ml of serum, respectively.

  2. P-glycoprotein expression and amyloid accumulation in human aging and Alzheimer's disease: preliminary observations.

    Science.gov (United States)

    Chiu, Catherine; Miller, Miles C; Monahan, Renée; Osgood, Doreen P; Stopa, Edward G; Silverberg, Gerald D

    2015-09-01

    P-glycoprotein (P-gp), part of the blood-brain barrier, limits drug access to the brain and is the target for therapies designed to improve drug penetration. P-gp also extrudes brain amyloid-beta (Aβ). Accumulation of Aβ is a hallmark of Alzheimer's disease (AD). Aβ accumulates in normal aging and in AD primarily due to decreased Aβ clearance. This is a preliminary report on the relative protein and messenger RNA expression of P-gp in human brains, ages 20-100 years, including AD subjects. In these preliminary studies, cortical endothelial P-gp expression decreased in AD compared with controls (p P-gp expression in human aging are similar to aging rats. Microvessel P-gp messenger RNA remained unchanged with aging and AD. Aβ plaques were found in 42.8% of normal subjects (54.5% of those older than 50 years). A qualitative analysis showed that P-gp expression is lower than the group mean in subjects older than 75 years but increased if younger. Decreased P-gp expression may be related to Aβ plaques in aging and AD. Downregulating P-gp to allow pharmaceuticals into the central nervous system may increase Aβ accumulation.

  3. A simple method for obtaining transferrins from human plasma and porcine serum: preparations and properties.

    Science.gov (United States)

    Wu, Lin; Wu, Jinhui; Zhang, Jian; Zhou, Yuanyuan; Ren, Guoyan; Hu, Yiqiao

    2008-05-01

    A simple method was described for the purification of serum transferrin (Tf) from human plasma and porcine serum with relative high yield and purity. The properties including purity, integrity, immunoreactivity and the receptor-binding ability of the proteins were studied by several assays, comprising spectrometry, SDS-PAGE, HPLC, Western blotting, urea electrophoresis, mass spectrometry and cytometry. Analysis from all the different aspects manifested that the proteins were of high purity. The two kinds of Tfs appeared to be iron-saturated as confirmed by their absorbance spectra and urea-PAGE mobility. The specific spectra of absorption of the two Tfs were both at around 465 nm. The relative molecular weights of human Tf (hTf) and porcine Tf (pTf) were determined by SDS-PAGE and further identified by MAIDI-TOF mass spectrometry with a result of 79,707 and 79,258, respectively. Immunoblotting assay showed that pTf could react with the anti-human Tf monoclonal antibody with a less level compared to hTf. FACS assays of their binding activities to Tf receptor-positive cell (K562 cell line) indicated that pTf could be recognized by the hTf receptor and internalized into cells, with a slightly less efficacy than hTf. All special property studies demonstrated that pTf was similar to hTf in physical and chemical characteristics, which gave a hint that pTf could substitute for hTf in some kinds of researches, such as using hTf as a carrier in drug targeting system.

  4. Reciprocal allosteric modulation of carbon monoxide and warfarin binding to ferrous human serum heme-albumin.

    Directory of Open Access Journals (Sweden)

    Alessio Bocedi

    Full Text Available Human serum albumin (HSA, the most abundant protein in human plasma, could be considered as a prototypic monomeric allosteric protein, since the ligand-dependent conformational adaptability of HSA spreads beyond the immediate proximity of the binding site(s. As a matter of fact, HSA is a major transport protein in the bloodstream and the regulation of the functional allosteric interrelationships between the different binding sites represents a fundamental information for the knowledge of its transport function. Here, kinetics and thermodynamics of the allosteric modulation: (i of carbon monoxide (CO binding to ferrous human serum heme-albumin (HSA-heme-Fe(II by warfarin (WF, and (ii of WF binding to HSA-heme-Fe(II by CO are reported. All data were obtained at pH 7.0 and 25°C. Kinetics of CO and WF binding to the FA1 and FA7 sites of HSA-heme-Fe(II, respectively, follows a multi-exponential behavior (with the same relative percentage for the two ligands. This can be accounted for by the existence of multiple conformations and/or heme-protein axial coordination forms of HSA-heme-Fe(II. The HSA-heme-Fe(II populations have been characterized by resonance Raman spectroscopy, indicating the coexistence of different species characterized by four-, five- and six-coordination of the heme-Fe atom. As a whole, these results suggest that: (i upon CO binding a conformational change of HSA-heme-Fe(II takes place (likely reflecting the displacement of an endogenous ligand by CO, and (ii CO and/or WF binding brings about a ligand-dependent variation of the HSA-heme-Fe(II population distribution of the various coordinating species. The detailed thermodynamic and kinetic analysis here reported allows a quantitative description of the mutual allosteric effect of CO and WF binding to HSA-heme-Fe(II.

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

    Science.gov (United States)

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

    2016-01-07

    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.

  6. Human cryptosporidiosis: detection of specific antibodies in the serum by an indirect immunofluorescence

    Directory of Open Access Journals (Sweden)

    Braz Lúcia M.A.

    1996-01-01

    Full Text Available Cryptosporidium sp., a coccidian parasite usually found in the faeces of cattle, has been recently implicated as an agent of human intestinal disease, mainly in immunocompromised patients. In the study realized, by an indirect immunofluorescence technique, specific immunoglobulins (IgG and IgM have been demonstrated in human serum against Cryptosporidium oocysts. Purified oocysts were used as antigens in the indirect immunofluorecence assay. After analyzing this test in sera from selected groups of patients, the frequency of both specific IgG and IgM of immunocompetent children who were excreting oocysts in their faeces was 62% and in children with negative excretion of oocysts was 20% and 40%, respectively. In adults infected with the human immunodeficiency virus (HIV and who were excreting Cryptosporidium in their stools, the frequency was 57% for IgG but only 2% for IgM. Twenty three percent of immunocompromised adults with not determined excretion of oocysts in their stools had anti-Cryptosporidium IgG in their sera. Children infected with human immunodeficiency virus had no IgM and only 14% had IgG detectable in their sera. The indirect immunoflorescence assay, when used with other parasitological techniques appears to be useful for retrospective population studies and for diagnosis of acute infection. The humoral immune response of HIV positive patients to this protozoan agent needs clarification.

  7. Serum Metabolomics Investigation of Humanized Mouse Model of Dengue Virus Infection.

    Science.gov (United States)

    Cui, Liang; Hou, Jue; Fang, Jinling; Lee, Yie Hou; Costa, Vivian Vasconcelos; Wong, Lan Hiong; Chen, Qingfeng; Ooi, Eng Eong; Tannenbaum, Steven R; Chen, Jianzhu; Ong, Choon Nam

    2017-07-15

    Dengue is an acute febrile illness caused by dengue virus (DENV) and a major cause of morbidity and mortality in tropical and subtropical regions of the world. The lack of an appropriate small-animal model of dengue infection has greatly hindered the study of dengue pathogenesis and the development of therapeutics. In this study, we conducted mass spectrometry-based serum metabolic profiling from a model using humanized mice (humice) with DENV serotype 2 infection at 0, 3, 7, 14, and 28 days postinfection (dpi). Forty-eight differential metabolites were identified, including fatty acids, purines and pyrimidines, acylcarnitines, acylglycines, phospholipids, sphingolipids, amino acids and derivatives, free fatty acids, and bile acid. These metabolites showed a reversible-change trend-most were significantly perturbed at 3 or 7 dpi and returned to control levels at 14 or 28 dpi, indicating that the metabolites might serve as prognostic markers of the disease in humice. The major perturbed metabolic pathways included purine and pyrimidine metabolism, fatty acid β-oxidation, phospholipid catabolism, arachidonic acid and linoleic acid metabolism, sphingolipid metabolism, tryptophan metabolism, phenylalanine metabolism, lysine biosynthesis and degradation, and bile acid biosynthesis. Most of these disturbed pathways are similar to our previous metabolomics findings in a longitudinal cohort of adult human dengue patients across different infection stages. Our analyses revealed the commonalities of host responses to DENV infection between humice and humans and suggested that humice could be a useful small-animal model for the study of dengue pathogenesis and the development of dengue therapeutics.IMPORTANCE Dengue virus is the most widespread arbovirus, causing an estimated 390 million dengue infections worldwide every year. There is currently no effective treatment for the disease, and the lack of an appropriate small-animal model of dengue infection has greatly increased

  8. Serum human chorionic gonadotropin is associated with angiogenesis in germ cell testicular tumors

    Directory of Open Access Journals (Sweden)

    Avilés-Salas Alejandro

    2009-08-01

    Full Text Available Abstract Background Germ cell testicular tumors have survival rate that diminishes with high tumor marker levels, such as human chorionic gonadotropin (hCG. hCG may regulate vascular neoformation through vascular endothelial growth factor (VEGF. Our purpose was to determine the relationship between hCG serum levels, angiogenesis, and VEGF expression in germ cell testicular tumors. Methods We conducted a retrospective study of 101 patients. Serum levels of hCG, alpha-fetoprotein (AFP, and lactate dehydrogenase were measured prior to surgery. Vascular density (VD and VEGF tissue expression were determined by immunohistochemistry and underwent double-blind analysis. Results Histologically, 46% were seminomas and 54%, non-seminomas. Median follow-up was 43 ± 27 months. Relapse was present in 7.5% and mortality in 11.5%. Factors associated with high VD included non-seminoma type (p = 0.016, AFP ≥ 14.7 ng/mL (p = 0.0001, and hCG ≥ 25 mIU/mL (p = 0.0001. In multivariate analysis, the only significant VD-associated factor was hCG level (p = 0.04. When hCG levels were stratified, concentrations ≥ 25 mIU/mL were related with increased neovascularization (p Conclusion This is the first study that relates increased serum hCG levels with vascularization in testicular germ cell tumors. Hence, its expression might play a role in tumor angiogenesis, independent of VEGF expression, and may explain its association with poor prognosis. hCG might represent a molecular target for therapy.

  9. Selenium in soil, grass, and human serum in the Zlatibor mountain area (Serbia): geomedical aspects.

    Science.gov (United States)

    Maksimović, Z; Rsumović, M; Jović, V; Kosanović, M; Jovanović, T

    1998-01-01

    The Zlatibor district in Serbia has lower mortality rates of malignant and cardiovascular diseases (CVD) compared with other regions in Serbia. To better understand the influence of the geochemical environment, we collected and analyzed soil from various bedrocks and the grass growing on them. We also analyzed spring and stream waters, including large water supply accumulations, for major chemical elements and examined the serum of healthy adults in the large area of Zlatibor for selenium (Se) and magnesium (Mg). Our studies included villages, small towns, and the town of Uzice. Our results showed a variable Se content in the soil over different bedrocks. In general, soil in this area has a higher Se content than in other regions of Serbia. The Se content of the grass is influenced by bedrock and soil mineralogy, but mostly by soil pH and the date of collection. For example, in late summer, grass contains twice as much Se than in spring. Mg2+HCO3(-)-type waters occur in the ultramafic massif of Zlatibor in a concentration of 44 to 68 mgMg/L. The serum Se values were higher in the Zlatibor area than in other regions of Serbia (62.6 +/- 14.9 microgSe/L; n = 158). The serum Mg content (22.7 +/- 2.2 mg/L; n = 158) was in the uppermost part of the reference range. Taking into account their biological role, the Se and Mg levels in the human population in the Zlatibor area could influence the lower mortality rates of cancer and CVD in this region compared with other regions in Serbia.

  10. Serum metabolic profiling of human gastric cancer based on gas chromatography/mass spectrometry

    Directory of Open Access Journals (Sweden)

    Hu Song

    2012-01-01

    Full Text Available Research on molecular mechanisms of carcinogenesis plays an important role in diagnosing and treating gastric cancer. Metabolic profiling may offer the opportunity to understand the molecular mechanism of carcinogenesis and help to non-invasively identify the potential biomarkers for the early diagnosis of human gastric cancer. The aims of this study were to explore the underlying metabolic mechanisms of gastric cancer and to identify biomarkers associated with morbidity. Gas chromatography/mass spectrometry (GC/MS was used to analyze the serum metabolites of 30 Chinese gastric cancer patients and 30 healthy controls. Diagnostic models for gastric cancer were constructed using orthogonal partial least squares discriminant analysis (OPLS-DA. Acquired metabolomic data were analyzed by the nonparametric Wilcoxon test to find serum metabolic biomarkers for gastric cancer. The OPLS-DA model showed adequate discrimination between cancer and non-cancer cohorts while the model failed to discriminate different pathological stages (I-IV of gastric cancer patients. A total of 44 endogenous metabolites such as amino acids, organic acids, carbohydrates, fatty acids, and steroids were detected, of which 18 differential metabolites were identified with significant differences. A total of 13 variables were obtained for their greatest contribution in the discriminating OPLS-DA model [variable importance in the projection (VIP value >1.0], among which 11 metabolites were identified using both VIP values (VIP >1 and the Wilcoxon test. These metabolites potentially revealed perturbations of glycolysis and of amino acid, fatty acid, cholesterol, and nucleotide metabolism of gastric cancer patients. These results suggest that gastric cancer serum metabolic profiling has great potential in detecting this disease and helping to understand its metabolic mechanisms.

  11. Development of {sup 68}Ga-labelled DTPA galactosyl human serum albumin for liver function imaging

    Energy Technology Data Exchange (ETDEWEB)

    Haubner, Roland [Innsbruck Medical University, Department of Nuclear Medicine, Innsbruck (Austria); Medizinische Universitaet Innsbruck, Universitaetsklinik fuer Nuklearmedizin, Innsbruck (Austria); Vera, David R.; Farshchi-Heydari, Salman [University of California, Department of Radiology, School of Medicine, and the UCSD Molecular Imaging Program, San Diego, CA (United States); Helbok, Anna; Rangger, Christine; Putzer, Daniel; Virgolini, Irene J. [Innsbruck Medical University, Department of Nuclear Medicine, Innsbruck (Austria)

    2013-08-15

    The hepatic asialoglycoprotein receptor is responsible for degradation of desialylated glycoproteins through receptor-mediated endocytosis. It has been shown that imaging of the receptor density using [{sup 99m}Tc]diethylenetriamine pentaacetic acid (DTPA) galactosyl human serum albumin ([{sup 99m}Tc]GSA) allows non-invasive determination of functional hepatocellular mass. Here we present the synthesis and evaluation of [{sup 68}Ga]GSA for the potential use with positron emission tomography (PET). Labelling of GSA with {sup 68}Ga was carried out using a fractionated elution protocol. For quality control thin-layer chromatography (TLC), high-performance liquid chromatography (HPLC) and size exclusion chromatography (SEC) techniques were evaluated. Stability of [{sup 68}Ga]GSA was studied in phosphate-buffered saline (PBS) and human serum. For in vivo evaluation [{sup 68}Ga]GSA distribution in Lewis rats was compared with [{sup 99m}Tc]GSA by using a dual isotope protocol. PET and planar imaging studies were performed using the same scaled molar dose of [{sup 68}Ga]GSA and [{sup 99m}Tc]GSA. Time-activity curves (TAC) for heart and liver were generated and corresponding parameters calculated (t50, t90). [{sup 68}Ga]GSA can be produced with high radiochemical purity. The best TLC methods for determining potential free {sup 68}Ga include 0.1 M sodium citrate as eluent. None of the TLC methods tested were able to determine potential colloids. This can be achieved by SEC. HPLC confirmed high radiochemical purity (>98 %). Stability after 120 min incubation at 37 C was high in PBS (>95 % intact tracer) and low in human serum ({proportional_to}27 % intact tracer). Biodistribution studies simultaneously injecting both tracers showed comparable liver uptake, whereas activity concentration in blood was higher for [{sup 68}Ga]GSA compared to [{sup 99m}Tc]GSA. The [{sup 99m}Tc]GSA TACs exhibited a small degree of hepatic metabolism compared to the [{sup 68}Ga]GSA curves. The mean

  12. Interaction of weakly bound antibiotics neomycin and lincomycin with bovine and human serum albumin: biophysical approach.

    Science.gov (United States)

    Keswani, Neelam; Choudhary, Sinjan; Kishore, Nand

    2010-07-01

    The thermodynamics of interaction of neomycin and lincomycin with bovine serum albumin (BSA) and human serum albumin (HSA) has been studied using isothermal titration calorimetry (ITC), in combination with UV-visible, steady state and time resolved fluorescence spectroscopic measurements. Neomycin is observed to bind weakly to BSA and HSA whereas lincomycin did not show any evidence for binding with the native state of these proteins, rather it interacts in the presence of surfactants. The ITC results suggest 1 : 1 binding stoichiometry for neomycin in the studied temperature range. The values of the van't Hoff enthalpy do not agree with the calorimetric enthalpy in the case of neomycin, suggesting conformational changes in the protein upon ligand binding, as well as with the rise in the temperature. Experiments at different ionic strengths, and in the presence of tetrabutyl ammonium bromide and surfactants suggest the predominant involvement of electrostatic interactions in the complexation process of neomycin with BSA and HSA, and non-specific interaction behaviour of lincomycin with these proteins.

  13. PEGylated Human Serum Albumin: Review of PEGylation, Purification and Characterization Methods

    Directory of Open Access Journals (Sweden)

    Parvin Akbarzadehlaleh

    2016-09-01

    Full Text Available Human serum albumin (HSA is a non-glycosylated, negatively charged protein (Mw: about 65-kDa that has one free cystein residue (Cys 34, and 17 disulfide bridges that these bridges have main role in its stability and longer biological life-time (15 to 19 days. As HSA is a multifunctional protein, it can also bind to other molecules and ions in addition to its role in maintaining colloidal osmotic pressure (COP in various diseases. In critical illnesses changes in the level of albumin between the intravascular and extravascular compartments and the decrease in its serum concentration need to be compensated using exogenous albumin; but as the size of HSA is an important parameter in retention within the circulation, therefore increasing its molecular size and hydrodynamic radius of HSA by covalent attachment of poly ethylene glycol (PEG, that is known as PEGylation, provides HSA as a superior volume expander that not only can prevent the interstitial edema but also can reduce the infusion frequency. This review focuses on various PEGylation methods of HSA (solid phase and liquid phase, and compares various methods to purifiy and characterize the pegylated form.

  14. PEGylated Human Serum Albumin: Review of PEGylation, Purification and Characterization Methods

    Science.gov (United States)

    Akbarzadehlaleh, Parvin; Mirzaei, Mona; Mashahdi-Keshtiban, Mahdiyeh; Shamsasenjan, Karim; Heydari, Hamidreza

    2016-01-01

    Human serum albumin (HSA) is a non-glycosylated, negatively charged protein (Mw: about 65-kDa) that has one free cystein residue (Cys 34), and 17 disulfide bridges that these bridges have main role in its stability and longer biological life-time (15 to 19 days). As HSA is a multifunctional protein, it can also bind to other molecules and ions in addition to its role in maintaining colloidal osmotic pressure (COP) in various diseases. In critical illnesses changes in the level of albumin between the intravascular and extravascular compartments and the decrease in its serum concentration need to be compensated using exogenous albumin; but as the size of HSA is an important parameter in retention within the circulation, therefore increasing its molecular size and hydrodynamic radius of HSA by covalent attachment of poly ethylene glycol (PEG), that is known as PEGylation, provides HSA as a superior volume expander that not only can prevent the interstitial edema but also can reduce the infusion frequency. This review focuses on various PEGylation methods of HSA (solid phase and liquid phase), and compares various methods to purifiy and characterize the pegylated form. PMID:27766215

  15. Quality assesment for the analysis of PCDDs/PCDFs in individual human serum samples

    Energy Technology Data Exchange (ETDEWEB)

    Perez, F. [IIQAB-CSIC, Barcelona (Spain). Dept. of Ecotechnologies, Lab. of Dioxins; Abad, E.; Llerena, J.J.; Caixach, J.; Rivera, J.

    2004-09-15

    The aim of this work was to optimise a relevant methodology for the ultratrace analysis of PCDDs/PCDFs in individual human serum samples. In order to carry out the study, different strategies including the elaboration of quality control samples, parallel sample analysis, control blanks and a number of quality assurance measures were implemented as analytical current practices. Some of the main drawbacks in the analysis of PCDDs/PCDFs in these kind of samples come from two conflicting aspects: the small sample size and the low levels expected to be found. Taking this into account, an unavoidable compromise between the sample amount and the minimum analytical requirements, mainly the detection limit (LOD), is mandatory. To reach this goal C{sub 18} solid phase extraction was used to remove the analytes from the matrix. Clean up was performed by solid-liquid adsorption chromatography using a variety of adsorbents. Instrumental analysis was achieved by high-resolution gas chromatography coupled to high-resolution mass spectrometry (HRGC/HRMS). Finally, the optimised methodology was applied to evaluate the potential impact in general population living in the surroundings of an obsolete municipal waste incinerator plant (MWI). Thus, more than 400 individuals serum samples potentially exposed to the emission of the incinerator and people not exposed were considered in this study.

  16. Biophysical analysis of the interaction of the serum protein human β2GPI with bacterial lipopolysaccharide

    Directory of Open Access Journals (Sweden)

    Anna Gries

    2014-01-01

    Full Text Available There are several human serum proteins for which no clear role is yet known. Among these is the abundant serum protein beta2-glycoprotein-I (β2GPI, which is known to bind to negatively charged phospholipids as well as to bacterial lipopolysaccharides (LPS, and was therefore proposed to play a role in the immune response. To understand the details of these interactions, a biophysical analysis of the binding of β2GPI to LPS and phosphatidylserine (PS was performed. The data indicate only a moderate tendency of the protein (1 to influence the LPS-induced cytokine production in vitro, (2 to react exothermally with LPS in a non-saturable way, and (3 to change its local microenvironment upon LPS association. Additionally, we found that the protein binds more strongly to phosphatidylserine (PS than to LPS. Furthermore, β2GPI converts the LPS bilayer aggregates into a stronger multilamellar form, and reduces the fluidity of the hydrocarbon moiety of LPS due to a rigidification of the acyl chains. From these data it can be concluded that β2GPI plays a role as an immune-modulating agent, but there is much less evidence for a role in immune defense against bacterial toxins such as LPS.

  17. [Isolation of isoforms of apolipoprotein CIII from human serum by chromatofocusing].

    Science.gov (United States)

    Fang, D; Gong, R; O, K

    1999-03-01

    This study aimed to isolate isoforms of apolipoprotein (apo) C III from human serum. 24-hour fasting serum from normal and hyperlipidemic subjects was pooled and subjected to ultracentrifugation at plasma density for 20 hours. Very low density lipoprotein (VLDL) was collected at density of d < 1.006 g/ml, and it was delipidated by ethanol and ether. The delipidated apo-VLDL was dissolved in a solution containing 7.2 mol/L urea and 20 mmol/L dithiothreitol. The insoluble apo B was removed by centrifugation. The soluble apo-VLDL was applied to PBE94 column, and eluted with elution buffer containing polybuffer 74 and 8 mol/L urea (1:8, pH4.0). After pooled, the eluted peaks of apolipoproteins were applied to column chromatography of hydroxylapatite to remove the polybuffer. The purified isoforms of apoC III and the purified apo C I, C II and E, were characterized by isoelectrofocusing and west blot. The results showed that the purified apoC III1, C III2, and C II were pure.

  18. Serum cytokine profiles of children with human enterovirus 71-associated hand, foot, and mouth disease.

    Science.gov (United States)

    Han, Jun; Wang, Ying; Gan, Xing; Song, Juan; Sun, Peng; Dong, Xiao-Ping

    2014-08-01

    Cytokine profiles may impact the pathogenicity and severity of hand, foot, and mouth disease caused by human enterovirus (HEV) 71. In 91 severe or mild HEV 71-associated hand, foot, and mouth disease children, serum was collected between days 2 and 10 or day >10. Serum cytokines including Type 1 T helper (Th1) cytokines: interleukin (IL)-2, interferon-gamma (IFN-γ), IL-12, and IL-18, Type 1 T helper (Th2) cytokines: IL-4, IL-10, IL-13, proinflammatory cytokines: IL-1α, IL-1β, IL-6, IL-8, IL-17, and tumor necrosis factor alpha (TNF-α), were assessed during the early stage and recovery. In the patients with mild illness, the peaks of IL-8 and IL-10 were observed on day 6 and that of IL-18 was on day 4. In the patients with severe illness, all cytokines spiked on day 3 and peaked on day 11. All cytokines except IL-6, IL-8, IL-18, and TNF-α were significantly correlated with immunoglobulin M levels by the end of the disease course. Cytokine profile variations between the patients with mild and severe illness may indicate prognosis and strain virulence, useful in clinical treatment of patients.

  19. Binding of ring-substituted indole-3-acetic acids to human serum albumin.

    Science.gov (United States)

    Soskić, Milan; Magnus, Volker

    2007-07-01

    The plant hormone, indole-3-acetic acid (IAA), and its ring-substituted derivatives have recently attracted attention as promising pro-drugs in cancer therapy. Here we present relative binding constants to human serum albumin for IAA and 34 of its derivatives, as obtained using the immobilized protein bound to a support suitable for high-performance liquid chromatography. We also report their octanol-water partition coefficients (logK(ow)) computed from retention data on a C(18) coated silica gel column. A four-parameter QSPR (quantitative structure-property relationships) model, based on physico-chemical properties, is put forward, which accounts for more than 96% of the variations in the binding affinities of these compounds. The model confirms the importance of lipophilicity as a global parameter governing interaction with serum albumin, but also assigns significant roles to parameters specifically related to the molecular topology of ring-substituted IAAs. Bulky substituents at ring-position 6 increase affinity, those at position 2 obstruct binding, while no steric effects were noted at other ring-positions. Electron-withdrawing substituents at position 5 enhance binding, but have no obvious effect at other ring positions.

  20. Interaction of imatinib mesylate with human serum transferrin: The comparative spectroscopic studies

    Science.gov (United States)

    Śliwińska-Hill, Urszula

    2017-02-01

    Imatinib mesylate (Imt) is a tyrosine kinase inhibitor mainly used in the treatment of Philadelphia chromosome-positive chronic myelogenous leukemia (Ph + CML). Human serum transferrin is the most abundant serum protein responsible for the transport of iron ions and many endogenous and exogenous ligands. In this study the mechanism of interactions between the imatinib mesylate and all states of transferrin (apo-Tf, Htf and holo-Tf) has been investigated by fluorescence, ultraviolet-visible (UV-vis), circular dichroism (CD) and zeta potential spectroscopic methods. Based on the experimental results it was proved that under physiological conditions the imatinib mesylate binds to the each form of transferrin with a binding constant c.a. 105 M- 1. The thermodynamic parameters indicate that hydrogen bonds and van der Waals were involved in the interaction of apo-Tf with the drug and hydrophobic and ionic strength participate in the reaction of Htf and holo-Tf with imatinib mesylate. Moreover, it was shown that common metal ions, Zn2 + and Ca2 + strongly influenced apo-Tf-Imt binding constant. The CD studies showed that there are no conformational changes in the secondary structure of the proteins. No significant changes in secondary structure of the proteins upon binding with the drug and instability of apo-Tf-Imt system are the desirable effects from pharmacological point of view.

  1. Development of immunoturbidimetric assays for fourteen human serum proteins on the Hitachi 912.

    Science.gov (United States)

    Ledue, Thomas B; Collins, Marilyn F; Ritchie, Robert F

    2002-05-01

    Many laboratories rely on dedicated nephelometers or turbidimeters and commercial reagent kits for the evaluation of serum proteins. However, with growing emphasis on cost containment, laboratories are forced to seek additional operational efficiencies by capitalizing on the use of existing analyzers whenever possible. In the present paper we describe the development of immunoturbidimetric assays for routine analysis of 14 human serum proteins (alpha1-antitrypsin, alpha2-macroglobulin, albumin, apolipoproteins Al and B, complement components 3 and 4, haptoglobin, immunoglobulins A, G, and M, orosomucoid, prealbumin, and transferrin) on the Hitachi 912, a general chemistry analyzer. With this system, we obtained excellent precision at levels corresponding to low, normal, and high physiologic concentrations of each protein (within-run imprecision CVs 0.97). We observed no significant interference from bilirubin (up to 718 micromol/l), hemoglobin (up to 8 g/l), triglyceride (up to 14.7 mmol/l) or rheumatoid factor (up to 4,140 IU/ml). Calibration for the 14 protein assays was stable for at least 7 days and onboard refrigerated reagents were stable for at least 3 months. The instrument's automated sample re-run feature minimized sample handling and helped to conserve specimens. In conclusion, the newly developed assays on the Hitachi 912 offer high throughput (>250 tests per hour) without the associated cost of a dedicated instrument for protein assays.

  2. Colocalization of Serum Amyloid A with Microtubules in Human Coronary Artery Endothelial Cells

    Directory of Open Access Journals (Sweden)

    Katja Lakota

    2011-01-01

    Full Text Available Serum amyloid A (SAA acts as a major acute phase protein and represents a sensitive and accurate marker of inflammation. Besides its hepatic origin, as the main source of serum SAA, this protein is also produced extrahepatically. The mRNA levels of SAA become significantly elevated following proinflammatory stimuli, as well as, are induced through their own positive feedback in human primary coronary artery endothelial cells. However, the intracellular functions of SAA are so far unknown. Colocalization of SAA with cytoskeletal filaments has previously been proposed, so we analyzed the colocalization of SAA with all three cytoskeletal elements: actin filaments, vimentin filaments, and microtubules. Immunofluorescent double-labeling analyses confirmed by PLA method revealed a strict colocalization of SAA with microtubules and a very infrequent attachment to vimentin while the distribution of actin filaments appeared clearly separated from SAA staining. Also, no significant colocalization was found between SAA and endomembranes labeled with the fluorescent lipid stain DiO6. However, SAA appears to be located also unbound in the cytosol, as well as inside the nucleus and within nanotubes extending from the cells or bridging neighboring cells. These different locations of SAA in endothelial cells strongly indicate multiple potential functions of this protein.

  3. Fractionation of human serum lipoproteins and simultaneous enzymatic determination of cholesterol and triglycerides.

    Science.gov (United States)

    Qureshi, Rashid Nazir; Kok, Wim Th; Schoenmakers, Peter J

    2009-11-03

    A method based on Asymmetric Flow Field-Flow Fractionation (AF4) was developed to separate different types of lipoproteins from human serum. The emphasis in the method optimization was on the possibilities to characterize the largest lipoprotein fractions (LDL and VLDL), which is usually not possible with the size-exclusion chromatography methods applied in routine analysis. Different channel geometries and flow programs were tested and compared. The use of a short fractionation channel was shown to give less sample dilution at the same fractionation power compared to a conventional, long channel. Different size selectivities were obtained with an exponential decay and a linear cross flow program. The ratio of the UV absorption signal to the light scattering signal was used to validate the relation between retention time and size of the fractionated particles. An experimental setup was developed for the simultaneous determination of the cholesterol and triglycerides distribution over the lipoprotein fractions, based on enzymatic reactions followed by UV detection at 500 nm. Coiled and knitted PTFE tubing reactors were compared. An improved peak sharpness and sensitivity were observed with the knitted tubing reactor. After optimization of the experimental conditions a satisfactory linearity and precision (2-3% rsd for cholesterol and 5-6% rsd for triglycerides) were obtained. Finally, serum samples, a pooled sample from healthy volunteers and samples of sepsis patients, were analyzed with the method developed. Lipoprotein fractionation and cholesterol and triglyceride distributions could be correlated with the clinical background of the samples.

  4. Fractionation of human serum lipoproteins and simultaneous enzymatic determination of cholesterol and triglycerides

    Energy Technology Data Exchange (ETDEWEB)

    Qureshi, Rashid Nazir [Polymer-Analysis Group, van' t Hoff Institute for Molecular Sciences, University of Amsterdam, Nieuwe Achtergracht 166, 1018WV Amsterdam (Netherlands); Kok, Wim Th., E-mail: W.Th.Kok@uva.nl [Polymer-Analysis Group, van' t Hoff Institute for Molecular Sciences, University of Amsterdam, Nieuwe Achtergracht 166, 1018WV Amsterdam (Netherlands); Schoenmakers, Peter J. [Polymer-Analysis Group, van' t Hoff Institute for Molecular Sciences, University of Amsterdam, Nieuwe Achtergracht 166, 1018WV Amsterdam (Netherlands)

    2009-11-03

    A method based on Asymmetric Flow Field-Flow Fractionation (AF4) was developed to separate different types of lipoproteins from human serum. The emphasis in the method optimization was on the possibilities to characterize the largest lipoprotein fractions (LDL and VLDL), which is usually not possible with the size-exclusion chromatography methods applied in routine analysis. Different channel geometries and flow programs were tested and compared. The use of a short fractionation channel was shown to give less sample dilution at the same fractionation power compared to a conventional, long channel. Different size selectivities were obtained with an exponential decay and a linear cross flow program. The ratio of the UV absorption signal to the light scattering signal was used to validate the relation between retention time and size of the fractionated particles. An experimental setup was developed for the simultaneous determination of the cholesterol and triglycerides distribution over the lipoprotein fractions, based on enzymatic reactions followed by UV detection at 500 nm. Coiled and knitted PTFE tubing reactors were compared. An improved peak sharpness and sensitivity were observed with the knitted tubing reactor. After optimization of the experimental conditions a satisfactory linearity and precision (2-3% rsd for cholesterol and 5-6% rsd for triglycerides) were obtained. Finally, serum samples, a pooled sample from healthy volunteers and samples of sepsis patients, were analyzed with the method developed. Lipoprotein fractionation and cholesterol and triglyceride distributions could be correlated with the clinical background of the samples.

  5. O{sub 2}-mediated oxidation of ferrous nitrosylated human serum heme-albumin is limited by nitrogen monoxide dissociation

    Energy Technology Data Exchange (ETDEWEB)

    Ascenzi, Paolo, E-mail: ascenzi@uniroma3.it [Interdepartmental Laboratory of Electron Microscopy, University Roma Tre, Via della Vasca Navale 79, I-00146 Roma (Italy); National Institute for Infectious Diseases I.R.C.C.S. ' Lazzaro Spallanzani' , Via Portuense 292, I-00149 Roma (Italy); Gullotta, Francesca; Gioia, Magda; Coletta, Massimo [Department of Experimental Medicine and Biochemical Sciences, University of Roma ' Tor Vergata' , Via Montpellier 1, I-00133 Roma (Italy); Interuniversity Consortium for the Research on the Chemistry of Metals in Biological Systems, Piazza Umberto I 1, I-87100 Bari (Italy); Fasano, Mauro [Department of Structural and Functional Biology, and Center of Neuroscience, University of Insubria, Via Alberto da Giussano 12a, I-21052 Busto Arsizio, VA (Italy)

    2011-03-04

    Research highlights: {yields} Human serum heme-albumin displays globin-like properties. {yields} O{sub 2}-mediated oxidation of ferrous nitrosylated human serum heme-albumin. {yields} Allosteric modulation of human serum heme-albumin reactivity. {yields} Rifampicin is an allosteric effector of human serum heme-albumin. {yields} Human serum heme-albumin is a ROS and NOS scavenger. -- Abstract: Human serum heme-albumin (HSA-heme-Fe) displays globin-like properties. Here, kinetics of O{sub 2}-mediated oxidation of ferrous nitrosylated HSA-heme-Fe (HSA-heme-Fe(II)-NO) is reported. Values of the first-order rate constants for O{sub 2}-mediated oxidation of HSA-heme-Fe(II)-NO (i.e., for ferric HSA-heme-Fe formation) and for NO dissociation from HSA-heme-Fe(II)-NO (i.e., for NO replacement by CO) are k = 9.8 x 10{sup -5} and 8.3 x 10{sup -4} s{sup -1}, and h = 1.3 x 10{sup -4} and 8.5 x 10{sup -4} s{sup -1}, in the absence and presence of rifampicin, respectively, at pH = 7.0 and T = 20.0 {sup o}C. The coincidence of values of k and h indicates that NO dissociation represents the rate limiting step of O{sub 2}-mediated oxidation of HSA-heme-Fe(II)-NO. Mixing HSA-heme-Fe(II)-NO with O{sub 2} does not lead to the formation of the transient adduct(s), but leads to the final ferric HSA-heme-Fe derivative. These results reflect the fast O{sub 2}-mediated oxidation of ferrous HSA-heme-Fe and highlight the role of drugs in modulating allosterically the heme-Fe-atom reactivity.

  6. Chronic ethanol consumption increases the levels of chemerin in the serum and adipose tissue of humans and rats

    Institute of Scientific and Technical Information of China (English)

    Rui-zhen REN; Xu ZHANG; Jin XU; Hai-qing ZHANG; Chun-xiao YU; Ming-feng CAO; Ling GAO; Qing-bo GUAN; Jia-jun ZHAO

    2012-01-01

    Chemerin is a new adipokine involved in adipogenesis and insulin resistance.Since ethanol affects the insulin sensitivity that is closely associated with adipokines.The aim of this study was to investigate the effects of ethanol on chemerin in humans and rats.Methods:In the human study,148 men who consumed alcohol for more than 3 years and 55 men who abstained from alcohol were included.Based on ethanol consumption per day,the drinkers were classified into 3 groups:low-dose (<15 g/d),middle-dose (15-47.9 g/d) and high-dose (≥48 g/d).Anthropometric measurementsand serum parameters were collected.In the rat study,27 male Wistar rats were randomly divided into 4 groups administered water or ethanol (0.5,2.5,or 5 g·kg-1·d-1) for 22 weeks.The chemerin levels in the sera,visceral adipose tissue (VAT) and liver were measured using ELISA.Results:In the high-dose group of humans and middle- and high-dose groups of rats,chronic ethanol consumption significantly increased the serum chemerin level.Both the middle- and high-dose ethanol significantly increased the chemerin level in the VAT of rats.In humans,triglyceride,fasting glucose,insulin and HOMA-IR were independently associated with chemerin.In rats,the serum chemerin level was positively correlated with chemerin in the VAT after adjustments for the liver chemerin (r=+0.768).High-dose ethanol significantly increased the body fat in humans and the VAT in rats.Conclusion:Chronic ethanol consumption dose-dependently increases the chemerin levels in the serum and VAT.The serum chemerin level is associated with metabolic parameters in humans.The increased serum chemerin level is mainly attributed to an elevation of chemerin in the VAT after the ethanol treatment.

  7. Behavior of human serum albumin on strong cation exchange resins: II. model analysis.

    Science.gov (United States)

    Voitl, Agnes; Butté, Alessandro; Morbidelli, Massimo

    2010-08-20

    Experiments with human serum albumin on a strong cation exchange resin exhibit a peculiar elution pattern: the protein elutes with two peaks in a modifier gradient. This behavior is modeled with a general rate model, where the two elution peaks are modeled with two binding conformations, one of which is at equilibrium conditions, while for the other, the adsorption process is rate limited. Isocratic experiments under nonadsorbing conditions were used to characterize the mass transfer process. The isotherm of both adsorption conformations as well as the kinetic of adsorption and desorption for the second conformation are functions of the modifier concentration. They are evaluated with linear modifier gradient experiments and step experiments with various adsorption times. All experimental features are well reproduced by the proposed modified general rate model.

  8. Thermodynamic parameters for binding of fatty acids to human serum albumin

    DEFF Research Database (Denmark)

    Pedersen, A O; Honoré, B; Brodersen, R

    1990-01-01

    Binding of laurate and myristate anions to human serum albumin has been studied over a range of temperatures, 5-37 degrees C, at pH 7.4. The binding curves indicate that the strength of binding of the first few molecules of fatty acid to albumin (r less than 5) decreases with increasing temperature...... constant, it was possible to calculate values for the changes in enthalpy and entropy during the initial binding step. For the medium-chain fatty acids, laurate and myristate, binding of the first molecule to albumin appeared to be enthalpic, with a tendency to an increasing contribution of entropy...... to binding energy with increasing chain length of the fatty acid. Udgivelsesdato: 1990-Jul-5...

  9. Proteomic Analysis of Outer Membrane Proteins from Salmonella Enteritidis Strains with Different Sensitivity to Human Serum

    Science.gov (United States)

    Dudek, Bartłomiej; Krzyżewska, Eva; Kapczyńska, Katarzyna; Rybka, Jacek; Pawlak, Aleksandra; Korzekwa, Kamila; Klausa, Elżbieta; Bugla-Płoskońska, Gabriela

    2016-01-01

    Differential analysis of outer membrane composition of S. Enteritidis strains, resistant to 50% normal human serum (NHS) was performed in order to find factors influencing the resistance to higher concentrations of NHS. Ten S. Enteritidis clinical strains, resistant to 50% NHS, all producing very long lipopolysaccharide, were subjected to the challenge of 75% NHS. Five extreme strains: two resistant and three sensitive to 75% NHS, were chosen for the further analysis of outer membrane proteins composition. Substantial differences were found in the levels of particular outer membrane proteins between resistant and sensitive strains, i.e. outer membrane protease E (PgtE) was present mainly in resistant strains, while sensitive strains possessed a high level of flagellar hook-associated protein 2 (FliD) and significantly higher levels of outer membrane protein A (OmpA). PMID:27695090

  10. Interaction of Human Serum Album and C60 Aggregates in Solution

    Directory of Open Access Journals (Sweden)

    Hailin Wang

    2011-08-01

    Full Text Available An important property of C60 in aquatic ecotoxicology is that it can form stable aggregates with nanoscale dimensions, namely nC60. Aggregation allows fullerenes to remain suspended for a long time, and the reactivity of individual C60 is substantially altered in this aggregate form. Herein, we investigated the interaction of nC60 and human serum album (HSA using the methods of fluorescence, fluorescence dynamics, circular dichroism (CD, and site marker competitive experiments. We proposed a binding model consistent with the available experimental results for the interactions of nC60 with HSA. During the interaction process, the structure and conformation of HSA were affected, leading to functional changes of drug binding sites of HSA.

  11. In vitro inhibition effect of some coumarin compounds on purified human serum paraoxonase 1 (PON1).

    Science.gov (United States)

    Gokce, Basak; Gencer, Nahit; Arslan, Oktay; Karatas, Mert Olgun; Alici, Bulent

    2016-08-01

    Human serum paraoxonase 1 (PON1; EC 3.1.8.1) is a high-density lipoprotein associated, calcium-dependent enzyme that hydrolyses aromatic esters, organophosphates and lactones and can protect the low-density lipoprotein against oxidation. In this study, in vitro effect of some hydroxy and dihydroxy ionic coumarin derivatives (1-20) on purified PON1 activity was investigated. Among these compounds, derivatives 11-20 are water soluble. In investigated compounds, compounds 6 and 13 were found the most active (IC50 = 35 and 34 µM) for PON1, respectively. The present study has demonstrated that PON1 activity is very highly sensitive to studied coumarin derivatives.

  12. Studies on the binding of vinpocetine to human serum albumin by molecular spectroscopy and modeling

    Institute of Scientific and Technical Information of China (English)

    Hua Jiang; Rong Rong Chen; Hong Cui Wang; Han Lin Pu

    2012-01-01

    The interaction between vinpocetine (VPC) and human serum albumin (HSA) in physiological buffer (pH 7.40) was investigated by fluorescence,FT-IR,UV-vis absorption and molecular modeling.VPC effectively quenched the intrinsic fluorescence of HSA via static quenching.The binding site number n and apparent binding constant Ka,corresponding thermodynamic parameters △G,△H and △S at different temperatures were calculated.The synchronous fluorescence and FT-IR spectra were used to investigate the structural change of HSA molecules with addition of VPC.Molecular modeling indicated that VPC could bind to the site I of HSA and hydrophobic interaction was the major acting force,which was in agreement with the binding mode study.

  13. Doping Human Serum Albumin with Retinoate Markedly Enhances Electron Transport Across the Protein

    CERN Document Server

    Amdursky, Nadav; Sheves, Mordechai; Cahen, David

    2012-01-01

    Electrons can migrate via proteins over distances that are considered long for non-conjugated systems. Proteins' nano-scale dimensions and the enormous flexibility of their structures and chemistry makes them fascinating subjects for investigating the mechanism of their electron transport (ETp) capacity. One particular attractive research direction is that of tuning their ETp efficiency by doping them with external small molecules. Here we report that solid-state ETp across human serum albumin (HSA) increases by more than two orders of magnitude upon retinoate (RA) binding to HSA. RA was chosen because optical spectroscopy has provided evidence for the non-covalent binding of at least three RA molecules to HSA and indications for their relative structural positions. The temperature dependence of ETp shows that both the activation energy and the distance-decay constant decrease with increasing RA binding to HSA. Furthermore, the observed transition from temperature-activated ETp above 190K to temperature-indep...

  14. [Management of fetuses in the late pregnancy by serial determinations of serum human placental lactogen levels].

    Science.gov (United States)

    Kaibara, M; Marumoto, Y; Taniguchi, I; Kobayashi, T

    1982-10-01

    Serum human placental lactogen levels (HPL) were measured serially during the last three weeks before full term deliveries of 69 normal and 60 high risk pregnant women with the method of latex agglutination (Gestefollow 'Eiken'). Except pregnancies complicated with diabetes mellitus, no fetal distress were observed when deliveries were made while HPL levels were increasing. The incidence of fetal distress was only 3.7 per cent when the range of variation of HPL levels was within 20 per cent for 3 weeks before delivery. On the contrary, the incidence of fetal distress increased to 29.4 per cent when infants were delivered after decreasing of HPL levels to less than 80 per cent of the highest HPL levels. It was also demonstrated that single determinations of HPL levels were not clinically useful in predicting fetal distress or fetal growth retardation.

  15. Fluorescence resonance energy transfer from tryptophan in human serum albumin to a bioactive indoloquinolizine system

    Indian Academy of Sciences (India)

    Paramita Das; Arabinda Mallick; Basudeb Haldar; Alok Chakrabarty; Nitin Chattopadhyay

    2007-03-01

    The interaction between a bioactive molecule, 3-acetyl-4-oxo-6,7-dihydro-12H indolo-[2,3-a] quinolizine (AODIQ), with human serum albumin (HSA) has been studied using steady-state absorption and fluorescence techniques. A 1 : 1 complex formation has been established and the binding constant () and free energy change for the process have been reported. The AODIQ-HSA complex results in fluorescence resonance energy transfer (FRET) from the tryptophan moiety of HSA to the probe. The critical energy-transfer distance (0) for FRET and the Stern-Volmer constant (sv) for the fluorescence quenching of the donor in the presence of the acceptor have been determined. Importantly, SV has been shown to be equal to the binding constant itself, implying that the fluorescence quenching arises only from the FRET process. The study suggests that the donor and the acceptor are bound to the same protein at different locations but within the quenching distance.

  16. Kinetics of fatty acid binding ability of glycated human serum albumin

    Indian Academy of Sciences (India)

    Eiji Yamazaki; Minoru Inagaki; Osamu Kurita; Tetsuji Inoue

    2005-09-01

    Kinetics of fatty acid binding ability of glycated human serum albumin (HSA) were investigated by fluorescent displacement technique with 1-anilino-8-naphtharene sulphonic acid (ANS method), and photometric detection of nonesterified-fatty-acid (NEFA method). Changing of binding affinities of glycated HSA toward oleic acid, linoleic acid, lauric acid, and caproic acid, were not observed by the ANS method. However, decreases of binding capacities after 55 days glycation were confirmed by the NEFA method in comparison to control HSA. The decrease in binding affinities was: oleic acid (84%), linoleic acid (84%), lauric acid (87%), and caproic acid (90%), respectively. The decreases were consistent with decrease of the intact lysine residues in glycated HSA. The present observation indicates that HSA promptly loses its binding ability to fatty acid as soon as the lysine residues at fatty acid binding sites are glycated.

  17. Binding of caffeine, theophylline, and theobromine with human serum albumin: A spectroscopic study

    Science.gov (United States)

    Zhang, Hong-Mei; Chen, Ting-Ting; Zhou, Qiu-Hua; Wang, Yan-Qing

    2009-12-01

    The interaction between three purine alkaloids (caffeine, theophylline, and theobromine) and human serum albumin (HSA) was investigated using UV/vis absorption, circular dichroism (CD), fluorescence, synchronous fluorescence, and three-dimensional fluorescence spectra techniques. The results revealed that three alkaloids caused the fluorescence quenching of HSA by the formation of alkaloid-HSA complex. The binding site number n and apparent binding constant KA, corresponding thermodynamic parameters the free energy change (Δ G), enthalpy change (Δ H), and entropy change (Δ S) at different temperatures were calculated. The hydrophobic interaction plays a major role in stabilizing the complex. The distance r between donor (HSA) and acceptor (alkaloids) was obtained according to fluorescence resonance energy transfer. The effect of alkaloids on the conformation of HSA was analyzed using circular dichroism (CD), UV/vis absorption, synchronous fluorescence and three-dimensional fluorescence spectra techniques.

  18. Covalent Modification of Human Serum Albumin by the Natural Sesquiterpene Lactone Parthenolide

    Directory of Open Access Journals (Sweden)

    Michael Plöger

    2015-04-01

    Full Text Available The reactivity of parthenolide (PRT, a natural sesquiterpene lactone from Tanacetum parthenium (Asteraceae, with human serum albumin (HSA was studied by UHPLC/+ESI-QqTOF MS analysis after tryptic digestion of albumin samples after incubation with this compound. It was found that the single free cysteine residue, C34, of HSA (0.6 mM reacted readily with PRT when incubated at approximately 13-fold excess of PRT (8 mM. Time-course studies with PRT and its 11β,13-dihydro derivative at equimolar ratios of the reactants revealed that PRT under the chosen conditions reacts preferably with C34 and does so exclusively via its α-methylene-γ-lactone moiety, while the epoxide structure is not involved in the reaction.

  19. [Determination of phenobarbital in human urine and serum using flow injection chemiluminescence].

    Science.gov (United States)

    Li, X; Niu, L C; He, X L; Song, Z H

    2012-01-01

    A sensitive chemiluminescence method, based on the enhancive effect of phenobarbital on the chemiluminescence reaction between luminol and dissolved oxygen in a flow injection system, was proposed for the determination of phenobarbital. The chemiluminescence intensity responded to the concentration of phenobarbital linearly ranging from 0.05 to 10 ng x ml(-1) with the detection limit of 0.02 ng x ml(-1) (3 sigma). At a flow rate of 2.0 ml x min(-1), a complete determination of phenobarbital, including sampling and washing, could be accomplished in 0.5 min, offering the sampling efficiency of 120 h(-1) accordingly. The method was applied successfully in an assay of PB for pharmaceutical preparations, human urine and serum without any pretreatment with recovery from 95.7 to 106.7% and RSDs of less than 3.0%.

  20. Covalent modification of human serum albumin by the natural sesquiterpene lactone parthenolide.

    Science.gov (United States)

    Plöger, Michael; Sendker, Jandirk; Langer, Klaus; Schmidt, Thomas J

    2015-04-09

    The reactivity of parthenolide (PRT), a natural sesquiterpene lactone from Tanacetum parthenium (Asteraceae), with human serum albumin (HSA) was studied by UHPLC/+ESI-QqTOF MS analysis after tryptic digestion of albumin samples after incubation with this compound. It was found that the single free cysteine residue, C34, of HSA (0.6 mM) reacted readily with PRT when incubated at approximately 13-fold excess of PRT (8 mM). Time-course studies with PRT and its 11β,13-dihydro derivative at equimolar ratios of the reactants revealed that PRT under the chosen conditions reacts preferably with C34 and does so exclusively via its α-methylene-γ-lactone moiety, while the epoxide structure is not involved in the reaction.

  1. Ligand binding strategies of human serum albumin: how can the cargo be utilized?

    Science.gov (United States)

    Varshney, Ankita; Sen, Priyankar; Ahmad, Ejaz; Rehan, Mohd; Subbarao, Naidu; Khan, Rizwan Hasan

    2010-01-01

    Human serum albumin (HSA), being the most abundant carrier protein in blood and a modern day clinical tool for drug delivery, attracts high attention among biologists. Hence, its unfolding/refolding strategies and exogenous/endogenous ligand binding preference are of immense use in therapeutics and clinical biochemistry. Among its fellow proteins albumin is known to carry almost every small molecule. Thus, it is a potential contender for being a molecular cargo/or nanovehicle for clinical, biophysical and industrial purposes. Nonetheless, its structure and function are largely regulated by various chemical and physical factors to accommodate HSA to its functional purpose. This multifunctional protein also possesses enzymatic properties which may be used to convert prodrugs to active therapeutics. This review aims to highlight current overview on the binding strategies of protein to various ligands that may be expected to lead to significant clinical applications.

  2. Study of caffeine binding to human serum albumin using optical spectroscopic methods

    Institute of Scientific and Technical Information of China (English)

    2009-01-01

    The binding of caffeine to human serum albumin (HSA) under physiological conditions has been stud-ied by the methods of fluorescence,UV-vis absorbance and circular dichroism (CD) spectroscopy. The mechanism of quenching of HSA fluorescence by caffeine was shown to involve a dynamic quenching procedure. The number of binding sites n and apparent binding constant Kb were measured by the fluorescence quenching method and the thermodynamic parameters △H,△G,△S were calculated. The results indicate that the binding is mainly enthalpy-driven,with van der Waals interactions and hydrogen bonding playing major roles in the reaction. The distance r between donor (HSA) and acceptor (caffeine) was obtained according to the Frster theory of non-radiative energy transfer. Synchronous fluorescence,CD and three-dimensional fluorescence spectroscopy showed that the microenvironment and conformation of HSA were altered during the reaction.

  3. Interaction of Hyperoside with Human Serum Albumin and Effect of Glucose on the Binding

    Directory of Open Access Journals (Sweden)

    Jie Yang

    2014-01-01

    Full Text Available The interaction of hyperoside (Hyp with human serum albumin (HSA and effect of glucose on the binding were studied in simulating physiological condition (pH 7.40. The results suggested that Hyp quenched the endogenous fluorescence of HSA via a static quenching process with the distance of 1.95 nm between Hyp and HSA. Hydrophobic forces played a major role in stabilizing the Hyp-HSA complex. Through synchronous fluorescence monitoring of conformation of HSA, we found that the binding to Hyp can change the microenvironment around tryptophan (Trp residues. Increasing in glucose concentration over a range from 0 to 9 mM decreased the binding ability of HSA to Hyp, implying that increasing in glucose concentration would increase the concentration of free Hyp.

  4. Investigation of ketoprofen binding to human serum albumin by spectral methods

    Science.gov (United States)

    Bi, Shuyun; Yan, Lili; Sun, Yantao; Zhang, Hanqi

    2011-01-01

    The binding of ketoprofen with human serum albumin (HSA) was studied by fluorescence and absorption spectroscopic methods. Quenching of fluorescence of HSA was found to be a static quenching process. At 288.15, 298.15, 308.15 and 318.15 K, the binding constants and binding sites were obtained. The effects of Cu 2+, Al 3+, Ca 2+, Pb 2+ and K + on the binding at 288.15 K were also studied. The thermodynamic parameters, Δ H, Δ G and Δ S were got and the main sort of acting force between ketoprofen and HSA was studied. Based on the Förster's theory of non-radiation energy transfer, the binding average distance, r, between the acceptor (ketoprofen) and the donor (HSA) was calculated.

  5. Influence of the galloyl moiety in tea catechins on binding affinity for human serum albumin.

    Science.gov (United States)

    Minoda, Kanako; Ichikawa, Tatsuya; Katsumata, Tomoharu; Onobori, Ken-ichi; Mori, Taiki; Suzuki, Yukiko; Ishii, Takeshi; Nakayama, Tsutomu

    2010-01-01

    The major catechins of green tea extract are (-)-epicatechin (EC), (-)-epigallocatechin (EGC), (-)-epicatechin gallate (ECg), and (-)-epigallocatechin gallate (EGCg). Recent research has indicated that catechins form complexes with human serum albumin (HSA) in blood, and differences in their binding affinity toward HSA are believed to modulate their bioavailability. In this study, we kinetically investigated the interaction between the catechins and HSA immobilized on a quartz-crystal microbalance (QCM). The association constants obtained from the frequency changes of QCM revealed interactions of ECg and EGCg with HSA that are 100 times stronger than those of EC and EGC. Furthermore, comparisons of these catechins by native-gel electrophoresis/blotting with redox-cycling staining revealed that, in a phosphate buffer, ECg and EGCg have a higher binding affinity toward HSA than EC and EGC. These observations indicate that catechins with a galloyl moiety have higher binding affinities toward HSA than catechins lacking a galloyl moiety.

  6. Binding properties of drospirenone with human serum albumin and lysozyme in vitro

    Science.gov (United States)

    Wang, Qing; Ma, Xiangling; He, Jiawei; Sun, Qiaomei; Li, Yuanzhi; Li, Hui

    2016-01-01

    The interaction of drospirenone (DP) with human serum albumin (HSA)/lysozyme (LYZ) was investigated using different optical techniques and molecular models. Results from the emission and time resolved fluorescence studies revealed that HSA/LYZ emission quenching with DP was initiated by static quenching mechanism. The LYZ-DP system was more easily influenced by temperature than the HSA-DP system. Displacement experiments demonstrated that the DP binding site was mainly located in site 1 of HSA. Based on the docking methods, DP was mainly bound in the active site hinge region where Trp-62 and Trp-63 are located. Conformation study showed that DP had different effects on the local conformation of HSA and LYZ molecules.

  7. Gold nanoparticles' blocking effect on UV-induced damage to human serum albumin

    Energy Technology Data Exchange (ETDEWEB)

    Calzolai, Luigi, E-mail: luigi.calzolai@jrc.ec.europa.eu; Laera, Stefania; Ceccone, Giacomo; Gilliland, Douglas [Institute for Health and Consumer Protection, European Commission, Joint Research Centre (Italy); Hussain, Rohanah; Siligardi, Giuliano [Diamond Light Source (United Kingdom); Rossi, Francois [Institute for Health and Consumer Protection, European Commission, Joint Research Centre (Italy)

    2013-01-15

    Ultraviolet radiation can cause the unfolding and destabilization of proteins. By using high energy photons from a synchrotron radiation source, we show that the UV-induced destabilization of human serum albumin (HSA) can be detected and monitored by measuring the circular dichroism spectrum of the protein. The high flux radiation source damages the HSA protein by causing a partial unfolding of the protein and a significant reduction in the amount of its secondary structure. Gold nanoparticles can effectively stop this UV-induced unfolding of HSA caused by synchrotron radiation. These phenomena could offer interesting applications to protect HSA protein from UV-induced damage and provide an alternative method to measure the relative stability of HSA.

  8. Serum Amyloid P Component (SAP) Interactome in Human Plasma Containing Physiological Calcium Levels.

    Science.gov (United States)

    Poulsen, Ebbe Toftgaard; Pedersen, Kata Wolff; Marzeda, Anna Maria; Enghild, Jan J

    2017-02-14

    The pentraxin serum amyloid P component (SAP) is secreted by the liver and found in plasma at a concentration of approximately 30 mg/L. SAP is a 25 kDa homopentamer known to bind both protein and nonprotein ligands, all in a calcium-dependent manner. The function of SAP is unclear but likely involves the humoral innate immune system spanning the complement system, inflammation, and coagulation. Also, SAP is known to bind to the generic structure of amyloid deposits and possibly to protect them against proteolysis. In this study, we have characterized the SAP interactome in human plasma containing the physiological Ca(2+) concentration using SAP affinity pull-down and co-immunoprecipitation experiments followed by mass spectrometry analyses. The analyses resulted in the identification of 33 proteins, of which 24 were direct or indirect interaction partners not previously reported. The SAP interactome can be divided into categories that include apolipoproteins, the complement system, coagulation, and proteolytic regulation.

  9. A Comparative Study on the Interaction of Sulfonamide and Nanosulfonamide with Human Serum Albumin

    Directory of Open Access Journals (Sweden)

    G. Rezaei Behbehani

    2013-01-01

    Full Text Available Binding parameters of the N-phenyl benzene sulfonyl hydrazide, sulfonamide, and nanosulfonamide interaction with human serum albumin were determined by calorimetry method. The obtained binding parameters indicated that sulfonamide in the second binding sites has higher affinity for binding than the first binding sites. The binding process of sulfonamide to HSA is both enthalpy and entropy driven. The associated equilibrium constants confirm that sulfonamide binds to HSA with high affinity (2.2×106 and 3.86105 M−1 for first and second sets of binding sites, resp.. The obtained results indicate that sulfonamide increases the HSA antioxidant property. Nanosulfonamide has much more affinity for HSA (3.6×106 M−1 than sulfonamide.

  10. Measurement of triglycerides concentration in human serum using near-infrared transmission spectroscopy and interval PLS

    Science.gov (United States)

    Huang, Furong; Yu, Jianhui; Li, Shiping

    2011-11-01

    In order to measurement of Triglycerides in human serum with reagent-less using near-infrared (NIR) spectroscopy. Interval partial least square (iPLS) was proposed as an effective variable selection approach for multivariate calibration. For this purpose, an independent sample set was employed to evaluate the prediction ability of the resulting model. The spectrum was split into different interval. Then, the informative region of Triglycerides (1654-1746nm), in which the PLS model has a low RMSEP with 0.157mmol/L and a high R with 0.967, is selected with 18 intervals. The results show that the informative region of Triglycerides can be obtained by iPLS and applied to design the simpler reagent-less NIR instruments for inexpensive Triglycerides measurement in future.

  11. DNA-duplex linker for AFM-SELEX of DNA aptamer against human serum albumin.

    Science.gov (United States)

    Takenaka, Musashi; Okumura, Yuzo; Amino, Tomokazu; Miyachi, Yusuke; Ogino, Chiaki; Kondo, Akihiko

    2017-02-15

    DNA-duplex interactions in thymines and adenins are used as a linker for the novel methodology of Atomic Force Microscope-Systematic Evolution of Ligands by EXpotential enrichment (AFM-SELEX). This study used the hydrogen bonds in 10 mer of both thymines (T10) and adenines (A10). Initially, the interactive force in T10-A10 was measured by AFM, which returned an average interactive force of approximately 350pN. Based on this result, DNA aptamers against human serum albumin could be selected in the 4th round, and 15 different clones could be sequenced. The lowest dissociation constant of the selected aptamer was identified via surface plasmon resonance, and it proved to be identical to that of the commercial aptamer. Therefore, specific hydrogen bonds in DNA can be useful linkers for AFM-SELEX. Copyright © 2016 Elsevier Ltd. All rights reserved.

  12. Multiple binding of bilirubin to human serum albumin and cobinding with laurate

    DEFF Research Database (Denmark)

    Sato, H; Honoré, B; Brodersen, R

    1988-01-01

    method, based upon a difference of light absorption spectrum for free and bound bilirubin. The observations were supplemented with previous data from an independent technique, measurement of oxidation rates of free bilirubin with hydrogen peroxide and peroxidase. A continuous isotherm was obtained......Numerical analysis of multiple binding of two ligands to one carrier has been accomplished, using the principle of several sets of acceptable binding constants, with bilirubin-laurate-albumin as an example. Binding of bilirubin to defatted human serum albumin was investigated by a spectroscopic....... Cobinding of bilirubin and laurate was studied, with up to 2 mol of each ligand per mole albumin, using the peroxidase method for determination of free equilibrium concentrations of bilirubin, and a dialysis rate technique for free laurate. The findings could be described in terms of a stoichiometric model...

  13. Heterogeneity in copper and glycan content of ceruloplasmin in human serum differs in health and disease

    DEFF Research Database (Denmark)

    Hansen, J.-E.S.; Heegaard, Peter M. H.; Jensen, S.P.

    1988-01-01

    Crossed immunoelectrophoresis of human serum revealed two heterogeneity types of ceruloplasmin with different electrophoretic migration. The two types both consisted of peptides with Mr 150 000, 100 000 and 45 000, which were interpreted as native ceruloplasmin and two hydrolytic fragments. The two...... types were different in copper content, and one type could reversibly be changed into the other. The glycan microheterogeneity of ceruloplasmin was analyzed by crossed affinommunoelectrophoresis with free Lens culinaris agglutinin (LCA) and wheat germ agglutinin (WGA). A third of the ceruloplasmin...... molecules, both high and low copper type, bound to LCA and two thirds to WGA. The heterogeneity and the microheterogeneity of ceruloplasmin in two groups of patient sera were compared to sera from healthy individuals. The ceruloplasmin type with respect to copper content was a much better factor than either...

  14. Radioimmunoassay of human homologous prolactin in serum with commercially available reagents. [/sup 125/I tracer technique

    Energy Technology Data Exchange (ETDEWEB)

    Kao, P.C.; Jiang, N.S.; Abboud, C.F.

    1977-09-01

    A clinically useful and reproducible radioimmunoassay for human homologous prolactin, established with commercially available reagents, was studied and validated. We present detailed conditions for iodination and purification of labeled prolactin and the optimal conditions for the assay. By the method, we found values (..mu..g/liter) as follows for serum prolactin: normal men, 8.9 +- 5.2 (mean +- SD); normal women, 11.8 +- 5.5; normal women taking contraceptive pills, 9.2 +- 5.0; pregnant women in the third trimester, 188 +- 69.5; patients with various diseases other than of the hypothalamic-pituitary axis, 9.3 +- 6.3; in some patients with amenorrhea and galactorrhea of diverse origin, 78.2 +- 87.4; and in some patients with surgically proven pituitary tumor, 1414 +- 1980. Results under provocative testing are also presented for a patient with normal hypothalamic-pituitary function.

  15. Investigation on a Potential Targeting Drug Delivery System Consisting of Folate, Mitoxantrone and Human Serum Albumin

    Institute of Scientific and Technical Information of China (English)

    ZHOU Qiu-Jua; BI Ya-Jing; XIANG Jun-Feng; TANG Ya-Lin; YANG Qian-Fan; XU Guang-Zhi

    2008-01-01

    A potential targeting drug delivery system consisting of folate (FA), the targeting molecule, human serum al- bumin (HSA), the carrier, and mitoxantrone (MTO), the medicine, has been designed. Data obtained by UV absorp-tion, fluorescence, and NMR techniques indicated the formation of ternary complexes and possible application to building a targeting drug delivery system by using FA, MTO and HSA. Furthermore, cytotoxicity assay indicated that the toxicity of the FA-HSA-MTO against PC-3 cell line was 79.95%, which was much higher than that of free MTO tested in totally the same conditions. About 30% increase of the toxicity should be owed to the targeting ef-fect of FA. Thus, the feasibility and validity of a novel targeting drug delivery system, FA-HSA-MTO, was con-firmed.

  16. Human Serum Albumin Hybrid Incorporating Synthetic Hemes :A Novel O2-Carrying Hemoprotein

    Institute of Scientific and Technical Information of China (English)

    2001-01-01

    Incorporation of synthetic heme (FeP) into recombinant human serum albumin(rHSA) provides an artificial hemoprotein(rHSA-FeP) which can bind and release oxygenreversibly under physiological conditions(in aqueous media, pH 7.3, 37 ℃) like hemoglobin(Hb) and myoglobin. An rHSA host absorbs maximally eight FeP molecules, and thesolution properties are almost identical to those of rHSA itself. The second-order structureand surface charge distribution of rHSA were always constant independent of the bindingnumbers of FeP. Its O2-binding ability satisfies the initial clinical requirements for red cellsubstitute. Although the NO-binding affinity is 8-fold high compared to the Hb's,administration of this fluid into rats showed negligible change in the blood pressure.Physiological responses to exchange transfusion with this rHSA-FeP into anaesthetized ratshave also been evaluated.

  17. Calorimetric and spectroscopic studies on the interaction of anticancer drug mitoxantrone with human serum albumin

    Energy Technology Data Exchange (ETDEWEB)

    Keswani, Neelam [Department of Chemistry, Indian Institute of Technology Bombay, Powai, Mumbai 400 076 (India); Kishore, Nand, E-mail: nandk@chem.iitb.ac.in [Department of Chemistry, Indian Institute of Technology Bombay, Powai, Mumbai 400 076 (India)

    2011-09-15

    Highlights: > Human serum albumin exhibits two binding sites for mitoxantrone. > Discrepancies in calorimetric and spectroscopic results clarify binding sites. > Effect of ionic strength on binding permitted detailed analysis of interactions. > Electrostatic interactions predominate in binding. > One binding site on protein does not have tryptophan in immediate vicinity. - Abstract: Binding of the anticancer drug mitoxantrone with the protein human serum albumin (HSA) has been studied by using isothermal titration calorimetry (ITC), in combination with fluorescence, UV-visible, and circular dichroism spectroscopy. The thermodynamic parameters of binding have been evaluated from ITC and spectroscopic results and compared. The ITC results demonstrate that the binding of mitoxantrone with HSA occurs according to two sets of binding sites on the protein as opposed to the fluorescence and UV-visible spectroscopic results. Blockage of one binding site on HSA for mitoxantrone in the presence of NaCl indicates strong involvement of electrostatic interactions in the binding of the drug with the protein. An insignificant temperature dependence of the association constant observed in fluorescence measurements suggests a very low enthalpy of binding which is in close agreement with the results obtained from ITC measurements. Fluorescence life time measurements suggest formation of a static complex between mitoxantrone and HSA. The discrepancies in the ITC and fluorescence results suggest that one of the binding sites on the protein for mitoxantrone does not contain tryptophan residue in its immediate vicinity. The calorimetric and spectroscopic results have provided quantitative information on the binding of mitoxantrone with HSA and suggest that the binding is dominated by electrostatic interactions.

  18. Cellulose-based diagnostic devices for diagnosing serotype-2 dengue fever in human serum.

    Science.gov (United States)

    Wang, Hsi-Kai; Tsai, Cheng-Han; Chen, Kuan-Hung; Tang, Chung-Tao; Leou, Jiun-Shyang; Li, Pi-Chun; Tang, Yin-Liang; Hsieh, Hsyue-Jen; Wu, Han-Chung; Cheng, Chao-Min

    2014-02-01

    Here, two types of cellulose-based in vitro diagnostic devices are demonstrated for the diagnosis of dengue virus infection in both buffer system and human serum: 1) paper-based ELISA for providing the semiquantitative information of the disease activity of serotype-2 dengue fever to healthcare persons (i.e., monitoring the disease activity with a specific serotype in single patients); 2) lateral flow immunoassays to screen for infection with serotype-2 dengue fever (i.e., rapid YES or NO diagnosis prepared for large populations, in terms of global public health). Paper-based ELISA (specific to serotype-2 dengue fever), which builds off of our previous studies and a revised previous ELISA procedure, owns multiple advantages: 1) high sensitivity (about 40 times higher than the current ELISA-based approaches, due to our therapeutic-based monoclonal antibody) and specificity (specific to dengue virus serotype-2 nonstructural protein-1 antigens); 2) tiny amount of sample and reagent used for single tests; 3) short operating duration (i.e., rapid diagnostic device); and, 4) inexpensiveness (appropriate for use in all developing and underdeveloped nations of the world). Due to the higher sensitivity and shorter operating duration of paper-based ELISA (compared with conventional ELISA, and lateral flow immunoassays also performed in this study), this study has not only been able to perform the diagnosis of dengue virus serotype-2 nonstructural protein-1 antigens in both buffer system and human serum but also to evaluate dengue virus serotype-2 envelope proteins in the buffer system, thus successfully achieving the first such use of these proteins as the target antigen for the development of diagnostic tools. These results provide a more comprehensive understanding for the genesis of dengue fever diagnostic tools (through antibody-antigen recognition).

  19. Persistent Organic Pollutants in Serum and Several Different Fat Compartments in Humans

    Directory of Open Access Journals (Sweden)

    George W. Yu

    2011-01-01

    Full Text Available Background. Chemicals that store in lipid-rich compartments have the potential for long-term disruption of metabolic and endocrine processes. Given the evidence that persistent organic pollutants (POPs also alter systemic metabolic, endocrine, and immune system functions, it follows that elevated chemical concentrations in intra-abdominal fat may alter function, through local chemical signaling, of visceral organs. Despite this potential, there has been little study defining POP concentrations in live human intra-abdominal fat. It is at present uncertain whether POPs distribute equally to all fat compartments, including fat in serum. Methods. Seven human subjects scheduled for elective surgery for benign lesions or cancer provided consent for removal of samples of subcutaneous and intra-abdominal fat and/or cancerous tissue. These samples were analyzed for 22 chlorinated pesticides and 10 polychlorinated biphenyl (PCB congeners by GC/ECD plus GC/MS. Results. In only two subjects were the patterns and relative concentrations of PCBs and pesticides about the same in all fat compartments. In the other subjects, there were major differences in levels in subcutaneous as compared to other compartments, but with some higher and some lower. While the pattern of PCBs in the various compartments matched that of the pesticides in some, it was opposite in others. Interpretation. These results demonstrate a complicated distribution of PCB congeners and pesticides in various lipid compartments. The difference may reflect various Kows, different rates of metabolism, and/or different lengths of exposure. But the results suggest that contaminant levels in serum or even subcutaneous fat do not necessarily indicate concentrations and patterns in other kinds of adipose tissue.

  20. Kinetics and thermodynamics of human serum albumin adsorption on silicon doped diamond like carbon

    Energy Technology Data Exchange (ETDEWEB)

    Ahmed, Mukhtar H., E-mail: ahmed-m@email.ulster.ac.uk; Byrne, John A.; McLaughlin, James

    2015-03-15

    To gain a better understanding of protein adsorption onto biomaterial surfaces is required for the control of biocompatibility and bioactivity. Various samples of diamond like carbon (DLC) and silicon-doped DLC were synthesised using plasma enhanced chemical vapour deposition (PECVD). The effects of surface morphology on the interaction of human serum albumin (HSA) with doped and undoped DLC films was investigated using spectroscopic ellipsometry (SE) and other surface analysis techniques. The results highlighted an increase in both contact angle and hydrophobicity with increasing silicon dopant levels. A reduction on the contact angle values. After adsorption of HSA, the films showed a reduction in the contact angle with a significant change in the cosΔ and this gap increased with increasing surface coverage of HSA. The adsorption kinetics of HSA were also investigated and revealed that the maximum adsorption occurred at pH 5.0 and the process involved chemisorption. The experimental isotherm data were analysed using the Langmuir and Freundlich‎ models. The amount of HSA adsorbed increased with contact time and reached saturation ‎after 30 min. The adsorption ‎process was found to be pseudo first order with respect to the bulk concentration and was dependent on both the concentration of protein and surface characteristics of the samples. The amount of adsorbed HSA was higher with higher levels of silicon doping of the DLC. Therefore, doping DLC may provide an approach to controlling the protein adsorption. - Graphical abstract: The average thickness layer of HSA measurement onto surfaces of DLC and Si-DLC. - Highlights: • Diamond Like Carbon (DLC) and Silicon doped DLC were synthesised and characterised. • Si-DLC increases the hydrophobicity and decreases the surface free energy. • Adsorption study using human serum albumin (HSA). • The adsorbed amount of HSA increases with increasing of Silicon content DLC. • Adsorption process follow pseudo

  1. Data set for mass spectrometric analysis of recombinant human serum albumin from various expression systems

    Directory of Open Access Journals (Sweden)

    Daryl G.S. Smith

    2015-09-01

    Full Text Available Human serum albumin (HSA is a versatile and important protein for the pharmaceutical industry (Fanali et al., Mol. Aspects Med. 33(3 (2012 209–290. Due to the potential transmission of pathogens from plasma sourced albumin, numerous expression systems have been developed to produce recombinant HSA (rHSA (Chen et al., Biochim. Biophys. Acta (BBA—Gen. Subj. 1830(12 (2013 5515–5525; Kobayashi, Biologicals 34(1 (2006 55–59. Based on our previous study showing increased glycation of rHSA expressed in Asian rice (Frahm et al., J. Phys. Chem. B 116(15 (2012 4661–4670, both supplier-to-supplier and lot-to-lot variability of rHSAs from a number of expression systems were evaluated using reversed phase liquid chromatography linked with MS and MS/MS analyses. The data are associated with the research article ‘Determination of Supplier-to-Supplier and Lot-to-Lot Variability in Glycation of Recombinant Human Serum Albumin Expressed in Oryza sativa’ where further analysis of rHSA samples with additional biophysical methods can be found (Frahm et al., PLoS ONE 10(9 (2014 e109893. We determined that all rHSA samples expressed in rice showed elevated levels of arginine and lysine hexose glycation compared to rHSA expressed in yeast, suggesting that the extensive glycation of the recombinant proteins is a by-product of either the expression system or purification process and not a random occurrence.

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

    Science.gov (United States)

    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.

  3. The Investigation of the Interaction between Lomefloxacin and Human Serume Albumin by Specteroscopic Methods

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    F. S. Goldouzian

    2012-03-01

    Full Text Available Mechanism of the binding of lomefloxacin (LMF with human serum albumin has been studied at physiological pH (7.4 using fluorescence spectroscopic technique. LMF is a third-generation fluoroquinolone antibiotic that exhibits striking potency against a broad spectrum of Gram-negative and Gram-positive bacteria through inhibition of DNA gyrase. Lomefloxacin is a drug that is excreted in urine and has very variable systemic absorption. Human serum albumin (HSA is the most important and abundant constituent of blood plasma and serves as a protein storage component. Recently, the three-dimensional structure of HSA was determined through X-ray crystallographic measurement. Fluorescence studies showed that (LMF has an ability to quench the intrinsic fluorescence of HSA through a static quenching  procedure  according to the Stern–Volmer equation .LMF showed two types of binding sites, the first having a very high affinity (1/72 ×107M-1 and a secondary binding site with an affinity two orders lower than the primary site. The number of binding sites for complex: HSA-LMF at 280 nm was calculated 1and0.5. The microenvironment of tryptophan and tyrosin residues and more hydrophobic of fluorophores microenvironment were changed and disturbed by the blue shift in maximum wavelength and decreased in fluorescence intensity in the presence of lomefloxacin revealed  decreased polarity of the fluorophores. The binding site for LMF is in a hydrophobic pocket in the sub-domain II A of HSA.

  4. Effects of titania nanotubes with or without bovine serum albumin loaded on human gingival fibroblasts

    Directory of Open Access Journals (Sweden)

    Liu X

    2014-03-01

    Full Text Available Xiangning Liu,1,* Xiaosong Zhou,2,* Shaobing Li,3 Renfa Lai,1 Zhiying Zhou,1 Ye Zhang,1 Lei Zhou3 1The First Affiliated Hospital of Jinan University, Guangzhou, 2Chemistry Science and Technology School, Zhanjiang Normal University, Zhanjiang, 3Guangdong Provincial Stomatological Hospital, Southern Medical University, Guangzhou, People's Republic of China *These authors contributed equally to this work Abstract: Modifying the surface of the transmucosal area is a key research area because this process positively affects the three functions of implants: attachment to soft tissue, inhibiting bacterial biofilm adhesion, and the preservation of the crestal bone. To exploit the potential of titania nanotube arrays (TNTs with or without using bovine serum albumin (BSA to modify the surface of a dental implant in contact with the transmucosal area, BSA was loaded into TNTs that were fabricated by anodizing Ti sheets; the physical characteristics of these arrays, including their morphology, chemical composition, surface roughness, contact angle, and surface free energy (SFE, were assessed. The effect of Ti surfaces with TNTs or TNTs-BSA on human gingival fibroblasts (HGFs was determined by analyzing cell morphology, early adhesion, proliferation, type I collagen (COL-1 gene expression, and the extracellular secretion of COL-1. The results indicate that early HGF adhesion and spreading behavior is positively correlated with surface characteristics, including hydrophilicity, SFE, and surface roughness. Additionally, TNT surfaces not only promoted early HGF adhesion, but also promoted COL-1 secretion. BSA-loaded TNT surfaces promoted early HGF adhesion, while suppressing late proliferation and COL-1 secretion. Therefore, TNT-modified smooth surfaces are expected to be applicable for uses involving the transmucosal area. Further study is required to determine whether BSA-loaded TNT surfaces actually affect closed loop formation of connective tissue because

  5. Serological proteome analysis of dogs with breast cancer unveils common serum biomarkers with human counterparts.

    Science.gov (United States)

    Zamani-Ahmadmahmudi, Mohamad; Nassiri, Seyed Mahdi; Rahbarghazi, Reza

    2014-03-01

    Canine mammary tumor is being touted as a model for investigating the human breast cancer. Breast cancer of the both species has similar biological behavior, histopathologic characteristics, and metastatic pattern. In this study, we used the serological proteome analysis to detect autoantigens that elicit a humoral response in dogs with mammary tumor in order to identify serum biomarkers with potential usefulness as diagnostic markers and to better understand molecular mechanisms underlying canine breast cancer development. Protein extract from a cell line was subject to 2DE followed by Western blotting using sera from 15 dogs with mammary tumor and sera from 15 healthy control dogs. Immunoreactive autoantigens were subsequently identified by the MALDI-TOF MS. Four autoantigens, including manganese-superoxide dismutase, triose phosphate isomerase, alpha-enolase, and phosphoglycerate mutase1, with significantly higher immunoreactivity in the tumor samples than in the normal samples were identified as biomarker candidates. Immunohistochemistry and Western blotting revealed higher expression of these biomarkers in the malignant tumors than in the normal or benign tumors. The autoantigens found in this study have been reported to elicit autoantibody response in the human breast cancer, indicating the similarity of breast cancer proteome profile in dogs with that in human beings.

  6. Methyl-triclosan binding to human serum albumin: multi-spectroscopic study and visualized molecular simulation.

    Science.gov (United States)

    Lv, Wenjuan; Chen, Yonglei; Li, Dayong; Chen, Xingguo; Leszczynski, Jerzy

    2013-10-01

    Methyl-triclosan (MTCS), a transformation product and metabolite of triclosan, has been widely spread in environment through the daily use of triclosan which is a commonly used anti-bacterial and anti-fungal substance in consumer products. Once entering human body, MTCS could affect the conformation of human serum albumin (HSA) by forming MTCS-HSA complex and alter function of protein and endocrine in human body. To evaluate the potential toxicity of MTCS, the binding mechanism of HSA with MTCS was investigated by UV-vis absorption, circular dichroism and Fourier transform infrared spectroscopy. Binding constants, thermodynamic parameters, the binding forces and the specific binding site were studied in detail. Binding constant at room tempreture (T = 298K) is 6.32 × 10(3)L mol(-1); ΔH(0), ΔS(0) and ΔG(0) were 22.48 kJ mol(-1), 148.16 J mol(-1)K(-1) and -21.68 kJ mol(-1), respectively. The results showed that the interactions between MTCS and HSA are mainly hydrophobic forces. The effects of MTCS on HSA conformation were also discussed. The binding distance (r = 1.2 nm) for MTCS-HSA system was calculated by the efficiency of fluorescence resonance energy transfer. The visualized binding details were also exhibited by molecular modeling method and the results could agree well with that from the experimental study.

  7. Serum antibody response to group II chaperonin from Methanobrevibacter oralis and human chaperonin CCT.

    Science.gov (United States)

    Hirai, Kimito; Maeda, Hiroshi; Omori, Kazuhiro; Yamamoto, Tadashi; Kokeguchi, Susumu; Takashiba, Shogo

    2013-06-01

    Both group I (HSP60) and group II (CCT) chaperonins are targets of autoantibodies. Autoimmune reactions to HSP60 have been well characterized, while immune reactions to group II chaperonin have not been clarified. Methanobrevibacter oralis is a suspected periodontal pathogen with group II chaperonin. In this study, serum responses to M. oralis chaperonin, human HSP60, and CCT subunits were examined using sera from patients with periodontitis and autoimmune diseases. In comparison with healthy controls, periodontitis patients showed significantly higher responses to CCT4 and CCT8 on dot blot analysis. Signals for CCT3 and CCT8 in autoimmune disease patients were significantly higher than in controls. Significant differences were also demonstrated by Western blotting in anti-CCT4 response in both patient groups. All subjects showed strong reactivity to M. oralis chaperonin and faint signals to human HSP60. Autoantibodies were raised against CCT rather than HSP60; and CCT3, CCT4, and CCT8 were shown to be the main targets. Host immune systems may be frequently exposed to chaperonins of Archaea in various habitats. Although further studies of the cross-reactivity between M. oralis chaperonin and human CCT are required, anti-CCT autoantibodies may be involved in the pathogenesis of periodontitis and autoimmune diseases.

  8. Detection of poxvirus in cattle associated with human cases in the State of Rio de Janeiro: preliminary report

    Directory of Open Access Journals (Sweden)

    Hermann Gonçalves Schatzmayr

    2000-10-01

    Full Text Available This preliminary report describes human and cow cases of poxvirus that recently ocurred in the State of Rio de Janeiro. The electron microscopic findings were consistent with parapoxviral and orthopoxviral infection. Orthopoxvirus strains were isolated from human and cow cases. Detailed viral characterization by means of genetical techniques is under investigation. Based on these informations, poxviral diseases should be also considered an emerging viral zoonosis that can affect human beings.

  9. Preliminary perspectives on DNA collection in anti-human trafficking efforts.

    Science.gov (United States)

    Katsanis, Sara H; Kim, Joyce; Minear, Mollie A; Chandrasekharan, Subhashini; Wagner, Jennifer K

    2014-01-01

    Forensic DNA methodologies have potential applications in the investigation of human trafficking cases. DNA and relationship testing may be useful for confirmation of biological relationship claims in immigration, identification of trafficked individuals who are missing persons, and family reunification of displaced individuals after mass disasters and conflicts. As these applications rely on the collection of DNA from non-criminals and potentially vulnerable individuals, questions arise as to how to address the ethical challenges of collection, security, and privacy of collected samples and DNA profiles. We administered a survey targeted to victims' advocates to gain preliminary understanding of perspectives regarding human trafficking definitions, DNA and sex workers, and perceived trust of authorities potentially involved in DNA collection. We asked respondents to consider the use of DNA for investigating adoption fraud, sex trafficking, and post-conflict child soldier cases. We found some key differences in perspectives on defining what qualifies as "trafficking." When we varied terminology between "sex worker" and "sex trafficking victim" we detected differences in perception on which authorities can be trusted. Respondents were supportive of the hypothetical models proposed to collect DNA. Most were favorable of DNA specimens being controlled by an authority outside of law enforcement. Participants voiced concerns focused on privacy, misuse of DNA samples and data, unintentional harms, data security, and infrastructure. These preliminary data indicate that while there is perceived value in programs to use DNA for investigating cases of human trafficking, these programs may need to consider levels of trust in authorities as their logistics are developed and implemented.

  10. Time-resolved fluoroimmunoassays of the complete set of secreted phospholipases A2 in human serum.

    Science.gov (United States)

    Nevalainen, Timo J; Eerola, Leena I; Rintala, Esa; Laine, V Jukka O; Lambeau, Gérard; Gelb, Michael H

    2005-04-15

    Time-resolved fluoroimmunoassays (TR-FIA) were developed for all human secreted phospholipases A(2) (PLA(2)), viz. group (G) IB, GIIA, GIID, GIIE, GIIF, GIII, GV, GX and GXIIA PLA(2) and the GXIIB PLA(2)-like protein. Antibodies were raised in rabbits against recombinant human PLA(2) proteins and used in sandwich-type TR-FIAs as both catching and detecting antibodies, the latter after labeling with Europium. The antibodies were non-cross-reactive. The analytical sensitivities were 1 microg/L for the TR-FIA for GIB PLA(2), 1 microg/L (GIIA), 35 microg/L (GIID), 3 microg/L (GIIE), 4 microg/L (GIIF), 14 microg/L (GIII), 11 microg/L (GV), 2 microg/L (GX), 92 microg/L (GXIIA) and 242 microg/L (GXIIB). All secreted PLA(2)s were assayed by these TR-FIAs in serum samples from 34 patients (23 men and 11 women, mean age 53.2 years) treated in an intensive care unit for septic infections, and in control samples from 28 volunteer blood donors (14 men and 14 women, mean age 57.0 years). Five serum samples (3 in the sepsis group and 2 in the blood donor group) gave high TR-FIA signals that were reduced to background (blank) levels by the addition of non-immune rabbit IgG to the sera. This reactivity was assumed to be due to the presence of heterophilic antibodies in these subjects. In all other subjects, including septic patients and healthy blood donors, the TR-FIA signals for GIID, GIIE, GIIF, GIII, GV, GX and GXIIA PLA(2) and the GXIIB PLA(2)-like protein were at background (blank) levels. Four patients in the sepsis group had pancreatic involvement and elevated concentration of GIB PLA(2) in serum (median 19.0 microg/L, range 13.1-33.7 microg/L, n = 4) as compared to the healthy blood donors (median 1.8 microg/L, range 0.8-3.4 microg/L, n = 28, P < 0.0001). The concentration of GIIA PLA(2) in the sera of septic patients (median 315.7 microg/L, range 15.9-979.6 microg/L, n = 34) was highly elevated as compared to that of the blood donors (median 1.8 microg/L, range 0

  11. Final report for CCQM-K107: total elements and selenomethionine in human serum

    Science.gov (United States)

    Goenaga Infante, Heidi

    2016-01-01

    Routine tests that measure the concentration of electrolytes in serum are needed for diagnosis and management of renal, endocrine, acid-base, water balance and other conditions such as screening D- and A-vitamin disorders, kidney insufficiency, bone diseases and leukaemia. The diagnostic concentration ranges for many such markers are narrow, requiring reference methods with small uncertainty. Serum concentration of total selenium (Se) is important in health studies but there is increasing interest in the speciation of selenium compounds in clinical samples such as serum and individual Se- Species are bio-indicators of Se status. The last CCQM IAWG key comparison for elements in the clinical area (CCQM-K14: Ca in human serum) was organized in 2003 and the previous key comparison (CCQM-K60) for Se and Se species used a wheat flour sample. Therefore, the CCQM IAWG agreed that CCQM-K107 and a parallel pilot study CCQM-P146 should be carried out. The candidate human serum sample used for both CCQM-K107 and P146 is of high complexity and contains approximately 1000-fold lower concentrations of selenium methionine (SeMet) than those encountered in the CCQM-K60 wheat flour. This significantly broadens the scope and degree of difficulty of earlier measurements in this field. A total of eleven institutes participated in CCQM-K107 (11 participants for total elements and 7 for SeMet). The performance of the majority of the K107 participants for all the measurands was very good, illustrating their ability to obtain accurate results for analytes such as electrolytes at mg kg-1 level, essential elements at µg kg-1 level and selenium species at µg kg-1 level in a complex biological fluid. The range of agreement between participants was within the interval of ± 0.1% for Ca and up to ± 1.8% for Fe. CMC claims based on total elements in this study may include other elements with similar core competencies (e.g. Se, Cu, Zn) in a wide range of biological materials (including liquids

  12. Oxytocin is hydrolyzed by an enzyme in human placenta that is identical to the oxytocinase of pregnancy serum.

    Science.gov (United States)

    Naruki, M; Mizutani, S; Goto, K; Tsujimoto, M; Nakazato, H; Itakura, A; Mizuno, K; Kurauchi, O; Kikkawa, F; Tomoda, Y

    1996-01-01

    The hydrolysis of oxytocin (OT) by human placental subcellular fractions and pregnant sera was studied in the presence of bestatin, a potent inhibitor of aminopeptidases, and the antibody against pregnant serum oxyotocinase (P-LAP)(EC 3.4 11.3) by measuring liberated amino acids by high performance liquid chromatography (HPLC). Our immunotitration study and the effect of bastatin on the oxytocin-degrading protease showed that the initiating and responsible protease in oxyotocin degradation in human placenta and pregnant serum is P-LAP.

  13. Impact of antibacterial drugs on human serum paraoxonase-1 (hPON1) activity:an in vitro study

    Institute of Scientific and Technical Information of China (English)

    Hakan Syt; Elif Duygu Kaya; kr Beydemir

    2014-01-01

    Objective:To investigate the in vitro effects of the antibacterial drugs, meropenem trihydrate, piperacillin sodium, and cefoperazone sodium, on the activity of human serum paraoxonase Methods: hPON1 was purified from human serum using simple chromatographic methods, including DEAE-Sephadex anion exchange and Sephadex G-200 gel filtration chromatography. Results:The three antibacterial drugs decreased in vitro hPON1 activity. Inhibition mechanisms meropenem trihydrate was noncompetitive while piperacillin sodium and cefoperazone sodium were competitive. Conclusions:Our results showed that antibacterial drugs significantly inhibit hPON1 activity, both in vitro, with rank order meropenem trihydrate piperacillin sodium cefoperazone sodium in vitro.

  14. Human dental pulp stem cells cultured in serum-free supplemented medium

    Directory of Open Access Journals (Sweden)

    Virginie eBonnamain

    2013-12-01

    Full Text Available Growing evidence show that human dental pulp stem cells (DPSCs could provide a source of adult stem cells for the treatment of neurodegenerative pathologies. In this study, DPSCs were expanded and cultured with a protocol generally used for the culture of neural stem/progenitor cells.Methodology: DPSC cultures were established from third molars. The pulp tissue was enzymatically digested and cultured in serum-supplemented basal medium for 12 hours. Adherent (ADH and non-adherent (non-ADH cell populations were separated according to their differential adhesion to plastic and then cultured in serum-free defined N2 medium with epidermal growth factor (EGF and basic fibroblast growth factor (bFGF. Both ADH and non-ADH populations were analyzed by FACS and/or PCR.Results: FACS analysis of ADH-DPSCs revealed the expression of the mesenchymal cell marker CD90, the neuronal marker CD56, the transferrin receptor CD71, and the chemokine receptor CXCR3, whereas hematopoietic stem cells markers CD45, CD133 and CD34 were not expressed. ADH-DPSCs expressed transcripts coding for the Nestin gene, whereas expression levels of genes coding for the neuronal markers β-III tubulin and NF-M, and the oligodendrocyte marker PLP-1 were donor dependent. ADH-DPSCs did not express the transcripts for GFAP, an astrocyte marker. Cells of the non-ADH population that grew as spheroids expressed Nestin, β-III tubulin, NF-M and PLP-1 transcripts. DPSCs migrated out of the spheroids exhibited an odontoblast-like morphology and expressed a higher level of DSPP and osteocalcin transcripts than ADH-DPSCs. Conclusion: Collectively, these data indicate that human DPSCs can be expended and cultured in serum-free supplemented medium with EGF and bFGF. ADH-DPSCs and non-ADH populations contained neuronal and/or oligodendrocyte precursors at different stages of commitment and interestingly, cells from spheroid structures seem to be more engaged into the odontoblastic lineage than the

  15. Impedimetric immunosensor for human serum albumin detection on a direct aldehyde-functionalized silicon nitride surface

    Energy Technology Data Exchange (ETDEWEB)

    Caballero, David, E-mail: caballero@unistra.fr [Nanobioengineering group-IBEC, Barcelona Science Park, C/ Baldiri Reixach 10-12, 08028 Barcelona (Spain); University of Barcelona, Department of Electronics, C/ Marti i Franques 1, 08028 Barcelona (Spain); Centro de Investigacion Biomedica en Red en Bioingenieria, Biomateriales y Nanomedicina (CIBER-BBN), 50018 Zaragoza (Spain); Martinez, Elena [Nanobioengineering group-IBEC, Barcelona Science Park, C/ Baldiri Reixach 10-12, 08028 Barcelona (Spain); Centro de Investigacion Biomedica en Red en Bioingenieria, Biomateriales y Nanomedicina (CIBER-BBN), 50018 Zaragoza (Spain); Bausells, Joan [Centre Nacional de Microelectronica (CNM-IMB), CSIC, Campus UAB, 08193 Bellaterra (Spain); Errachid, Abdelhamid, E-mail: abdelhamid.errachid-el-salhi@univ-lyon1.fr [Nanobioengineering group-IBEC, Barcelona Science Park, C/ Baldiri Reixach 10-12, 08028 Barcelona (Spain); Universite Claude Bernard - Lyon 1, LSA - UMR 5180, 43 Bd du 11 novembre 1918, 69622 Villeurbanne Cedex (France); Samitier, Josep [Nanobioengineering group-IBEC, Barcelona Science Park, C/ Baldiri Reixach 10-12, 08028 Barcelona (Spain); University of Barcelona, Department of Electronics, C/ Marti i Franques 1, 08028 Barcelona (Spain); Centro de Investigacion Biomedica en Red en Bioingenieria, Biomateriales y Nanomedicina (CIBER-BBN), 50018 Zaragoza (Spain)

    2012-03-30

    Highlights: Black-Right-Pointing-Pointer An impedimetric label-free immunosensor was developed for the specific detection of human serum albumin proteins. Black-Right-Pointing-Pointer Anti-HSA antibodies were covalently immobilized on silicon nitride surfaces using a direct functionalization methodology. Black-Right-Pointing-Pointer Silicon nitride offers multiple advantages compared to other common materials. Black-Right-Pointing-Pointer The proposed sensor has high sensitivity and good selectivity for the detection of HSA proteins. - Abstract: In this work we report the fabrication and characterization of a label-free impedimetric immunosensor based on a silicon nitride (Si{sub 3}N{sub 4}) surface for the specific detection of human serum albumin (HSA) proteins. Silicon nitride provides several advantages compared with other materials commonly used, such as gold, and in particular in solid-state physics for electronic-based biosensors. However, few Si{sub 3}N{sub 4}-based biosensors have been developed; the lack of an efficient and direct protocol for the integration of biological elements with silicon-based substrates is still one of its the main drawbacks. Here, we use a direct functionalization method for the direct covalent binding of monoclonal anti-HSA antibodies on an aldehyde-functionalized Si-p/SiO{sub 2}/Si{sub 3}N{sub 4} structure. This methodology, in contrast with most of the protocols reported in literature, requires less chemical reagents, it is less time-consuming and it does not need any chemical activation. The detection capability of the immunosensor was tested by performing non-faradaic electrochemical impedance spectroscopy (EIS) measurements for the specific detection of HSA proteins. Protein concentrations within the linear range of 10{sup -13}-10{sup -7} M were detected, showing a sensitivity of 0.128 {Omega} {mu}M{sup -1} and a limit of detection of 10{sup -14} M. The specificity of the sensor was also addressed by studying the

  16. Identification of high immunoreactive proteins from Streptococcus agalactiae isolates recognized by human serum antibodies.

    Science.gov (United States)

    Brzychczy-Wloch, Monika; Gorska, Sabina; Brzozowska, Ewa; Gamian, Andrzej; Heczko, Piotr B; Bulanda, Malgorzata

    2013-12-01

    The aim of the studies was to identify immunogenic proteins of Streptococcus agalactiae (group B streptococcus; GBS) isolates. Investigation of the immunoreactivity with human sera allowed us to determine major immunogenic proteins which might be potential candidates for the development of vaccine. For the study, we have selected 60 genetically different, well-characterized GBS clinical isolates. The proteins immunoreactivity with 24 human sera from patients with GBS infections, carriers, and control group without GBS was detected by SDS-PAGE and Western blotting. As a result, some major immunogenic proteins were identified, of which four proteins with molecular masses of about 45 to 50 kDa, which exhibited the highest immunoreactivity features, were analyzed by LC-MS/MS. The proteins were identified by comparative analysis of peptides masses using MASCOT and statistical analysis. The results showed known molecules such as enolase (47.4 kDa), aldehyde dehydrogenase (50.6 kDa), and ones not previously described such as trigger factor (47 kDa) and elongation factor Tu (44 kDa). The preliminary results indicated that some GBS proteins that elicit protective immunity hold promise not only as components in a vaccine as antigens but also as carriers or adjuvants in polysaccharide conjugate vaccines, but more studies are needed.

  17. A comparative study of some physico-chemical properties of human serum albumin samples from different sources--I : Some physico-chemical properties of isoionic human serum albumin solutions

    NARCIS (Netherlands)

    Dröge, J.H.M.; Janssen, L.H.M.; Wilting, J.

    1982-01-01

    Human serum albumin samples from different sources were investigated. The fatty acid content of the albumin before and after deionization on a mixed bed ion-exchange column varied from sample to sample. When an albumin sample from one source was deionized under standard conditions the amount of fatt

  18. Preliminary analyses of scenarios for potential human interference for repositories in three salt formations

    Energy Technology Data Exchange (ETDEWEB)

    1985-10-01

    Preliminary analyses of scenarios for human interference with the performance of a radioactive waste repository in a deep salt formation are presented. The following scenarios are analyzed: (1) the U-Tube Connection Scenario involving multiple connections between the repository and the overlying aquifer system; (2) the Single Borehole Intrusion Scenario involving penetration of the repository by an exploratory borehole that simultaneously connects the repository with overlying and underlying aquifers; and (3) the Pressure Release Scenario involving inflow of water to saturate any void space in the repository prior to creep closure with subsequent release under near lithostatic pressures following creep closure. The methodology to evaluate repository performance in these scenarios is described and this methodology is applied to reference systems in three candidate formations: bedded salt in the Palo Duro Basin, Texas; bedded salt in the Paradox Basin, Utah; and the Richton Salt Dome, Mississippi, of the Gulf Coast Salt Dome Basin.

  19. Report: Heparin-induced thrombocytopenia associated with cardiopulmonary bypass: Preliminary attempt with recombinant human thrombopoietin therapy.

    Science.gov (United States)

    Yuan, Shi-Min

    2015-09-01

    Recombinant human thrombopoietin (rhTPO) is popularly used for the treatment of chemotherapy-induced thrombocytopenia. However, rhTPO therapy for heparin-induced thrombocytopenia relating to cardiopulmonary bypass has not been previously described. A young patient developed heparin-induced thrombocytopenia during open-heart surgery. Postoperative rhTPO therapy (15000 units injection hypodermatica once daily for consecutive 3 days) made a quick platelet recovery without any side effects. Heparin-induced thrombocytopenia associated with cardiopulmonary bypass is more likely to be benign, and is curable to rhTPO therapy. The preliminary rhTPO administration of heparin-induced thrombocytopenia in association with cardiopulmonary bypass shows satisfactory pharmaceutical effects with lower dose, shorter duration treatment and shorter platelet increase time and recovery time in comparison with those for the treatment of chemotherapy-induced thrombocytopenia. rhTPO therapy does not produce any side effects and it could avoid or minimize necessary blood product infusions.

  20. Selective and sensitive detection of free bilirubin in blood serum using human serum albumin stabilized gold nanoclusters as fluorometric and colorimetric probe.

    Science.gov (United States)

    Santhosh, Mallesh; Chinnadayyala, Somasekhar R; Kakoti, Ankana; Goswami, Pranab

    2014-09-15

    We report here a fluorescence quenching based non-enzymatic method for sensitive and reliable detection of free bilirubin in blood serum samples using human serum albumin (HSA) stabilized gold nanoclusters (HSA-AuNCs) as fluorescent probe. The fluorescence of the nanoclusters was strongly quenched by bilirubin in a concentration dependent manner by virtue of the inherent specific interaction between bilirubin and HSA. A strong binding constant of 0.55×10(6) L mole(-1) between the HSA-AuNC and bilirubin was discerned. The nano clusters each with size ~1.0 nm (in diameter) and a core of Au18 were homogeneously distributed in HSA molecules as revealed from the respective high resolution transmission electron microscopic and mass spectroscopic studies. The fluorescence quenching phenomena which obeyed a simple static quenching mechanism, was utilized for interference free detection of bilirubin with minimum detection limit (DL) of 248±12 nM (S/N=3). The fluorescence response of HSA-AuNCs against bilirubin was practically unaltered over a wide pH (6-9) and temperature (25-50 °C) range. Additionally, peroxidase-like catalytic activity of these nanoclusters was exploited for colorimetric detection of bilirubin in serum sample with a DL of 200±19 nM by following the decrease in absorbance (at λ440 nm) of the reaction and its rate constant (Kp) of 2.57±0.63 mL μg(-1) min(-1). Both these fluorometric and colorimetric methods have been successfully used for detection of free bilirubin in blood serum samples.

  1. TALEN-mediated modification of the bovine genome for large-scale production of human serum albumin.

    Science.gov (United States)

    Moghaddassi, Shaida; Eyestone, Will; Bishop, Colin E

    2014-01-01

    As an initial step towards creating genetically modified cattle as a biopharming source of recombinant human serum albumin (rHSA), we report modification of the bovine albumin (bA) locus by transcription activator-like effector nuclease (TALEN)-stimulated homology-directed repair (HDR). Pedigreed bovine fibroblasts were co-transfected with TALENs and an 11.5-kb human serum albumin (HSA) minigene donor construct, designed to simultaneously disrupt and replace bovine serum albumin (BSA) expression with controlled rHSA expression in both the liver and the milk. Targeted integration of the HSA minigene was confirmed in transfected fibroblasts at a frequency of approximately 11% and transgenic bovine embryos were produced from targeted fibroblasts using somatic cell nuclear transfer (SCNT). The research delineated here lays the foundation for the future generation of transgenic rHSA cattle with the potential to provide a large-scale, reliable, and quality-controlled source of rHSA.

  2. Study the interactions between human serum albumin and two antifungal drugs: fluconazole and its analogue DTP.

    Science.gov (United States)

    Zhang, Shao-Lin; Yao, Huankai; Wang, Chenyin; Tam, Kin Y

    2014-11-01

    Binding affinities of fluconazole and its analogue 2-(2,4-dichlorophenyl)-1,3-di(1H-1,2,4-triazol-yl)-2-propanol (DTP) to human serum albumin (HSA) were investigated under approximately human physiological conditions. The obtained result indicated that HSA could generate fluorescent quenching by fluconazole and DTP because of the formation of non-fluorescent ground-state complexes. Binding parameters calculated from the Stern-Volmer and the Scatchard equations showed that fluconazole and DTP bind to HSA with binding affinities of the order 10(4)L/mol. The thermodynamic parameters revealed that the binding was characterized by negative enthalpy and positive entropy changes, suggesting that the binding reaction was exothermic. Hydrogen bonds and hydrophobic interaction were found to be the predominant intermolecular forces stabilizing the drug-protein. The effect of metal ions on the binding constants of fluconazole-HSA complex suggested that the presence of Mg(2+) and Zn(2+) ions could decrease the free drug level and extend the half-life in the systematic circulation. Docking experiments revealed that fluconazole and DTP binds in HSA mainly by hydrophobic interaction with the possibility of hydrogen bonds formation between the drugs and the residues Arg 222, Lys 199 and Lys 195 in HSA.

  3. Interaction of oridonin with human serum albumin by isothermal titration calorimetry and spectroscopic techniques.

    Science.gov (United States)

    Li, Xiangrong; Yang, Zhenhua

    2015-05-05

    Oridonin has been traditionally and widely used for treatment of various human diseases due to its uniquely biological, pharmacological and physiological functions. In this study, the interaction between oridonin and human serum albumin (HSA) was investigated using isothermal titration calorimetry (ITC), in combination with fluorescence spectroscopy and UV-vis absorption spectroscopy. We found that the hydrogen bond and van der Waals force are the major binding forces in the binding of oridonin to HSA. The binding of oridonin to HSA is driven by favorable enthalpy and unfavorable entropy. Oridonin can quench the fluorescence of HSA through a static quenching mechanism. The binding constant between oridonin and HSA is moderate and the equilibrium fraction of unbound oridonin f(u) > 60%. Binding site I is found to be the primary binding site for oridonin. Additionally, oridonin may induce conformational changes of HSA and affect its biological function as the carrier protein. The results of the current study suggest that oridonin can be stored and transported from the circulatory system to reach its target organ to provide its therapeutic effects. But its side-effect in the clinics cannot be overlook. The study provides an accurate and full basic data for clarifying the binding mechanism of oridonin with HSA and is helpful for understanding its effect on protein function during the blood transportation process and its biological activity in vivo. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

  4. Evaluation of human serum of severe rheumatoid arthritis by confocal Raman spectroscopy

    Science.gov (United States)

    Carvalho, C. S.; Raniero, L.; Santo, A. M. E.; Pinheiro, M. M.; Andrade, L. E. C.; Cardoso, M. A. G.; Junior, J. S.; Martin, A. A.

    2010-02-01

    Rheumatoid Arthritis is a systemic chronic inflammatory disease, recurrent and systemic, initiated by autoantibodies and maintained by inflammatory mechanisms cellular applicants. The evaluation of this disease to promote early diagnosis, need an associations of many tools, such as clinical, physical examination and thorough medical history. However, there is no satisfactory consensus due to its complexity. In the present work, confocal Raman spectroscopy was used to evaluate the biochemical composition of human serum of 40 volunteers, 24 patients with rheumatoid arthritis presenting clinical signs and symptoms, and 16 healthy donors. The technique of latex agglutination for the polystyrene covered with human immunoglobulin G and PCR (protein c-reactive) was performed for confirmation of possible false-negative results within the groups, facilitating the statistical interpretation and validation of the technique. This study aimed to verify the changes for the characteristics Raman peaks of biomolecules such as immunoglobulins amides and protein. The results were highly significant with a good separation between groups mentioned. The discriminant analysis was performed through the principal components and correctly identified 92% of the donors. Based on these results, we observed the behavior of arthritis autoimmune, evident in certain spectral regions that characterize the serological differences between the groups.

  5. Human primary brain tumor cell growth inhibition in serum-free medium optimized for neuron survival.

    Science.gov (United States)

    Brewer, Gregory J; LeRoux, Peter D

    2007-07-09

    Glioblastoma is the most common primary brain tumor in adults from which about 15,000 patients die each year in the United States. Despite aggressive surgery, radiotherapy and chemotherapy, median survival remains only 1 year. Here we evaluate growth of primary human brain tumor cells in a defined nutrient culture medium (Neuregen) that was optimized for neuron regeneration. We hypothesized that Neuregen would inhibit tumor cell growth because of its ability to inhibit gliosis in rat brain. Tumor tissue was collected from 18 patients including 10 males and 8 females (mean age 60+/-12 years) who underwent craniotomy for newly diagnosed, histologically confirmed brain tumors. The tissue was shipped overnight in Hibernate transport medium. Tumor cells were isolated and plated in Neurobasal/serum or Neuregen on culture plastic. After 1 week, growth in Neuregen was significantly less in 9/10 glioblastoma multiforme cases, 5/5 meningioma cases and 3/3 cases of brain metastasis. Analysis of deficient formulations of Neuregen and formulations to which selected components were added back implicate no single active component. However, individual cases were sensitive to corticosterone, selenium, ethanolamine, fatty acids and/or antioxidants. Therefore, a defined culture medium that promotes neuron regeneration inhibits the growth of human primary glioblastoma, meningioma and metastatic tumor cells in culture. The possible in vivo efficacy of Neuregen for treatment of brain tumor resections remains to be determined.

  6. Potential toxicity of sulfanilamide antibiotic: Binding of sulfamethazine to human serum albumin

    Energy Technology Data Exchange (ETDEWEB)

    Chen, Jiabin [State Key Laboratory of Pollution Control and Resources Reuse, College of Environmental Science and Engineering, Tongji University, Shanghai, 200092 (China); Zhou, Xuefei [Key Laboratory of Yangtze River Water Environment for Ministry of Education, College of Environmental Science and Engineering, Tongji University, Shanghai, 200092 (China); Zhang, Yalei, E-mail: zhangyalei2003@163.com [State Key Laboratory of Pollution Control and Resources Reuse, College of Environmental Science and Engineering, Tongji University, Shanghai, 200092 (China); Gao, Haiping [Key Laboratory of Yangtze River Water Environment for Ministry of Education, College of Environmental Science and Engineering, Tongji University, Shanghai, 200092 (China)

    2012-08-15

    Antibiotics are widely used in daily life but their abuse has posed a potential threat to human health. The interaction between human serum albumin (HSA) and sulfamethazine (SMZ) was investigated by capillary electrophoresis, fluorescence spectrometry, and circular dichroism. The binding constant and site were determined to be 1.09 Multiplication-Sign 10{sup 4} M{sup -1} and 1.14 at 309.5 K. The thermodynamic determination indicated that the interaction was driven by enthalpy change, where the electrostatic interaction and hydrogen bond were the dominant binding force. The binding distance between SMZ and tryptophan residue of HSA was obtained to be 3.07 nm according to Foerster non-radioactive energy transfer theory. The site marker competition revealed that SMZ bound into subdomain IIA of HSA. The binding of SMZ induced the unfolding of the polypeptides of HSA and transferred the secondary conformation of HSA. The equilibrium dialysis showed that only 0.13 mM SMZ decreased vitamin B{sub 2} by 38% transported on the HSA. This work provides a new quantitative evaluation method for antibiotics to cause the protein damage. -- Highlights: Black-Right-Pointing-Pointer Various techniques characterized the interactions between SMZ and HSA. Black-Right-Pointing-Pointer The electrostatic interaction and hydrogen bond dominated in the interaction. Black-Right-Pointing-Pointer SMZ induced the conformation change of HSA. Black-Right-Pointing-Pointer SMZ affected the transportation function of HSA.

  7. Determination of energies and sites of binding of PFOA and PFOS to human serum albumin.

    Science.gov (United States)

    Salvalaglio, Matteo; Muscionico, Isabella; Cavallotti, Carlo

    2010-11-25

    Structure and energies of the binding sites of perfluorooctanoic acid (PFOA) and perfluorooctane sulfonate (PFOS) to human serum albumin (HSA) were determined through molecular modeling. The calculations consisted of a compound approach based on docking, followed by molecular dynamics simulations and by the estimation of the free binding energies adopting WHAM-umbrella sampling and semiempirical methodologies. The binding sites so determined are common either to known HSA fatty acids sites or to other HSA sites known to bind to pharmaceutical compounds such as warfarin, thyroxine, indole, and benzodiazepin. Among the PFOA binding sites, five have interaction energies in excess of -6 kcal/mol, which become nine for PFOS. The calculated binding free energy of PFOA to the Trp 214 binding site is the highest among the PFOA complexes, -8.0 kcal/mol, in good agreement with literature experimental data. The PFOS binding site with the highest energy, -8.8 kcal/mol, is located near the Trp 214 binding site, thus partially affecting its activity. The maximum number of ligands that can be bound to HSA is 9 for PFOA and 11 for PFOS. The calculated data were adopted to predict the level of complexation of HSA as a function of the concentration of PFOA and PFOS found in human blood for different levels of exposition. The analysis of the factors contributing to the complex binding energy permitted to outline a set of guidelines for the rational design of alternative fluorinated surfactants with a lower bioaccumulation potential.

  8. Profiling serum bile acid glucuronides in humans: gender divergences, genetic determinants and response to fenofibrate

    Science.gov (United States)

    Trottier, Jocelyn; Perreault, Martin; Rudkowska, Iwona; Levy, Cynthia; Dallaire-Theroux, Amélie; Verreault, Mélanie; Caron, Patrick; Staels, Bart; Vohl, Marie-Claude; Straka, Robert J.; Barbier, Olivier

    2014-01-01

    Glucuronidation, catalyzed by UDP-glucuronosyltransferase (UGT) enzymes detoxifies cholestatic bile acids (BAs). We aimed at i) characterizing the circulating BA-glucuronide (-G) pool composition in humans, ii) evaluating how sex and UGT polymorphisms influence this composition, and iii) analyzing the effects of lipid-lowering drug fenofibrate on the circulating BA-G profile in 300 volunteers and 5 cholestatic patients. Eleven BA-Gs were determined in pre- and post-fenofibrate samples. Men exhibited higher BA-G concentrations, and various genotype/BA-G associations were discovered in relevant UGT genes. The chenodeoxycholic acid-3G concentration was associated with the UGT2B7 802C>T polymorphism. Glucuronidation assays confirmed the predominant role of UGT2B7 and UGT1A4 in CDCA-3G formation. Fenofibrate exposure increased the serum levels of 5 BA-G species, including CDCA-3G, and up-regulated expression of UGT1A4, but not UGT2B7, in hepatic cells. This study demonstrates that fenofibrate stimulates BA glucuronidation in humans, and thus reduces bile acid toxicity in the liver. PMID:23756370

  9. HPLC separation of human serum albumin isoforms based on their isoelectric points.

    Science.gov (United States)

    Turell, Lucía; Botti, Horacio; Bonilla, Lucía; Torres, María José; Schopfer, Francisco; Freeman, Bruce A; Armas, Larissa; Ricciardi, Alejandro; Alvarez, Beatriz; Radi, Rafael

    2014-01-01

    Human serum albumin (HSA) is the most abundant protein in plasma. Cys34, the only free Cys residue, is the predominant plasma thiol and a relevant sacrificial antioxidant. Both in vivo circulating HSA and pharmaceutical preparations are heterogeneous with respect to the oxidation state of Cys34. In this work, we developed an external pH gradient chromatofocusing procedure that allows the analysis of the oxidation status of HSA in human plasma and biopharmaceutical products based on the different apparent isoelectric points and chemical properties of the redox isoforms. Specifically, reduced-mercury blocked HSA (HSA-SHg(+)), HSA with Cys34 oxidized to sulfenic acid (HSA-SOH) and HSA oxidized to sulfinate anion (HSA-SO2(-)) can be separated with resolutions of 1.4 and 3.1 (first and last pair) and hence quantified and purified. In addition, an N-terminally degraded isoform (HSA3-585) in different redox states can be resolved as well. Confirmation of the identity of the chromatofocusing isolated isoforms was achieved by high resolution whole protein MS. It is proposed that the chromatofocusing procedure can be used to produce more exact and complete descriptions of the redox status of HSA in vivo and in vitro. Finally, the scalability capabilities of the chromatofocusing procedure allow for the preparation of highly pure standards of several redox isoforms of HSA. Copyright © 2013 Elsevier B.V. All rights reserved.

  10. Serum amyloid A is found on ApoB-containing lipoproteins in obese humans with diabetes.

    Science.gov (United States)

    Jahangiri, Anisa; Wilson, Patricia G; Hou, Tianfei; Brown, Aparna; King, Victoria L; Tannock, Lisa R

    2013-05-01

    In murine models of obesity/diabetes, there is an increase in plasma serum amyloid A (SAA) levels along with redistribution of SAA from high-density lipoprotein (HDL) to apolipoprotein B (apoB)-containing lipoprotein particles, namely, low-density lipoprotein and very low-density lipoprotein. The goal of this study was to determine if obesity is associated with similar SAA lipoprotein redistribution in humans. Three groups of obese individuals were recruited from a weight loss clinic: healthy obese (n = 14), metabolic syndrome (MetS) obese (n = 8), and obese with type 2 diabetes (n = 6). Plasma was separated into lipoprotein fractions by fast protein liquid chromatography, and SAA was measured in lipid fractions using enzyme-linked immunosorbent assay and Western blotting. Only the obese diabetic group had SAA detectable in apoB-containing lipoproteins, and SAA reverted back to HDL with active weight loss. In human subjects, SAA is found in apoB-containing lipoprotein particles only in obese subjects with type 2 diabetes, but not in healthy obese or obese subjects with MetS. Copyright © 2012 The Obesity Society.

  11. Human Serum Albumin Conjugates of 7-Ethyl-10-hydroxycamptothecin (SN38) for Cancer Treatment

    Science.gov (United States)

    Sepehri, Nima; Rouhani, Hasti; Gharghabi, Mehdi; Tavassolian, Faranak; Amini, Mohsen; Ostad, Seyed Nasser

    2014-01-01

    SN38 (7-ethyl-10-hydroxy-comptothecin) is a potent metabolite of irinotecan, which has been approved for treatment of metastatic colorectal cancer. Considering the notable potency of SN38, it has been introduced as an anticancer candidate. In this study, human serum albumin (HSA) conjugates of SN38 were formulated to overcome the solubility problem beside improving the active form stability and tumor tissue targeting. In this target, two different molar ratios of conjugates (SN38 : HSA 15 : 1 and 60 : 1) were prepared by derivatization of 20-hydroxyl group of SN38 with glycine, followed by addition of succinyl group to glycine through which HSA was covalently attached. The conjugates with particle size of about 100 nm revealed enhanced water solubility and were relatively stable in neutral and acidic solutions. For SN38-HSA-15 and SN38-HSA-60 IC50 values were compared with irinotecan in HT-29 human colon cancer cells. Furthermore, biodistribution studies of SN38-HSA conjugate resulted in proper blood concentration level within 4 h. Moreover, blood cytotoxicity assay revealed no toxicity effect on liver and spleen. Collectively, our present investigation offers a water-soluble form of SN38 attached to HSA and suggests using favorable properties as a promising anticancer agent for further preclinical and clinical investigations. PMID:24895635

  12. Human Serum Albumin Conjugates of 7-Ethyl-10-hydroxycamptothecin (SN38 for Cancer Treatment

    Directory of Open Access Journals (Sweden)

    Nima Sepehri

    2014-01-01

    Full Text Available SN38 (7-ethyl-10-hydroxy-comptothecin is a potent metabolite of irinotecan, which has been approved for treatment of metastatic colorectal cancer. Considering the notable potency of SN38, it has been introduced as an anticancer candidate. In this study, human serum albumin (HSA conjugates of SN38 were formulated to overcome the solubility problem beside improving the active form stability and tumor tissue targeting. In this target, two different molar ratios of conjugates (SN38 : HSA 15 : 1 and 60 : 1 were prepared by derivatization of 20-hydroxyl group of SN38 with glycine, followed by addition of succinyl group to glycine through which HSA was covalently attached. The conjugates with particle size of about 100 nm revealed enhanced water solubility and were relatively stable in neutral and acidic solutions. For SN38-HSA-15 and SN38-HSA-60 IC50 values were compared with irinotecan in HT-29 human colon cancer cells. Furthermore, biodistribution studies of SN38-HSA conjugate resulted in proper blood concentration level within 4 h. Moreover, blood cytotoxicity assay revealed no toxicity effect on liver and spleen. Collectively, our present investigation offers a water-soluble form of SN38 attached to HSA and suggests using favorable properties as a promising anticancer agent for further preclinical and clinical investigations.

  13. Determination of human albumin in serum and urine samples by constant-energy synchronous fluorescence method.

    Science.gov (United States)

    Madrakian, Tayyebeh; Bagheri, Habibollah; Afkhami, Abbas

    2015-08-01

    A sensitive spectrofluorimetric method using constant-energy synchronous fluorescence technique is proposed for the determination of human albumin without separation. In this method, no reagent was used for enhancement of the fluorescence signal of albumin in the solution. Effects of some parameters, such as energy difference between excitation and emission monochromators (ΔE), emission and excitation slit widths and scan rate of wavelength were studied and the optimum conditions were established. For this purpose factorial design and response surface method were employed for optimization of the effective parameters on the fluorescence signal. The results showed that the scan rate of the wavelength has no significant effect on the analytical signal. The calibration curve was linear in the range 0.1-220.0 µg mL(-1) of albumin with a detection limit of 7.0 × 10(-3)  µg mL(-1). The relative standard deviations (RSD) for six replicate measurements of albumin were calculated as 2.2%, 1.7% and 1.3% for 0.5, 10.0 and 100.0 µg mL(-1) albumin, respectively. Furthermore the proposed method has been employed for the determination of albumin in human serum and urine samples.

  14. Quantitation of 4,4′-methylene diphenyl diisocyanate human serum albumin adducts

    Directory of Open Access Journals (Sweden)

    Leah G. Luna

    2014-01-01

    Full Text Available 4,4′-Methylene diphenyl diisocyanate (herein 4,4′-MDI is used in the production of polyurethane foams, elastomers, coatings, adhesives and the like for a wide range of commercial products. Occupational exposure to MDI levels above current airborne exposure limits can elicit immune mediated hypersensitivity reactions such as occupational asthma in sensitive individuals. To accurately determine exposure, there has been increasing interest in developing analytical methods to measure internal biomarkers of exposure to MDI. Previous investigators have reported methodologies for measuring MDI diamine metabolites and MDI-Lysine (4,4′-MDI-Lys adducts. The purpose of this study was to develop and validate an ultra performance liquid chromatography isotope dilution tandem mass spectrometry (UPLC-ID/MS/MS quantitation method via a signature peptide approach to enable biomonitoring of 4,4′-MDI adducted to human serum albumin (HSA in plasma. A murine, anti-4,4′-MDI monoclonal IgM antibody was bound to magnetic beads and utilized for enrichment of the MDI adducted HSA. Following enrichment, trypsin digestion was performed to generate the expected 414 site (primary site of adduction 4,4′-MDI-adducted HSA signature peptide that was quantified by UPLC-ID/MS/MS. An Agilent 6530 UPLC/quadrupole time of flight MS (QTOF system was utilized for intact adducted protein analysis and an Agilent 6490 UPLC/MS/MS system operated in multiple reaction monitoring (MRM mode was utilized for quantification of the adducted signature peptide biomarker both for in chemico and worker serum samples. Worker serum samples were initially screened utilizing the previously developed 4,4′-MDI-Lys amino acid method and results showed that 12 samples were identified as quantifiable for 4,4′-MDI-Lys adducts. The signature peptide adduct approach was applied to the 12 worker samples identified as quantifiable for 4,4′-MDI-Lys adducts. Results indicated no positive results

  15. Early serum human chorionic gonadotropin (hCG) trends after medication abortion.

    Science.gov (United States)

    Pocius, Katherine D; Maurer, Rie; Fortin, Jennifer; Goldberg, Alisa B; Bartz, Deborah

    2015-06-01

    Despite increased reliance on human chorionic gonadotropin (hCG) for early pregnancy monitoring, there is limited information about hCG trends soon after medication abortion. The purpose of this study was to determine if there is a predictable decline in serum hCG values shortly after medication abortion. This is a retrospective study of women with early intrauterine pregnancies who underwent medication abortion with mifepristone and misoprostol and had a serum hCG level on Day 1 (day of mifepristone) and a repeat value on Day 2 to 6. The percent hCG decline was calculated from baseline to repeat measure, with repeat values from the same patient accounted for through repeated measure analysis of variance. Eighty-eight women with a mean gestational age of 5.5 weeks and median baseline hCG of 5220 IU met study criteria over a 3-year period. The mean decline (±SD) in hCG from the Day 1 baseline value was 56.9%±29.5% on Day 3, 73.5%±38.6% on Day 4, 86.1%±8.8% on Day 5, and 92.9%±3.4% on Day 6. Eighty-two women (93% of the cohort) had a complete abortion without further intervention. The least square means hCG decline among these women was 57.6% [95% confidence interval (CI): 50.3-64.9%] on Day 3, 78.9% (95% CI: 75.0-82.8%) on Day 4 and 86.2% (95% CI: 81.3-91.1%) on Day 5. There is a rapid decline in serum hCG within the first few days after early medication abortion. Further research is needed to delineate how soon after medication abortion this decline may be specific enough to confirm abortion completion. This study provides the largest cohort of patients followed with serial hCG values in the first few days after medication abortion. Our findings demonstrate the trend in hCG decline in this population, which may be predictable by Day 5. Copyright © 2015 Elsevier Inc. All rights reserved.

  16. Concentrations of organochlorine pesticides in pooled human serum by age and gender.

    Science.gov (United States)

    Thomas, Aleysha; Toms, Leisa-Maree Leontjew; Harden, Fiona A; Hobson, Peter; White, Nicole M; Mengersen, Kerrie L; Mueller, Jochen F

    2017-04-01

    Organochlorine pesticides (OCPs) have been used for many decades in Australia with cessation of selected persistent and bioaccumulative OCPs ranging from the 1970s to as recently as 2007. The specific aims of this study were to use samples representative of an Australian population to assess age and gender differences in the concentration of OCPs in human blood sera and to investigate temporal trends in these chemicals. Serum was collected from de-identified, surplus pathology samples over five time periods (2002/03, 2006/07, 2008/09, 2010/11 and 2012/13), with 183 serum pools made from 12,175 individual samples; 26 pools in 2002/03, 85 pools in 2006/07 and 24 pools each in 2008/09, 2010/11 and 2012/13. Samples were analyzed for hexachlorobenzene (HCB), β-hexachlorocyclohexane (β-HCH), γ -hexachlorocyclohexane (lindane) (γ-HCH), oxy-chlordane, trans-nonachlor, p,p'-DDE, o,p'-DDT, p,p'-DDT and Mirex. Stratification criteria included gender and age (0-4; 5-15; 16-30; 31-45; 46-60; and >60 years) with age additionally stratified by adults >16 years and children 0-4 and 5-15 years. All pools from all collection periods had detectable concentrations of OCPs with a detection frequency of >60% for HCB, β-HCH, trans-nonachlor, p,p'-DDT and p,p'-DDE. The overall OCP concentrations increased with age with the highest concentrations in the >60 years groups. Females did not have higher mean OCP concentrations than males except for HCB concentrations (p=0.0006). Temporal trends showed overall decreasing serum concentrations by collection period with the exception of an increase in OCP concentrations between 2006/07 and 2008/09. Excluding this data point, HCB decreased from year to year by 7-76%; β-HCH concentrations decreased by 14 - 38%; trans-nonachlor concentrations decreased by 10 - 65%; p,p'-DDE concentrations decreased by 6 - 52%; and p,p'-DDT concentrations decreased by 7 - 30%. The results indicate that OCP concentrations have decreased over time as is to be

  17. Mechanisms by Which Salt Concentration Moderates the Dynamics of Human Serum Transferrin.

    Science.gov (United States)

    Abdizadeh, Haleh; Atilgan, Ali Rana; Atilgan, Canan

    2017-05-11

    The dynamical and thermodynamic behavior of human transferrin (hTf) protein in saline aqueous solution of various concentrations is studied. hTf is an essential transport protein circulating iron in the blood and delivering it to tissues. It displays highly pH dependent cooperativity between the two lobes, each carrying an iron, and forms a tight complex with the receptor during endocytosis, eventually recycled to the serum after iron release. Molecular dynamics simulations are used to investigate the effects of the amount of salt on protein conformation and dynamics to analyze the structure-function relationship in free hTf at serum pH. To monitor the ionic strength dependence, four different ionic concentrations, 0, 50, 130, and 210 mM NaCl for two protonation states of the iron coordination site is considered. Two mechanisms by which salt affects hTf are disclosed. In the totally closed state where iron coordinating tyrosines are deprotonated, the addition of even 50 mM of salt alters the electrostatic potential distribution around the protein, opening energetic pathways for tyrosine protonation from nearby charged residues as a required first step for iron release. Once domain opening is observed, conformational plasticity renders the iron binding site more accessible by the solvent. At this second stage of iron release, R124 in the N-lobe is identified as kinetically significant anion binding site that accommodates chloride ions and allosterically communicates with the iron binding residues. Opening motions are maximized at 150 mM IS in the N-lobe, and at 210 mM in the C-lobe. The extra mobility in the latter is thought to preclude binding of hTf to its receptor. Thus, the physiological IS is optimal for exposing iron for release from hTf. However, the calculated binding affinities of iron show that even in the most open conformations, iron dissociation needs to be accompanied by chelators.

  18. Biochemical and structural characterization of recombinant human serum transferrin from rice (Oryza sativa L.).

    Science.gov (United States)

    Steere, Ashley N; Bobst, Cedric E; Zhang, Deshui; Pettit, Steve C; Kaltashov, Igor A; Huang, Ning; Mason, Anne B

    2012-11-01

    The Fe(3+) binding protein human serum transferrin (hTF) is well known for its role in cellular iron delivery via the transferrin receptor (TFR). A new application is the use of hTF as a therapy and targeted drug delivery system for a number of diseases. Recently, production of hTF in plants has been reported; such systems provide a relatively inexpensive, animal-free (eliminating potential contamination by animal pathogens) method to produce large amounts of recombinant proteins for such biopharmaceutical applications. Specifically, the production of Optiferrin (hTF produced in rice, Oryza sativa, from InVitria) has been shown to yield large amounts of functional protein for use in culture medium for cellular iron delivery to promote growth. In the present work we describe further purification (by gel filtration) and characterization of hTF produced in rice (purified Optiferrin) to determine its suitability in biopharmaceutical applications. The spectral, mass spectrometric, urea gel and kinetic analysis shows that purified Optiferrin is similar to recombinant nonglycosylated N-His tagged hTF expressed by baby hamster kidney cells and/or serum derived glycosylated hTF. Additionally, in a competitive immunoassay, iron-loaded Optiferrin is equivalent to iron-loaded N-His hTF in its ability to bind to the soluble portion of the TFR immobilized in an assay plate. As an essential requirement for any functional hTF, both lobes of purified Optiferrin bind Fe(3+) tightly yet reversibly. Although previously shown to be capable of delivering Fe(3+) to cells, the kinetics of iron release from iron-loaded Optiferrin™/sTFR and iron-loaded N-His hTF/sTFR complexes differ somewhat. We conclude that the purified Optiferrin might be suitable for consideration in biopharmaceutical applications. Copyright © 2012 Elsevier Inc. All rights reserved.

  19. Investigation on the binding activities of citalopram with human and bovine serum albumins

    Energy Technology Data Exchange (ETDEWEB)

    Lin, Jingjing; Liu, Yan, E-mail: liuyan@fjirsm.ac.cn; Chen, Mingmao; Huang, Huayin; Song, Ling, E-mail: songling@fjirsm.ac.cn

    2014-02-15

    The binding interactions of citalopram (CIT), an efficient antidepressant, with human serum albumin (HSA) and bovine serum albumin (BSA) were investigated by a series of spectroscopic methods including fluorescence, UV–vis absorption, circular dichroism (CD) and {sup 1}H nuclear magnetic resonance ({sup 1}H NMR). The fluorescence quenching and UV–vis absorption studies reveal that CIT could form complexes with both HSA and BSA. The CIT–BSA complex exhibits higher binding affinity than CIT–HSA complex. The thermodynamic study further suggests that the interactions between CIT and SAs are mainly driven by hydrophobic forces and hydrogen bonds. The {sup 1}H NMR analysis indicates that the participation of different functional groups of CIT is unequal in the complexation of CIT–HSA and CIT–BSA. Site marker competitive experiments show that the interactions between CIT and SAs primarily locate at sub-domain II A (site I). The effects of CIT on the conformation of SAs are further analyzed via synchronous fluorescence, three-dimensional fluorescence and CD spectra techniques. The results prove that the presence of CIT decreases the α-helical content of both SAs and induces the slight unfolding of the polypeptides of protein. Additionally, the conformational change of BSA induced by CIT is larger than that of HSA. -- Highlights: • The difference of binding activity between CIT–BSA and CIT–HSA is first reported. • Use spectroscopic, thermodynamic, and NMR methods. • CIT exhibits higher binding affinity to BSA than to HSA. • The binding forces between CIT and SA have been investigated. • The complexation of CIT–SA induces the conformational change of SA.

  20. Interplay of Multiple Interaction Forces: Binding of Norfloxacin to Human Serum Albumin.

    Science.gov (United States)

    Paul, Bijan K; Ghosh, Narayani; Mukherjee, Saptarshi

    2015-10-15

    Herein, the binding interaction of a potential chemotherapeutic antibacterial drug norfloxacin (NOF) with a serum transport protein, human serum albumin (HSA), is investigated. The prototropic transformation of the drug (NOF) is found to be remarkably modified following interaction with the protein as manifested through significant modulations of the photophysics of the drug. The predominant zwitterionic form of NOF in aqueous buffer phase undergoes transformation to the cationic form within the protein-encapsulated state. This implies the possible role of electrostatic interaction force in NOF-HSA binding. This postulate is further substantiated from the effect of ionic strength on the interaction process. To this end, the detailed study of the thermodynamics of the drug-protein interaction process from isothermal titration calorimetric (ITC) experiments is found to unfold the signature of electrostatic as well as hydrophobic interaction forces underlying the binding process. Thus, interplay of more than one interaction forces is argued to be responsible for the overall drug-protein binding. The ITC results reveal an important finding in terms of enthalpy-entropy compensation (EEC) characterizing the NOF-HSA binding. The effect of drug-binding on the native protein conformation has also been evaluated from circular dichroism (CD) spectroscopy which unveils partial rupture of the protein secondary structure. In conjunction to this, the functionality of the native protein (in terms of esterase-like activity) is found to be lowered as a result of binding with NOF. The AutoDock-based docking simulation unravels the probable binding location of NOF within the hydrophilic subdomain IA of HSA. The present program also focuses on exploring the dynamical aspects of the drug-protein interaction scenario. The rotational-relaxation dynamics of the protein-bound drug reveals the not-so-common "dip-and-rise" pattern.

  1. Influence of fatty acids on the binding of warfarin and phenprocoumon to human serum albumin with relation to anticoagulant therapy

    DEFF Research Database (Denmark)

    Vorum, H; Honoré, B

    1996-01-01

    Warfarin and phenprocoumon binding to human serum albumin was studied by equilibrium dialysis. The first stoichiometric binding constant was 1.89 x 10(5) M-1 for warfarin and 2.40 x 10(5) M-1 for phenprocoumon. The affinity of warfarin was markedly increased on addition of up to 3 mol mol-1 albumin...

  2. Targeting naproxen coupled to human serum albumin to nonparenchymal cells reduces endotoxin-induced mortality in rats with biliary cirrhosis

    NARCIS (Netherlands)

    Albrecht, C.; Meijer, D.K F; Lebbe, C; Sägesser, H; Melgert, B.N; Poelstra, Klaas; Reichen, J

    1997-01-01

    Endotoxin is thought to play a major role in cirrhotic liver disease, Cyclo-oxygenase inhibitors were shown to be partially protective against endotoxin but cannot be used in cirrhotic patients because of renal side-effects, We argued that administration of naproxen (NAP) linked to human serum album

  3. Targeting naproxen coupled to human serum albumin to nonparenchymal cells reduces endotoxin-induced mortality in rats with biliary cirrhosis

    NARCIS (Netherlands)

    Albrecht, C.; Meijer, D.K F; Lebbe, C; Sägesser, H; Melgert, B.N; Poelstra, Klaas; Reichen, J

    1997-01-01

    Endotoxin is thought to play a major role in cirrhotic liver disease, Cyclo-oxygenase inhibitors were shown to be partially protective against endotoxin but cannot be used in cirrhotic patients because of renal side-effects, We argued that administration of naproxen (NAP) linked to human serum

  4. Italian network for human biomonitoring of metals: preliminary results from two regions

    Directory of Open Access Journals (Sweden)

    Beatrice Bocca

    2010-01-01

    Full Text Available The Italian program for human biomonitoring (HBM of chemical elements, PROgram for Biomonitoring of the Exposure (PROBE, started in 2008 with the aim to provide the knowledge about risk assessment of the Italian population following the environmental exposure to metals. The project is implemented through a HBM campaign for the production of data on 19 metals in the blood and serum of subjects living in different Italian Regions. The metals studied are: antimony, beryllium, cadmium, chromium, cobalt, iridium, lead, manganese, mercury, molybdenum, nickel, palladium, platinum, rhodium, thallium, tin, tungsten, uranium and vanadium. The first phase of the project has included the development and validation of laboratory protocols for the collection of fluids and quantification of metals. The second phase provides the HBM data expressed as the reference values (RVs for the Italian population, i.e., as the level of metals in the general population not occupationally exposed. In this paper, the experimental protocols used for the maintenance of high standards of analysis and the RVs for metals in serum of inhabitants of two Italian Regions (Calabria and Umbria are described.

  5. Xeno-free culture condition for human bone marrow and umbilical cord matrix-derived mesenchymal stem/stromal cells using human umbilical cord blood serum

    Science.gov (United States)

    Esmaeli, Azadeh; Moshrefi, Mojgan; Shamsara, Ali; Eftekhar-vaghefi, Seyed Hasan; Nematollahi-mahani, Seyed Noureddin

    2016-01-01

    Background: Fetal bovine serum (FBS) is widely used in cell culture laboratories, risk of zoonotic infections and allergic side effects create obstacles for its use in clinical trials. Therefore, an alternative supplement with proper inherent growth-promoting activities is demanded. Objective: To find FBS substitute, we tested human umbilical cord blood serum (hUCS) for proliferation of human umbilical cord matrix derived mesenchymal stem cells (hUC-MSCs) and human bone marrow-derived mesenchymal cells (hBM-MSCs). Materials and Methods: Umbilical cord blood of healthy neonates, delivered by Caesarian section, was collected and the serum was separated. hUC-MSCs and hBM-MSCs were isolated and characterized by assessment of cell surface antigens by flow cytometry, alkaline phosphatase activity and osteogenic/adipogenic differentiation potential. The cells were then cultured in Iscove's Modified Dulbecco's Medium (IMDM) by conventional methods in three preparations: 1- with hUCS, 2- with FBS, and 3- without serum supplements. Cell proliferation was measured using WST-1 assay, and cell viability was assessed by trypan blue staining. Results: The cells cultured in hUCS and FBS exhibited similar morphology and mesenchymal stem cells properties. WST-1 proliferation assay data showed no significant difference between the proliferation rate of either cells following hUCS and FBS supplementation. Trypan blue exclusion dye test also revealed no significant difference for viability between hUCS and FBS groups. A significant difference was detected between the proliferation rate of stem cells cultured in serum-supplemented medium compared with serum-free medium. Conclusion: Our results indicate that human umbilical cord serum can effectively support proliferation of hBM-MSCS and hUC-MSCs in vitro and can be used as an appropriate substitute for FBS, especially in clinical studies. PMID:27738658

  6. Immunofunctional assay of human growth hormone (hGH) in serum: A possible consensus for quantitative hGH measurement

    OpenAIRE

    Strasburger, Christian J.; Wu, Zida; Pflaum, Claus-Dieter; Dressendörfer, Regina A.

    1996-01-01

    Confirmation of the diagnosis of GH deficiency in adults and children involves provocative testing for human (h) GH. Different commercially available immunoassays yield largely discrepant results in the measurement of GH levels in human serum. These discrepancies result in doubtful relevance of cut-off levels proposed for GH provocative testing. We have developed an immunofunctional assay method that allows quantitation of only those GH forms in circulation that possess both binding sites of ...

  7. Umbilical Cord Blood Platelet Lysate as Serum Substitute in Expansion of Human Mesenchymal Stem Cells.

    Science.gov (United States)

    Shirzad, Negin; Bordbar, Sima; Goodarzi, Alireza; Mohammad, Monire; Khosravani, Pardis; Sayahpour, Froughazam; Baghaban Eslaminejad, Mohamadreza; Ebrahimi, Marzieh

    2017-10-01

    The diverse clinical applications for human mesenchymal stem cells (hMSCs) in cellular therapy and regenerative medicine warrant increased focus on developing adequate culture supplements devoid of animal-derived products. In the present study, we have investigated the feasibility of umbilical cord blood-platelet lysate (UCB-PL) as a standard substitute for fetal bovine serum (FBS) and human peripheral blood-PL (PB-PL). In this experimental study, platelet concentrates (PC) from UCB and human PB donors were frozen, melted, and sterilized to obtain PL. Quality control included platelet cell counts, sterility testing (viral and microbial), total protein concentrations, growth factor levels, and PL stability. The effects of UCB-PL and PB-PL on hMSCs proliferation and differentiation into osteocytes, chondrocytes, and adipocytes were studied and the results compared with FBS. UCB-PL contained high levels of protein content, platelet-derived growth factor- AB (PDGF-AB), and transforming growth factor (TGF) compared to PB-PL. All growth factors were stable for at least nine months post-storage at -70˚C. hMSCs proliferation enhanced following treatment with UCB-PL. With all three supplements, hMSCs could differentiate into all three lineages. PB-PL and UCB-PL both were potent in hMSCs proliferation. However, PB promoted osteoblastic differentiation and UCB-PL induced chondrogenic differentiation. Because of availability, ease of use and feasible standardization of UCB-PL, we have suggested that UCB-PL be used as an alternative to FBS and PB-PL for the cultivation and expansion of hMSCs in cellular therapy.

  8. Genetics meets metabolomics: a genome-wide association study of metabolite profiles in human serum.

    Science.gov (United States)

    Gieger, Christian; Geistlinger, Ludwig; Altmaier, Elisabeth; Hrabé de Angelis, Martin; Kronenberg, Florian; Meitinger, Thomas; Mewes, Hans-Werner; Wichmann, H-Erich; Weinberger, Klaus M; Adamski, Jerzy; Illig, Thomas; Suhre, Karsten

    2008-11-01

    The rapidly evolving field of metabolomics aims at a comprehensive measurement of ideally all endogenous metabolites in a cell or body fluid. It thereby provides a functional readout of the physiological state of the human body. Genetic variants that associate with changes in the homeostasis of key lipids, carbohydrates, or amino acids are not only expected to display much larger effect sizes due to their direct involvement in metabolite conversion modification, but should also provide access to the biochemical context of such variations, in particular when enzyme coding genes are concerned. To test this hypothesis, we conducted what is, to the best of our knowledge, the first GWA study with metabolomics based on the quantitative measurement of 363 metabolites in serum of 284 male participants of the KORA study. We found associations of frequent single nucleotide polymorphisms (SNPs) with considerable differences in the metabolic homeostasis of the human body, explaining up to 12% of the observed variance. Using ratios of certain metabolite concentrations as a proxy for enzymatic activity, up to 28% of the variance can be explained (p-values 10(-16) to 10(-21)). We identified four genetic variants in genes coding for enzymes (FADS1, LIPC, SCAD, MCAD) where the corresponding metabolic phenotype (metabotype) clearly matches the biochemical pathways in which these enzymes are active. Our results suggest that common genetic polymorphisms induce major differentiations in the metabolic make-up of the human population. This may lead to a novel approach to personalized health care based on a combination of genotyping and metabolic characterization. These genetically determined metabotypes may subscribe the risk for a certain medical phenotype, the response to a given drug treatment, or the reaction to a nutritional intervention or environmental challenge.

  9. Comparison of extraction and quantification methods of perfluorinated compounds in human plasma, serum, and whole blood

    Energy Technology Data Exchange (ETDEWEB)

    Reagen, William K. [3M Environmental Laboratory, 3M Center, Building 0260-05-N-17, St. Paul, MN 55144-1000 (United States)], E-mail: wkreagen@mmm.com; Ellefson, Mark E. [3M Environmental Laboratory, 3M Center, Building 0260-05-N-17, St. Paul, MN 55144-1000 (United States); Kannan, Kurunthachalam [Wadsworth Center, New York State Department of Health and Department of Environmental Health Sciences (United States); State University of New York at Albany, NY 12201-0509 (United States); Giesy, John P. [Department of Veterinary Biomedical Sciences and Toxicology Centre, University of Saskatchewan, 44 Campus Drive, Saskatoon, SK (Canada); Department of Biology and Chemistry, Center for Coastal Pollution and Conservation, City University of Hong Kong, Tat Chee Avenue, Kowloon, Hong Kong (China); Zoology Department, National Food Safety and Toxicology Center, Center for Integrative Toxicology, Michigan State University, E. Lansing, MI (United States); School of Environment, Nanjing University, Nanjing (China)

    2008-11-03

    Perfluorinated compounds are ubiquitous in the environment and have been reported to occur in human blood. Accurate risk assessments require accurate measurements of exposures, but identification and quantification of PFCs in biological matrices can be affected by both ion suppression and enhancement in liquid chromatography-tandem mass spectrometry techniques (LC/MS-MS). A study was conducted to quantify potential biases in LC/MS-MS quantification methods. Using isotopically labeled perfluorooctanoic acid ([{sup 13}C{sub 2}]-PFOA), perfluorononanoic acid ([{sup 13}C{sub 2}]-PFNA), and ammonium perfluorooctanesulfonate ([{sup 18}O{sub 2}]-PFOS) spiked tissues, ion-pairing extraction, solid-phase extraction, and protein precipitation sample preparation techniques were compared. Analytical accuracy was assessed using both solvent calibration and matrix-matched calibration for quantification. Data accuracy and precision of 100 {+-} 15% was demonstrated in both human sera and plasma for all three sample preparation techniques when matrix-matched calibration was used in quantification. In contrast, quantification of ion-pairing extraction data using solvent calibration in combination with a surrogate internal standard resulted in significant analytical biases for all target analytes. The accuracy of results, based on solvent calibration was highly variable and dependent on the serum and plasma matrices, the specific target analyte [{sup 13}C{sub 2}]-PFOA, [{sup 13}C{sub 2}]-PFNA, or [{sup 18}O{sub 2}]-PFOS, the target analyte concentration, the LC/MS-MS instrumentation used in data generation, and the specific surrogate internal standard used in quantification. These results suggest that concentrations of PFCs reported for human blood using surrogate internal standards in combination with external solvent calibration can be inaccurate unless biases are accounted for in data quantification.

  10. Genetics meets metabolomics: a genome-wide association study of metabolite profiles in human serum.

    Directory of Open Access Journals (Sweden)

    Christian Gieger

    2008-11-01

    Full Text Available The rapidly evolving field of metabolomics aims at a comprehensive measurement of ideally all endogenous metabolites in a cell or body fluid. It thereby provides a functional readout of the physiological state of the human body. Genetic variants that associate with changes in the homeostasis of key lipids, carbohydrates, or amino acids are not only expected to display much larger effect sizes due to their direct involvement in metabolite conversion modification, but should also provide access to the biochemical context of such variations, in particular when enzyme coding genes are concerned. To test this hypothesis, we conducted what is, to the best of our knowledge, the first GWA study with metabolomics based on the quantitative measurement of 363 metabolites in serum of 284 male participants of the KORA study. We found associations of frequent single nucleotide polymorphisms (SNPs with considerable differences in the metabolic homeostasis of the human body, explaining up to 12% of the observed variance. Using ratios of certain metabolite concentrations as a proxy for enzymatic activity, up to 28% of the variance can be explained (p-values 10(-16 to 10(-21. We identified four genetic variants in genes coding for enzymes (FADS1, LIPC, SCAD, MCAD where the corresponding metabolic phenotype (metabotype clearly matches the biochemical pathways in which these enzymes are active. Our results suggest that common genetic polymorphisms induce major differentiations in the metabolic make-up of the human population. This may lead to a novel approach to personalized health care based on a combination of genotyping and metabolic characterization. These genetically determined metabotypes may subscribe the risk for a certain medical phenotype, the response to a given drug treatment, or the reaction to a nutritional intervention or environmental challenge.

  11. Fetal calf serum heat inactivation and lipopolysaccharide contamination influence the human T lymphoblast proteome and phosphoproteome

    Directory of Open Access Journals (Sweden)

    Rahman Hazir

    2011-11-01

    Full Text Available Abstract Background The effects of fetal calf serum (FCS heat inactivation and bacterial lipopolysaccharide (LPS contamination on cell physiology have been studied, but their effect on the proteome of cultured cells has yet to be described. This study was undertaken to investigate the effects of heat inactivation of FCS and LPS contamination on the human T lymphoblast proteome. Human T lymphoblastic leukaemia (CCRF-CEM cells were grown in FCS, either non-heated, or heat inactivated, having low ( Results A total of four proteins (EIF3M, PRS7, PSB4, and SNAPA were up-regulated when CCRF-CEM cells were grown in media supplemented with heat inactivated FCS (HE as compared to cells grown in media with non-heated FCS (NHE. Six proteins (TCPD, ACTA, NACA, TCTP, ACTB, and ICLN displayed a differential phosphorylation pattern between the NHE and HE groups. Compared to the low concentration LPS group, regular levels of LPS resulted in the up-regulation of three proteins (SYBF, QCR1, and SUCB1. Conclusion The present study provides new information regarding the effect of FCS heat inactivation and change in FCS-LPS concentration on cellular protein expression, and post-translational modification in human T lymphoblasts. Both heat inactivation and LPS contamination of FCS were shown to modulate the expression and phosphorylation of proteins involved in basic cellular functions, such as protein synthesis, cytoskeleton stability, oxidative stress regulation and apoptosis. Hence, the study emphasizes the need to consider both heat inactivation and LPS contamination of FCS as factors that can influence the T lymphoblast proteome.

  12. A soluble form of the high affinity IgE receptor, Fc-epsilon-RI, circulates in human serum.

    Directory of Open Access Journals (Sweden)

    Eleonora Dehlink

    Full Text Available Soluble IgE receptors are potential in vivo modulators of IgE-mediated immune responses and are thus important for our basic understanding of allergic responses. We here characterize a novel soluble version of the IgE-binding alpha-chain of Fc-epsilon-RI (sFcεRI, the high affinity receptor for IgE. sFcεRI immunoprecipitates as a protein of ∼40 kDa and contains an intact IgE-binding site. In human serum, sFcεRI is found as a soluble free IgE receptor as well as a complex with IgE. Using a newly established ELISA, we show that serum sFcεRI levels correlate with serum IgE in patients with elevated IgE. We also show that serum of individuals with normal IgE levels can be found to contain high levels of sFcεRI. After IgE-antigen-mediated crosslinking of surface FcεRI, we detect sFcεRI in the exosome-depleted, soluble fraction of cell culture supernatants. We further show that sFcεRI can block binding of IgE to FcεRI expressed at the cell surface. In summary, we here describe the alpha-chain of FcεRI as a circulating soluble IgE receptor isoform in human serum.

  13. Control of lipopolysaccharide-high-density lipoprotein interactions by an acute-phase reactant in human serum.

    Science.gov (United States)

    Tobias, P S; McAdam, K P; Soldau, K; Ulevitch, R J

    1985-10-01

    We have recently described several phenomena involving the interactions of lipopolysaccharides (LPS) from Salmonella minnesota Re595 (Re595-LPS) with rabbit serum, which are different in and unique to acute-phase serum as compared with normal serum (P.S. Tobias and R.J. Ulevitch, J. Immunol. 131:1913-1916, 1983). To determine whether these phenomena could also be observed in acute-phase human serum (APHS), we used APHS obtained from volunteers injected with etiocholanolone. As observed in acute-phase rabbit serum, we found that (i) in APHS, Re595-LPS forms a protein complex with a density of 1.3 g/cm3 which does not form in normal human serum (NHS), (ii) in APHS, the t1/2 for LPS-high-density lipoprotein (HDL) complexation is at least a factor of 10 slower than the t1/2 for LPS-HDL complexation in NHS, (iii) when Re595-LPS serum mixtures are dialyzed against a low salt buffer, Re595-LPS precipitates in less soluble form from APHS than from NHS, and (iv) the precipitate from Re595-LPS-APHS mixtures includes a protein with a molecular weight of approximately 60,000 which does not precipitate from Re595-LPS-NHS mixtures or from NHS or APHS alone. These indications of an altered status of LPS in NHS and APHS suggest that one or more acute-phase reactants interact with Re595-LPS to modify its rate of binding to HDL.

  14. Use of biotin targeted methotrexate–human serum albumin conjugated nanoparticles to enhance methotrexate antitumor efficacy

    Directory of Open Access Journals (Sweden)

    Taheri A

    2011-09-01

    Full Text Available Azade Taheri1, Rassoul Dinarvand1,2, Faranak Salman Nouri1, Mohammad Reza Khorramizadeh3, Atefeh Taheri Borougeni4, Pooria Mansoori5, Fatemeh Atyabi1,21Department of Pharmaceutics, Faculty of Pharmacy, Tehran University of Medical Sciences, Tehran, Iran; 2Nanotechnology Research Centre, Faculty of Pharmacy, Tehran University of Medical sciences, Tehran, Iran; 3Department of Pathobiology, School of Public Health, Tehran University of Medical Sciences, Tehran, Iran; 4Department of Oral and Maxillofacial Pathology, Faculty of Dentistry, Tehran University of Medical Sciences, Tehran, Iran; 5Faculty of Medicine, Ahvaz Jundishapur University of Medical Sciences, Ahvaz, IranAbstract: Biotin molecules could be used as suitable targeting moieties in targeted drug delivery systems against tumors. To develop a biotin targeted drug delivery system, we employed human serum albumin (HSA as a carrier. Methotrexate (MTX molecules were conjugated to HSA. MTX-HSA nanoparticles (MTX-HSA NPs were prepared from these conjugates by cross-linking the HSA molecules. Biotin molecules were then conjugated on the surface of MTX-HSA NPs. The anticancer efficacy of biotin targeted MTX-HSA NPs was evaluated in mice bearing 4T1 breast carcinoma. A single dose of biotin targeted MTX-HSA NPs showed stronger in vivo antitumor activity than non-targeted MTX-HSA NPs and free MTX. By 7 days after treatment, average tumor volume in the biotin targeted MTX-HSA NPs-treated group decreased to 17.6% of the initial tumor volume when the number of attached biotin molecules on MTX-HSA-NPs was the highest. Average tumor volume in non-targeted MTX-HSA NPs-treated mice grew rapidly and reached 250.7% of the initial tumor volume. Biotin targeted MTX-HSA NPs increased the survival of tumor-bearing mice to 47.5 ± 0.71 days and increased their life span up to 216.7%. Mice treated with biotin targeted MTX-HSA NPs showed slight body weight loss (8% 21 days after treatment, whereas non-targeted MTX

  15. Preparation and characterization of conjugates of (modified) human serum albumin and liposomes : Drug carriers with an intrinsic anti-HIV activity

    NARCIS (Netherlands)

    Kamps, JAAM; Swart, PJ; Morselt, HWM; Pauwels, R; DeBethune, MP; DeClercq, E; Meijer, DKF; Scherphof, GL

    1996-01-01

    Human serum albumin (HSA) derivatized with cis-aconitic anhydride (Aco-HSA) that was earlier shown to inhibit replication of human immunodeficiency virus type 1 (HIV-1), was covalently coupled to conventional liposomes, consisting of phosphatidylcholine, cholesterol and

  16. Preanalytical and analytical variation of surface-enhanced laser desorption-ionization time-of-flight mass spectrometry of human serum

    DEFF Research Database (Denmark)

    Albrethsen, Jakob; Bøgebo, Rikke; Olsen, Jesper

    2006-01-01

    BACKGROUND: Surface-enhanced laser desorption-ionization time-of-flight (SELDI-TOF) mass spectrometry of human serum is a potential diagnostic tool in human diseases. In the present study, the preanalytical and analytical variation of SELDI-TOF mass spectrometry of serum was assessed in healthy...

  17. Modulation of polymorphonuclear leukocytes function by incubation with human serum from oxidant-challenged individuals

    Indian Academy of Sciences (India)

    E Hoffer; T Machamid; A Tabak; Y Baum; A Tamir; Y Lerman

    2003-02-01

    Polymorphonuclear leukocytes (PMN) from healthy donors were tested for stimulated release of superoxide anions after being incubated with serum of welders and of a group of unexposed individuals. These two groups were further subdivided either according to age or to smoking habits. The experiments showed that stimulated superoxide production from PMN was inhibited ( < 0.05) by serum from young smokers as compared to that of young nonsmokers, both from the unexposed group. Incubation of PMN with serum from elderly nonsmoking individuals decreased superoxide production as compared to incubation with serum from young nonsmoking individuals, both from the unexposed group. A decrease in superoxide production by incubation with serum of welders as compared to that of unexposed individuals was significant only when the comparison was carried out between the young, non-smoking subgroups. These findings suggest that age, smoking, and exposure to oxidants induce appearance in serum of factors that affect the PMN function.

  18. Comparison of Architect i2000sr and Cobas e601 Systems for Determining Serum Human Chorionic Gonadotropin-Beta.

    Science.gov (United States)

    Guan, Xiaoyong; Sun, Yifan; Zhang, Hongyu; Liang, Ka; Long, Kang; Li, Jin; Tang, Shifu; Liu, Chunming

    2016-09-01

    Human chorionic gonadotropin-beta (β-hCG) is an important index used to monitor embryonic development following embryo transfer. Architect i2000sr and Cobas e601 are widely used automated immunoassay systems used to measure serum β-hCG concentrations; however, the correlations between serum β-hCG levels measured with these two immunoassays and the accuracy of the immunoassays have not been fully evaluated. Serum β-hCG levels were measured in 133 serum samples using the Architect i2000sr and Cobas e601 automated immunoassay systems. Passing-Bablok regression analysis was used to compare the correlation in serum β-hCG levels obtained using the two immunoassays. A Bland-Altman plot analysis was used to identify mean ratios and 95% CIs of the mean ratios of the β-hCG results between the two immunoassays. In this graphical method the mean ratios between the two techniques were plotted against the averages of the two techniques. The total coefficients of variations (CVs) of serum β-hCG ranged from 3.12 - 4.66% for Cobas e601 and 3.18 - 4.99% for Architect i2000sr. The measured value of serum β-hCG detected by the two immunoassays was statistically significant (p coefficient r was 0.9628. At a high concentration of serum β-hCG (> 10000 IU/L, n = 81), the correlation coefficient r was 0.8076. The Bland-Altman plot analysis showed that the measured value of serum β-hCG detected by Architect i2000sr was about 1.25 times higher than that of Cobas e601. The mean ratio was 1.12 at a low concentration of serum β-hCG, and it was 1.33 at a high concentration. Architect i2000sr and Cobas e601 have good concordance for determining serum β-hCG. However, the β-hCG values measured with Architect i2000sr were 25% higher than those obtained using Cobas e601.

  19. Direct determination of lead in human urine and serum samples by electrothermal atomic absorption spectrometry and permanent modifiers

    OpenAIRE

    Andrada,Daniel; Pinto,Frederico G.; Magalhães, Cristina Gonçalves; Nunes,Berta R.; Franco,Milton B.; Silva,José Bento Borba da

    2006-01-01

    The object of the present study was the development of alternative methods for the direct determination of lead in undigested samples of human urine and serum by electrothermal atomic absorption spectrometry (ET AAS). Thus, some substances have been investigated to act as chemical modifiers. Volumes of 20 µL of diluted samples, 1 + 1, v/v for urine and 1 + 4, v/v for serum, with HNO3 1% v/v and 0.02% v/v of cetil trimethyl ammonium chloride (CTAC) were prepared directly in the autosampler cup...

  20. Applicability of (99m) Tc-Labeled Human Serum Albumin Scintigraphy in Dogs With Protein-Losing Enteropathy.

    Science.gov (United States)

    Engelmann, N; Ondreka, N; von Pückler, K; Mohrs, S; Sicken, J; Neiger, R

    2017-03-01

    Diagnosis of protein loss into the gastrointestinal tract using noninvasive techniques is challenging. In people, scintigraphy not only is a sensitive tool to confirm protein-losing enteropathy (PLE), but it also allows for localization of protein loss. To investigate the feasibility of (99m) Tc-labeled human serum albumin (HSA) scintigraphy in dogs with PLE in comparison with control dogs. A total of 8 clinically healthy control research dogs and 7 client-owned dogs with gastrointestinal clinical signs and hypoalbuminemia (serum albumin concentration Internal Medicine published by Wiley Periodicals, Inc. on behalf of the American College of Veterinary Internal Medicine.

  1. Purification and preliminary X-ray crystallographic studies of β-microseminoprotein from human seminal plasma

    Science.gov (United States)

    Kumar, Vijay; Roske, Yvette; Singh, Nagendra; Heinemann, Udo; Singh, Tej P.; Yadav, Savita

    2009-01-01

    β-Microseminoprotein (β-MSP) is a small cysteine-rich protein with a molecular mass of 10 kDa. It was first isolated from human seminal plasma and has subsequently been identified from several species. Comparison of the amino-acid sequences of β-MSP proteins suggests that the protein is a rapidly evolving protein. The function of β-MSP is poorly understood. Furthermore, no crystal structure has been reported of any β-MSP; therefore, determination of the crystal structure of β-MSP is the foremost task in order to understand the function of this protein completely. Here, the purification, crystallization and preliminary X-ray diffraction analysis of β-MSP from human seminal plasma are described. The protein was purified using anion-exchange and size-exclusion chromatography and the purified protein was crystallized using 0.1 M ammonium sulfate, 0.1 M HEPES buffer pH 7.0 and 20%(w/v) PEG 3350. The crystals belonged to the tetragonal space group P4322 and contained three β-MSP molecules in the asymmetric unit. X-ray intensity data were collected to 2.4 Å resolution. PMID:19407392

  2. A standardized method for quantitating the complement-mediated immune complex solubilizing capacity of human serum

    DEFF Research Database (Denmark)

    Baatrup, G; Peterson, I; Svehag, S E;

    1983-01-01

    A standardized radioassay for measuring the complement-mediated immune complex solubilizing capacity (CMSC) and the initial kinetics of the solubilization (IKS) reaction is described. The total complement (C)-mediated solubilizing capacity was determined after incubation of diluted serum and 125I......-BSA-anti-BSA. Percentage C-mediated solubilization (CMS) was measured after centrifugation by determining the distribution of radioactivity. The dependency of CMSC upon factors such as serum dilution and buffer system used, amount of IC added to serum, serum storage conditions and centrifugation conditions...

  3. Receptor research on xenohormone effects of human serum extracts containing the actual mixture of perfluorinated alkyl acids: a short review

    DEFF Research Database (Denmark)

    Bjerregaard-Olesen, Christian; Bonefeld-Jørgensen, Eva Cecilie

    2015-01-01

    risk of breast cancer and other adverse health effects such as lower birth weight and longer time to pregnancy which might be related to disruptions of various hormonal systems. For instance, direct cell exposure studies in vitro suggest that some PFAAs can transactivate the estrogen receptor (ER......), antagonize the androgen receptor (AR) and has the potential to interfere with Thyroid Hormone and Aryl-hydrocarbon Receptor functions. Moreover, the PFAAs also showed cellular oxidative stress potential. Humans are exposed to an array of PFAAs, and the quantity and combination of these PFAAs in human serum...... of the actual serum PFAA mixture on both steroid hormone actions as well as other hormonal systems e.g. thyroid hormone function. In the current review we will discuss how our recently developed PFAA extraction method might be used in future research to assess the endocrine impact of PFAAs on human health....

  4. Oxidation Enhances Human Serum Albumin Thermal Stability and Changes the Routes of Amyloid Fibril Formation

    Science.gov (United States)

    Sancataldo, Giuseppe; Vetri, Valeria; Foderà, Vito; Di Cara, Gianluca; Militello, Valeria; Leone, Maurizio

    2014-01-01

    Oxidative damages are linked to several aging-related diseases and are among the chemical pathways determining protein degradation. Specifically, interplay of oxidative stress and protein aggregation is recognized to have a link to the loss of cellular function in pathologies like Alzheimer's and Parkinson's diseases. Interaction between protein and reactive oxygen species may indeed induce small changes in protein structure and lead to the inhibition/modification of protein aggregation process, potentially determining the formation of species with different inherent toxicity. Understanding the temperate relationship between these events can be of utmost importance in unraveling the molecular basis of neurodegeneration. In this work, we investigated the effect of hydrogen peroxide oxidation on Human Serum Albumin (HSA) structure, thermal stability and aggregation properties. In the selected conditions, HSA forms fibrillar aggregates, while the oxidized protein undergoes aggregation via new routes involving, in different extents, specific domains of the molecule. Minute variations due to oxidation of single residues affect HSA tertiary structure leading to protein compaction, increased thermal stability, and reduced association propensity. PMID:24416244

  5. The effect of Berberine on the secondary structure of human serum albumin

    Science.gov (United States)

    Li, Ying; He, WenYing; Tian, Jianniao; Tang, Jianghong; Hu, Zhide; Chen, Xingguo

    2005-05-01

    The presence of several high affinity binding sites on human serum albumin (HSA) makes it a possible target for many drugs. This study is designed to examine the effect of Berberine (an ancient Chinese drug used for antimicrobial, antiplasmodial, antidiarrheal and cardiovascular) on the solution structure of HSA using fluorescence, Fourier transform infrared (FT-IR), circular dichroism (CD) spectroscopic methods. The fluorescence spectroscopic results show that the fluorescence intensity of HSA was significantly decreased in the presence of Berberine. The Scatchard's plots indicated that the binding of Berberine to HSA at 296, 303, 318 K is characterized by one binding site with the binding constant is 4.071(±0.128)×10 4, 3.741(±0.089)×10 4, 3.454(±0.110)×10 4 M -1, respectively. The protein conformation is altered (FT-IR and CD data) with reductions of α-helices from 54 to 47% for free HSA to 45-32% and with increases of turn structure5% for free HSA to 18% in the presence of Berberine. The binding process was exothermic, enthalpy driven and spontaneous, as indicated by the thermodynamic analyses, Berberine bound to HSA was mainly based on hydrophobic interaction and electrostatic interaction cannot be excluded from the binding. Furthermore, the displace experiments indicate that Berberine can bind to the subdomain IIA, that is, high affinity site (site II).

  6. Multiple binding modes of ibuprofen in human serum albumin identified by absolute binding free energy calculations

    KAUST Repository

    Evoli, Stefania

    2016-11-10

    Human serum albumin possesses multiple binding sites and transports a wide range of ligands that include the anti-inflammatory drug ibuprofen. A complete map of the binding sites of ibuprofen in albumin is difficult to obtain in traditional experiments, because of the structural adaptability of this protein in accommodating small ligands. In this work, we provide a set of predictions covering the geometry, affinity of binding and protonation state for the pharmaceutically most active form (S-isomer) of ibuprofen to albumin, by using absolute binding free energy calculations in combination with classical molecular dynamics (MD) simulations and molecular docking. The most favorable binding modes correctly reproduce several experimentally identified binding locations, which include the two Sudlow\\'s drug sites (DS2 and DS1) and the fatty acid binding sites 6 and 2 (FA6 and FA2). Previously unknown details of the binding conformations were revealed for some of them, and formerly undetected binding modes were found in other protein sites. The calculated binding affinities exhibit trends which seem to agree with the available experimental data, and drastically degrade when the ligand is modeled in a protonated (neutral) state, indicating that ibuprofen associates with albumin preferentially in its charged form. These findings provide a detailed description of the binding of ibuprofen, help to explain a wide range of results reported in the literature in the last decades, and demonstrate the possibility of using simulation methods to predict ligand binding to albumin.

  7. Pharmaceutical aspects of the recombinant human serum albumin dimer: structural characteristics, biological properties, and medical applications.

    Science.gov (United States)

    Taguchi, Kazuaki; Chuang, Victor Tuan Giam; Maruyama, Toru; Otagiri, Masaki

    2012-09-01

    Human serum albumin is the most abundant protein in the blood. It is clinically used in the treatment of severe hypoalbuminemia and as a plasma expander. The use of albumins as a carrier for drugs is currently being developed, and some are now in the preclinical and clinical trial stages. The main technologies for utilizing an albumin as a drug carrier are protein fusion, polymerization and surface modification, and so on. Among these technologies, albumin dimerization has wide clinical applications as a plasma expander as well as a drug carrier. Despite the fact that many reports have appeared on drugs using an albumin dimer as a carrier, our knowledge of the characteristics of the albumin dimer itself is incomplete. In this review, we summarize the structural characteristics of recombinant albumin dimers produced by two methods, namely, chemical linkage with 1,6-bis(maleimido)hexane and genetically linked with an amino acid linker, and the physicochemical characteristics and biological properties of these preparations. Finally, the potential for pharmaceutical applications of albumin dimers in clinical situations is discussed.

  8. Probing the Effect of Ag2S Quantum Dots on Human Serum Albumin Using Spectral Techniques

    Directory of Open Access Journals (Sweden)

    Yiying Fu

    2017-01-01

    Full Text Available The understanding of the interaction between protein and quantum dots (QDs has significant implications for biological applications of QDs. Herein, we studied the effect of Ag2S QDs on human serum albumin (HSA using UV-Vis absorption spectra and fluorescence spectroscopy and found that the fluorescence intensity of HSA was gradually decreased with increasing Ag2S QDs concentrations. By using the Stern-Volmer equation for the fluorescence quenching constant (KSV of the response of Ag2S QDs to HSA as well as thermodynamic equations, the values of thermodynamic enthalpy change (ΔHθ, entropy change (ΔSθ, and free energy change (ΔGθ were calculated to be −10.79 KJ·mol−1, 37.80 J·mol−1·K−1, and −22.27 KJ·mol−1, respectively. The results indicate that Ag2S QDs exert an obvious static fluorescence quenching effect on HSA and electrostatic interaction plays a key role in the binding process. Furthermore, Raman spectral analysis reveals that Ag2S QDs alter the external environment of tyrosine and tryptophan or the C-H bending of HSA but not the α-helical content.

  9. Glycation of human serum albumin in diabetes: impacts on the structure and function.

    Science.gov (United States)

    Cao, Hui; Chen, Tingting; Shi, Yujun

    2015-01-01

    Diabetes mellitus is one of the most serious diseases in the world. The levels of glycated proteins in the blood of diabetics are higher than that of non-diabetic subjects. The glycation of proteins is believed to link to the occurrence of diabetic complications and related diseases. This review focuses on the influence of glycation of human serum albumin (HSA) on its structure and function. The glycation leads to change the HSA conformation, which will further influence its ligand binding properties. The levels of glycated HSA in hyperglycemic conditions showed a significant relationship to the germination of serious complications for diabetics, especially by affecting various cells functions. The conclusion from individual report is contradictory to each other; therefore, it is very difficult to give an univocal comment on the impact of glycation on the binding behaviors of HSA for small molecules. The influence of glycation of HSA on the binding affinities for small molecules is decided by the assay, the structures of small molecules, as well as the degree of glycation. However, the glycation of HSA is believed to reduce the binding affinities for acidic drugs such as polyphenols and phenolic acids.

  10. Effect of (-)-epigallocatechin-3-gallate on glucose-induced human serum albumin glycation.

    Science.gov (United States)

    Li, M; Hagerman, A E

    2015-01-01

    (-)-Epigallocatechin-3-gallate (EGCg) is a naturally occurring polyphenol found in plant-based foods and beverages such as green tea. Although EGCg can eliminate carbonyl species produced by glucose autoxidation and thus can inhibit protein glycation, it is also reported to be a pro-oxidant that stimulates protein glycation in vitro. To better understand the balance between antioxidant and pro-oxidant features of EGCg, we evaluated EGCg-mediated bioactivities in a human serum albumin (HSA)/glucose model by varying three different parameters (glucose level, EGCg concentration, and time of exposure to EGCg). Measurements of glycation-induced fluorescence, protein carbonyls, and electrophoretic mobility showed that the level of HSA glycation was positively related to the glucose level over the range 10-100 mM during a 21-day incubation at 37°C and pH: 7.4. Under mild glycemic pressure (10 mM), long exposure to EGCg enhanced HSA glycation, while brief exposure to low concentrations of EGCg did not. Under high glycemic pressure (100 mM glucose), long exposure to EGCg inhibited glycation. For the first time we showed that brief exposure to EGCg reversed glycation-induced fluorescence, indicating a restorative effect. In conclusion, our research identified glucose level, EGCg concentration, and time of exposure as critical factors dictating EGCg bioactivities in HSA glycation. EGCg did not affect HSA glycation under normal physiological conditions but had a potential therapeutic effect on HSA severely damaged by glycation.

  11. Cranberry phytochemicals inhibit glycation of human hemoglobin and serum albumin by scavenging reactive carbonyls.

    Science.gov (United States)

    Liu, Haiyan; Liu, Hanwei; Wang, Wei; Khoo, Christina; Taylor, James; Gu, Liwei

    2011-08-01

    Protein glycation caused by sugars and reactive carbonyls is a contributing factor to diabetic complications, aging, and other chronic diseases. The objective of this study was to investigate the inhibitory effects of cranberry phytochemicals on protein glycation. Cranberries, purified to yield sugar-free phytochemical powder, were fractionated into ethyl acetate and water fractions. Water fraction was further separated into water fraction I, II, and III on a Sephadex LH-20 column. Cranberry phytochemical powder and its fractions significantly inhibited the formation of glycated hemoglobin. The concentrations of cranberry phytochemicals required to inhibit 50% of albumin glycation (EC(50)) in albumin-glucose assay were lower than that of aminoguanidine except for water fraction I. Cranberry phytochemicals inhibited glycation of human serum albumin mediated by methylglyoxal, but the EC(50) were higher than that of aminoguanidine. Carbonyl scavenging assay showed that water fraction II scavenged 89.3% of methylglyoxal at 6 h of reaction. Fractions enriched with procyanidins showed higher antiglycation activities, suggesting procyanidins were the major active components. The hypothesis whether cranberry procyanidins scavenged reactive carbonyls by forming adducts was tested. Epicatechin was used as a model compound to react with methylglyoxal and glyoxal at pH 7.4. Five adducts were detected and their structures were tentatively identified using HPLC-ESI-MS/MS.

  12. Characterization of Klebsiella terrigena strains from humans: haemagglutinins, serum resistance, siderophore synthesis, and serotypes.

    Science.gov (United States)

    Podschun, R; Fischer, A; Ullmann, U

    2000-08-01

    Klebsiella terrigena is very rarely isolated from humans; as yet, its clinical significance is uncertain. The aim of the present study was to evaluate whether this species is able to express putative virulence factors. A total of 72 faecal (n = 50) and clinical (n = 22) K. terrigena isolates was investigated and compared with faecal and clinical strains of K. pneumoniae. Mannose-sensitive haemagglutination (MSHA) was observed less often in K. terrigena (64-74%) than in K. pneumoniae strains. In contrast, the incidence of mannose-resistant haemagglutinin indicative of type 3 pili (MR/K-HA) (77-94%), serum resistance properties (10-23%), and production of enterobactin (100%) was similar in both species. None of the K. terrigena isolates were able to synthesize aerobactin; however, the frequency of aerobactin synthesis in K. pneumoniae was also only 5%. Serotyping showed capsular types K5 and K70 to be predominant. The virulence-associated serotype K2 was common in both K. terrigena and K. pneumoniae isolates. Taken together, the present results suggest that K. terrigena and K. pneumoniae are indistinguishable with respect to the expression of virulence factors.

  13. Study on a noninvasive method for rapid screening Human Serum albumin injectables by Raman spectroscopy

    Directory of Open Access Journals (Sweden)

    Yu Zhao

    2017-01-01

    Full Text Available Human serum albumin (HSA injectable product is a severely afflicted area on drug safety due to its high price and restricted supply. Raman spectroscopy performances high specificity on HSA detection and it is even possible to determine HSA injectable products noninvasively. In this study, we developed a noninvasive rapid screening method for of HSA injectable products by using portable Raman spectrometer. Qualitative models were established by using principal component analysis combined with classical least squares (PCA-CLS algorithm, while quantitative model was established by using partial least squares (PLS algorithm. Model transfer in different instruments of both the same and different apparatus modules was further discussed in this paper. A total of 34 HSA injectable samples collected from markets were used for verification. The identification results showed 100% accuracy and the predicted concentrations of those identified as true HSA were consistent with their labeled concentrations. The quantitative results also indicated that model transfer was excellent in the same apparatus modules of Raman spectrometer at all concentration levels, and still good enough in the different apparatus modules although the relative standard deviation (RSD value showed a little increasing trend at low HSA concentration level. In conclusion, the method was proved to be feasible and efficient for screening HSA injections, especially on its screening speed and the consideration of glass containers. Moreover, with inspiring results on the model transfer, the method could be used as a universal screening mean to different Raman instruments.

  14. Human immunodeficiency virus type 1-related lipoatrophy and lipohypertrophy are associated with serum concentrations of leptin.

    Science.gov (United States)

    Nagy, G Sonia; Tsiodras, Sotirios; Martin, Lizabeth D; Avihingsanon, Anchalee; Gavrila, Alina; Hsu, William C; Karchmer, Adolf W; Mantzoros, Christos S

    2003-03-15

    The relationship between the adipocyte-derived hormone leptin, insulin resistance, and fat redistribution in patients with human immunodeficiency virus (HIV) infection has not been established. We classified a cohort of HIV type 1 (HIV-1)-infected patients with >or=6 months of antiretroviral exposure as having no lipodystrophy (51 patients [43% of the cohort]), lipoatrophy (23 patients [19% of the cohort]), mixed lipodystrophy (29 patients [24% of the cohort]), or lipohypertrophy (17 patients [14% of the cohort]), on the basis of physical examination, anthropometric measurements, and the findings of dual-emission x-ray absorptiometry and computed tomography. Measurements of insulin resistance were higher for patients with each category of lipodystrophy, compared with those observed for patients with no lipodystrophy (Plipohypertrophy (9.10+/-6.86 ng/mL), and significantly different from those in patients without lipodystrophy (3.14+/-2.30 ng/mL; both P<.01). In this cohort of antiretroviral-experienced HIV-infected patients, a low serum level of leptin was independently associated with insulin resistance in patients with lipoatrophy, after controlling for total and regional body fat.

  15. Probing the binding of fluoxetine hydrochloride to human serum albumin by multispectroscopic techniques

    Science.gov (United States)

    Katrahalli, Umesha; Jaldappagari, Seetharamappa; Kalanur, Shankara S.

    2010-01-01

    The interaction between human serum albumin (HSA) and fluoxetine hydrochloride (FLX) have been studied by using different spectroscopic techniques viz., fluorescence, UV-vis absorption, circular dichroism and FTIR under simulated physiological conditions. Fluorescence results revealed the presence of static type of quenching mechanism in the binding of FLX to HSA. The values of binding constant, K of FLX-HSA were evaluated at 289, 300 and 310 K and were found to be 1.90 × 10 3, 1.68 × 10 3 and 1.45 × 10 3 M -1, respectively. The number of binding sites, n was noticed to be almost equal to unity thereby indicating the presence of a single class of binding site for FLX on HSA. Based on the thermodynamic parameters, Δ H0 and Δ S0 nature of binding forces operating between HSA and FLX were proposed. Spectral results revealed the conformational changes in protein upon interaction. Displacement studies indicated the site I as the main binding site for FLX on HSA. The effect of common ions on the binding of FLX to HSA was also investigated.

  16. Interactions Between Sirolimus and Anti-Inflammatory Drugs: Competitive Binding for Human Serum Albumin

    Science.gov (United States)

    Khodaei, Arash; Bolandnazar, Soheila; Valizadeh, Hadi; Hasani, Leila; Zakeri-Milani, Parvin

    2016-01-01

    Purpose: The aim of the present study was investigating the effects of three anti-inflammatory drugs, on Sirolimus protein biding. The binding site of Sirolimus on human serum albumin (HSA) was also determined. Methods: Six different concentrations of Sirolimus were separately exposed to HSA at pH 7.4 and 37°C. Ultrafiltration method was used for separating free drug; then free drug concentrations were measured by HPLC. Finally, Sirolimus protein binding parameters was calculated using Scatchard plots. The same processes were conducted in the presence of NSAIDs at lower concentration of albumin and different pH conditions. To characterize the binding site of Sirolimus on albumin, the free concentration of warfarin sodium and Diazepam, site I and II specific probes, bound to albumin were measured upon the addition of increasing Sirolimus concentrations. Results: Based on the obtained results presence of Diclofenac, Piroxicam and Naproxen, could significantly decrease the percentage of Sirolimus protein binding. The Binding reduction was the most in the presence of Piroxicam. Sirolimus-NSAIDs interactions were increased in higher pH values and also in lower albumin concentrations. Probe displacement study showed that Sirolimus may mainly bind to site I on albumin molecule. Conclusion: More considerations in co-administration of NSAIDs and Sirolimus is recommended. PMID:27478785

  17. Temperature dependence of binding and catalysis for human serum arylesterase/paraoxonase.

    Science.gov (United States)

    Debord, Jean; Bollinger, Jean-Claude; Harel, Michel; Dantoine, Thierry

    2014-02-01

    The influence of temperature upon the hydrolysis of phenyl acetate, catalysed by purified human serum arylesterase/paraoxonase (E. C. 3.1.8.1), was studied in the temperature range 10 °C-40 °C by spectrophotometry in TRIS buffer, pH 8.0, using both initial rate analysis and progress curve analysis. The kinetic parameters (catalytic constant k(cat); Michaelis constant K(m); product inhibition constant K(p)) were determined by nonlinear regression. All parameters increased with temperature, but the ratios k(cat)/K(m) and K(p)/K(m) remained practically constant. Binding of both substrate and reaction product (phenol) was exothermic. A negative entropic term accounted for about 50% of the enthalpy change for both the binding and catalytic steps. Thermodynamic analysis suggested that: (1) the rate-limiting step is the nucleophilic attack of the carbonyl group of the substrate by a water molecule, (2) the active site is preorganized with no induced fit, (3) the enzyme-bound calcium plays an important role in stabilizing both the substrate and the transition state. The practical implications of these results are discussed.

  18. Adsorption of an amphiphilic penicillin onto human serum albumin: characterisation of the complex.

    Science.gov (United States)

    Ruso, J M; Taboada, P; Varela, L M; Attwood, D; Mosquera, V

    2001-08-30

    The complex formed by the interaction of the amphiphilic penicillin drug nafcillin and human serum albumin (HSA) in water at 25 degrees C has been characterised using a range of physicochemical techniques. Measurements of the solution conductivity and the electrophoretic mobility of the complexes have shown an ionic adsorption of the drug on the protein surface leading to a surface saturation at a nafcillin concentration of 0.012 mmol kg(-1) and subsequent formation of drug micelles in solutions of higher nafcillin concentration. Measurements of the size of the complex and the thickness of the adsorbed layer by static and dynamic light scattering have shown a gradual change in hydrodynamic radius of the complex with increasing drug concentration typical of a saturation rather than a denaturation process, the magnitude of the change being insufficient to account for any appreciable extension or unfolding of the HSA molecule. The interaction potential between the HSA/nafcillin complexes, and the stability of the complexes were determined from the dependence of diffusion coefficients on protein concentration by application of the DLVO colloidal stability theory. The results indicate decreasing stability of the colloidal dispersion of the drug/protein complexes with an increase in the concentration of added drug.

  19. Conformational changes in human serum albumin induced by sodium perfluorooctanoate in aqueous solutions.

    Science.gov (United States)

    Messina, Paula V; Prieto, Gerardo; Ruso, Juan M; Sarmiento, Félix

    2005-08-18

    Conformational changes in the bulk solution and at the air-aqueous interface of human serum albumin (HSA) induced by changes in concentration of sodium perfluorooctanoate (C(7)F(15)COO(-)Na(+)) were studied by difference spectroscopy, zeta-potential data, and axisymmetric drop shape analysis. zeta-potential was used to monitor the formation of the HSA-C(7)F(15)COO(-)Na(+) complex and the surface charge of the complex. The conformational transition of HSA in the bulk solution was followed as a function of denaturant concentration by absorbance measurements at 280 nm. The data were analyzed to obtain values for the Gibbs energies of the transition in water (DeltaG(0)(W)) and in a hydrophobic environment (DeltaG(0)(hc)) pertaining to saturated protein-surfactant complexes. The conformational changes that surfactants induce in HSA molecules alter its absorption behavior at the air-water interface. Dynamic surface measurements were used to evaluate this behavior. At low [C(7)F(15)COO(-)Na(+)], proteins present three adsorption regimes: induction time, monolayer saturation, and interfacial gelation. When surfactant concentration increases and conformational changes in the bulk solution occur, the adsorption regimes disappear. HSA molecules in an intermediate conformational state migrate to the air-water interface and form a unique monolayer. At high [C(7)F(15)COO(-)Na(+)], the adsorption of denatured molecules exhibits a behavior analogous to that of dilute solutions.

  20. Immune response to acetaldehyde-human serum albumin adduct among healthy subjects related to alcohol intake.

    Science.gov (United States)

    Romanazzi, Valeria; Schilirò, Tiziana; Carraro, Elisabetta; Gilli, Giorgio

    2013-09-01

    Acetaldehyde (AA) is the main metabolic product in ethanol metabolism, although it can also derive from sources of airborne pollution. As a typical aldehyde, AA is able to react with a variety of molecular targets, including DNA and protein. This property justifies the hypothesis of a immune reaction against this kind of adduct, to be studied by a seroprevalence screening approach. In this study, the correlation between drinking habits and the amount of circulating AA-human serum albumin adduct (AA-HSA) was evaluated in a group of healthy subjects, non alcohol-addicted. Daily ethanol intake (grams) was inferred for each subject using the information collected through a questionnaire, and AA-HSA antibodies (AA-HSA ab) analyses were performed using the Displacement Assay on whole blood samples. The findings showed a correlation between ethanol intake and immune response to molecular adduct. These results underscore the evaluation of AA-HSA ab amount as a suitable molecular marker for alcohol intake that can be applied in future investigations on a large scale for prevention screening.

  1. Comparative Interactions of Dihydroquinazolin Derivatives with Human Serum Albumin Observed via Multiple Spectroscopy

    Directory of Open Access Journals (Sweden)

    Yi Wang

    2017-02-01

    Full Text Available The interactions of dihydroquinazolines with human serum albumin (HSA were studied in pH 7.4 aqueous solution via fluorescence, circular dichroism (CD and Fourier transform infrared (FTIR spectroscopic techniques. In this work, 6-chloro-1-(3,3-dimethyl-butanoyl-2(unsubstitutedphenyl-2,3-dihydroquinazolin-4(1H-one (PDQL derivatives were designed and synthesized to study the impact of five similar substituents (methyl, methoxy, cyano, trifluoromethyl and isopropyl on the interactions between PDQL and HSA using a comparative methodology. The results revealed that PDQL quenched the intrinsic fluorescence of HSA through a static quenching process. Displacement experiments with site-specific markers revealed that PDQL binds to HSA at site II (subdomain IIIA and that there may be only one binding site for PDQL on HSA. The thermodynamic parameters indicated that hydrophobic interactions mainly drove the interactions between PDQL and HSA. The substitution using five similar groups in the benzene ring could increase the interactions between PDQL and HSA to some extent through the van der Waals force or hydrogen bond effects in the proper temperature range. Isopropyl substitution could particularly enhance the binding affinity, as observed via comparative studies

  2. Characteristics of magnetic Fe3O4 nanoparticles encapsulated with human serum albumin

    Institute of Scientific and Technical Information of China (English)

    2006-01-01

    Magnetic nanoparticles (Fe3O4) were prepared by chemical precipitation method using Fe2+ and Fe3+salts with sodium hydroxide in the nitrogen atmosphere. Fe3O4 nanoparticles were coated with human serum albumin(HSA) for magnetic resonance imaging as contrast agent. Characteristics of magnetic particles coated or uncoated were carried out using scanning electron microscopy and X-ray diffraction. Zeta potentials, package effects and distributions of colloid particles were measured to confirm the attachment of HSA on magnetic particles. Effects of Fe3O4 nanoparticles coated with HSA on magnetic resonance imaging were investigated with rats. The experimental results show that the adsorption of HSA on magnetic particles is very favorable to dispersing of magnetic Fe3O4 particles, while the sizes of Fe3O4 particles coated are related to the molar ratio of Fe3O4 to HSA. The diameters of the majority of particles coated are less than 100 nm. Fe3O4 nanoparticle coated with HSA has a good biocompatibility and low toxicity. This new contrast agent has some effects on the nuclear magnetic resonance imaging of liver and the lowest dosage is 20 μmol/kg for the demands of diagnosis.

  3. Study of interaction between human serum albumin and three phenanthridine derivatives: Fluorescence spectroscopy and computational approach

    Science.gov (United States)

    Liu, Jianming; Yue, Yuanyuan; Wang, Jing; Yan, Xuyang; Liu, Ren; Sun, Yangyang; Li, Xiaoge

    2015-06-01

    Over the past decades, phenanthridine derivatives have captured the imagination of many chemists due to their wide applications. In the present work, the interaction between phenanthridine derivatives benzo [4,5]imidazo[1,2-a]thieno[2,3-c]quinoline (BTQ), benzo[4,5]imidazo[1,2-a]furo[2,3-c]quinoline (BFQ), 5,6-dimethylbenzo[4,5]imidazo[1,2-a]furo[2,3-c]quinoline (DFQ) and human serum albumin (HSA) were investigated by molecular modeling techniques and spectroscopic methods. The results of molecular modeling simulations revealed that the phenanthridine derivatives could bind on both site I in HSA. Fluorescence data revealed that the fluorescence quenching of HSA by phenanthridine derivatives were the result of the formation of phenanthridine derivatives-HSA complex, and the binding intensity between three phenanthridine derivatives and HSA was BTQ > BFQ > DFQ. Thermodynamics confirmed that the interaction were entropy driven with predominantly hydrophobic forces. The effects of some biological metal ions and toxic ions on the binding affinity between phenanthridine derivatives and HSA were further examined.

  4. Development of human serum albumin conjugated with near-infrared dye for photoacoustic tumor imaging

    Science.gov (United States)

    Kanazaki, Kengo; Sano, Kohei; Makino, Akira; Takahashi, Atsushi; Deguchi, Jun; Ohashi, Manami; Temma, Takashi; Ono, Masahiro; Saji, Hideo

    2014-09-01

    Photoacoustic (PA) imaging has emerged as a noninvasive diagnostic method which detects ultrasonic waves thermoelastically induced by optical absorbers irradiated with laser. For tumor diagnosis, PA contrast agent has been proposed to enhance the PA effect for detecting tumors sensitively. Here, we prepared a human serum albumin (HSA) conjugated with indocyanine green (ICG) as a PA contrast agent allowing enhanced permeability and retention effect for sensitive tumor imaging. The feasibility of PA imaging with HSA-ICG to detect allografted tumors was evaluated in tumor-bearing mice. In vivo fluorescence imaging and radiolabeled biodistribution study showed that the biodistribution dramatically changed as the number of ICG bound to HSA increased, and the maximum accumulation of ICG was achieved when around three ICG molecules were loaded on an HSA. In vivo PA imaging demonstrated a tumor-selective and dose-dependent increase of PA signal intensity in mice injected with HSA-ICG (R2=0.88, 387% increase for HSA-ICG, 104 nmol ICG). In conclusion, HSA-ICG clearly visualized the allografted tumors with high tumor-to-background ratios having high quantitative and spatial resolution for the sensitive PA imaging of tumors. HSA-ICG could be useful as a favorable contrast agent for PA tumor imaging for the management of cancer.

  5. Spectroscopic studies on the interaction of cinnamic acid and its hydroxyl derivatives with human serum albumin

    Science.gov (United States)

    Min, Jiang; Meng-Xia, Xie; Dong, Zheng; Yuan, Liu; Xiao-Yu, Li; Xing, Chen

    2004-04-01

    Cinnamic acid and its derivatives possess various biological effects in remedy of many diseases. Interaction of cinnamic acid and its hydroxyl derivatives, p-coumaric acid and caffeic acid, with human serum albumin (HSA), and concomitant changes in its conformation were studied using fluorescence and Fourier transform infrared spectroscopic methods. Fluorescence data revealed the presence of one binding site on HSA for cinnamic acid and its hydroxyl derivatives, and their binding constants ( KA) are caffeic acid> p-coumaric acid> cinnamic acid when Cdrug/ CHSA ranging from 1 to 10. The changes of the secondary structure of HSA after interacting with the three drugs are estimated, respectively by combining the curve-fitting results of amid I and amid III bands. The α-helix structure has a decrease of ≈9, 5 and 3% after HSA interacted with caffeic acid, p-coumaric acid and cinnamic acid, respectively. It was found that the hydroxyls substituted on aromatic ring of the drugs play an important role in the changes of protein's secondary structure. Combining the result of fluorescence quenching and the changes of secondary structure of HSA after interaction with the three drugs, the drug-HSA interaction mode was discussed.

  6. Genetically engineered mannosylated-human serum albumin as a versatile carrier for liver-selective therapeutics.

    Science.gov (United States)

    Hirata, Kenshiro; Maruyama, Toru; Watanabe, Hiroshi; Maeda, Hitoshi; Nakajou, Keisuke; Iwao, Yasunori; Ishima, Yu; Katsumi, Hidemasa; Hashida, Mitsuru; Otagiri, Masaki

    2010-07-01

    Human serum albumin (HSA), a non-glycosylated protein, is widely employed as carrier for drug delivery systems. A series of recombinant, mannosylated-HSA mutants (Man-rHSAs: D63N, A320T and D494N) and their triple mutant (TM-rHSA: D63N/A320T/D494N) were prepared, that can be selectively delivered to the liver via mannose receptor (MR) on the liver non-parenchymal cells. A pharmacokinetic analysis of (111)In-Man-rHSAs in mice showed that they were rapidly cleared from the blood circulation, and were largely taken up by the liver rapidly in the order: TM-rHSA>D494N>A320T=D63N, consistent with their degree of mannosylation. In vivo competition experiments with an excess amount of chemically modified Man-BSA or mannan suggested that the hepatic uptake of TM-rHSA was selectively mediated by MR on Kupffer cells. Lastly, a TM-rHSA-NO conjugate, S-nitrosylated TM-rHSA, effectively delivered NO to the liver and then exhibited a significant inhibitory effect against hepatic ischemia/reperfusion injury model rats, accompanied by the induction of heme oxygenase-1.

  7. Human serum albumin binding to silica nanoparticles--effect of protein fatty acid ligand.

    Science.gov (United States)

    Ang, Joo Chuan; Henderson, Mark J; Campbell, Richard A; Lin, Jhih-Min; Yaron, Peter N; Nelson, Andrew; Faunce, Thomas; White, John W

    2014-06-07

    Neutron reflectivity shows that fatted (F-HSA) and defatted (DF-HSA) versions of human serum albumin behave differently in their interaction with silica nanoparticles premixed in buffer solutions although these proteins have close to the same surface excess when the silica is absent. In both cases a silica containing film is quickly established at the air-water interface. This film is stable for F-HSA at all relative protein-silica concentrations measured. This behaviour has been verified for two small silica nanoparticle radii (42 Å and 48 Å). Contrast variation and co-refinement have been used to find the film composition for the F-HSA-silica system. The film structure changes with protein concentration only for the DF-HSA-silica system. The different behaviour of the two proteins is interpreted as a combination of three factors: increased structural stability of F-HSA induced by the fatty acid ligand, differences in the electrostatic interactions, and the higher propensity of defatted albumin to self-aggregate. The interfacial structures of the proteins alone in buffer are also reported and discussed.

  8. Mn(II) binding to human serum albumin: a ¹H-NMR relaxometric study.

    Science.gov (United States)

    Fanali, Gabriella; Cao, Yu; Ascenzi, Paolo; Fasano, Mauro

    2012-12-01

    Human serum albumin (HSA) displays several metal binding sites, participating to essential and toxic metal ions disposal and transport. The major Zn(II) binding site, called Site A, is located at the I/II domain interface, with residues His67, Asn99, His247, and Asp249 contributing with five donor atoms to the metal ion coordination. Additionally, one water molecule takes part of the octahedral coordination geometry. The occurrence of the metal-coordinated water molecule allows the investigation of the metal complex geometry by water (1)H-NMR relaxation, provided that the diamagnetic Zn(II) is replaced by the paramagnetic Mn(II). Here, the (1)H-NMR relaxometric study of Mn(II) binding to HSA is reported. Mn(II) binding to HSA is modulated by Zn(II), pH, and myristate through competitive inhibition and allosteric mechanisms. The body of results indicates that the primary binding site of Zn(II) corresponds to the secondary binding site of Mn(II), i.e. the multimetal binding site A. Excess Zn(II) completely displaces Mn(II) from its primary site suggesting that the primary Mn(II) site corresponds to the secondary Zn(II) site. This uncharacterized site is functionally-linked to FA1; moreover, metal ion binding is modulated by myristate and pH. Noteworthy, water (1)H-NMR relaxometry allowed a detailed analysis of thermodynamic properties of HSA-metal ion complexes.

  9. Chiral recognition of metalaxyl enantiomers by human serum albumin: evidence from molecular modeling and photophysical approach.

    Science.gov (United States)

    Ding, Fei; Li, Xiu-Nan; Diao, Jian-Xiong; Sun, Ye; Zhang, Li; Sun, Ying

    2012-06-01

    Metalaxyl is an acylamine fungicide, belonging to the most widely known member of the amide group. This task is aimed to scrutinize binding region and spatial structural change of principal vector human serum albumin (HSA) complex with (R)-/(S)-metalaxyl by exploiting molecular modeling, steady-state and time-resolved fluorescence, and circular dichroism (CD) approaches. According to molecular modeling, (R)-metalaxyl is situated within subdomains IIA and IIIA and the affinity of site I with (R)-metalaxyl is greater than site II, whereas (S)-metalaxyl is only located at subdomain IIA and the affinity of (S)-metalaxyl with site I is superior compared with that with (R)-metalaxyl. This coincides with the competitive ligand binding, guanidine hydrochloride-induced unfolding of protein, and hydrophobic 8-anilino-1-naphthalenesulfonic acid experiments; the acting forces between (R)-/(S)-metalaxyl and HSA are hydrophobic, π-π interactions, and hydrogen bonds, as derived from molecular modeling. Fluorescence emission manifested that the complex of (R)-/(S)-metalaxyl to HSA is the formation of adduct with an affinity of 10(4) M(-1), which corroborates the time-resolved fluorescence that the static type was operated. Furthermore, the changes of far-UV CD spectra evidence the polypeptide chain of HSA partially unfolded after conjugation with (R)-/(S)-metalaxyl. Through this work, we envisage that it can offer central clues on the biodistribution, absorption, and bioaccumulation of (R)-/(S)-metalaxyl. Copyright © 2012 Wiley Periodicals, Inc.

  10. Investigation of Interaction Between Ozagrel and Human Serum Albumin by Spectroscopic and Electrochemical Methods

    Science.gov (United States)

    Li, S.; Wang, Li; Hao, J.; Wang, L.; Tong, Y.-J.; Fu, Z.-Q.; Zhang, A.-P.

    2017-01-01

    The interaction between ozagrel and human serum albumin (HSA) was investigated by fl uorescence spectroscopy, UV-Vis absorption spectroscopy, cyclic voltammetry (CV), differential pulse voltammetry (DPV), and Fourier transform infrared spectroscopy (FTIR) under simulative physiological conditions. The results of CV, DPV and fl uorescence titration revealed that ozagrel bound to HSA. The enthalpy change (ΔH) and the entropy change (ΔS) were derived to be positive values, indicating that the hydrophobic force played the main role in the binding of ozagrel with HSA. The binding distance between ozagrel and HSA was 1.75 nm. Upon binding with ozagrel, the conformation and the secondary structure of HSA molecules were changed. The percentage of α-helix and β-sheet structures decreased by 7.25% and 4.58%, respectively, while the percentage of a β-turn structure increased by 2.67%. The effect of common ions on the binding of ozagrel with HSA was also examined. This study will give an insight into the evaluation of the drug's stabi-lity during transport and its releasing effi ciency at the target site under simulative physiological conditions.

  11. [Direct proteome profiling of human blood serum in the experiment with 5-day dry immersion].

    Science.gov (United States)

    Pastushkova, L Kh; Pakharukova, N A; Trifonova, O P; Dobrokhotov, I V; Valeeva, O A; Larina, I M

    2011-01-01

    Purpose of the investigation was to determine changes in blood plasma proteome in healthy human subjects (n = 14, 19 to 26 y.o.) in an experiment with dry immersion (DI). Plasma samples were drawn 7 and 2 days before the exposure, on DI days 2, 3 and 5, and on days 1, 3, 7 and 15 after the experiment. Previous to direct MALDI-TOF mass-spectrometric profiling, serum samples were pre-fractionated and enriched with magnetic particles MB WCX (WCX--a weak cation exchanger) on ClinProt (Bruker Daltonics). In each spectrum, 175 MS-peaks were detected on average within the mass range from 1000 to 17,000 Da with the signal/noise ratio = 5. Student's criterion (p experiment. Significant increases of the peak area of apolipoprotein CI (reduced form with segregated threonine and proline) and C4 enzymes of the complement system, and fibrinogen on the first day after the experiment can be related to changes in motor activities of the subjects.

  12. Fibrillar disruption by AC electric field induced oscillation: A case study with human serum albumin.

    Science.gov (United States)

    Sen, Shubhatam; Chakraborty, Monojit; Goley, Snigdha; Dasgupta, Swagata; DasGupta, Sunando

    2017-07-01

    The effect of oscillation induced by a frequency-dependent alternating current (AC) electric field to dissociate preformed amyloid fibrils has been investigated. An electrowetting-on-dielectric type setup has been used to apply the AC field of varying frequencies on preformed fibrils of human serum albumin (HSA). The disintegration potency has been monitored by a combination of spectroscopic and microscopic techniques. The experimental results suggest that the frequency of the applied AC field plays a crucial role in the disruption of preformed HSA fibrils. The extent of stress generated inside the droplet due to the application of the AC field at different frequencies has been monitored as a function of the input frequency of the applied AC voltage. This has been accomplished by assessing the morphology deformation of the oscillating HSA fibril droplets. The shape deformation of the oscillating droplets is characterized using image analysis by measuring the dynamic changes in the shape dependent parameters such as contact angle and droplet footprint radius and the amplitude. It is suggested that the cumulative effects of the stress generated inside the HSA fibril droplets due to the shape deformation induced hydrodynamic flows and the torque induced by the intrinsic electric dipoles of protein due to their continuous periodic realignment in presence of the AC electric field results in the destruction of the fibrillar species. Copyright © 2017. Published by Elsevier B.V.

  13. Serum PEDF levels are decreased in a spontaneous animal model for human autoimmune uveitis.

    Science.gov (United States)

    Zipplies, Johanna K; Hauck, Stefanie M; Schoeffmann, Stephanie; Amann, Barbara; Stangassinger, Manfred; Ueffing, Marius; Deeg, Cornelia A

    2009-02-01

    Identification of biomarkers is of critical relevance toward improving diagnosis and therapy of autoimmune disorders. Serum markers are a desirable choice as sera are easily accessible and the development of assays for routine clinical detection prompts feasible. Autoimmune uveitis, a recurrent disease affecting the eye, is characterized by returning inflammatory attacks of the inner eye followed by variable periods of quiescent stages. Spontaneous equine recurrent uveitis (ERU) is the equine equivalent and serves as a model for the human disease. To identify potential biomarker candidates, we first systematically compared the proteomes of individual ERU cases with healthy controls by proteomic profiling using 2-D difference-gel-electrophoresis (2-D DIGE) followed by tandem mass spectrometry. A total of seven differentially expressed proteins were identified. Besides the upregulation of IgG and the significant lower expression of albumin, Antithrombin III, and Vitamin D binding protein, we found complement components C1q and C4, to be downregulated in uveitic state. Interestingly, Pigment epithelium-derived factor (PEDF), a marker already detected by 2DE differential proteome analysis in ERU target tissues, vitreous and retina, was found to be also significantly downregulated in sera. The lower expression of PEDF in sera of horses with uveitis could be verified in a cohort of 116 ERU cases and 115 healthy controls. Our findings of a significant lower PEDF expression in ERU cases also in the periphery of the eye proves PEDF as a promising uveitis biomarker.

  14. Photosensitizer-Conjugated Human Serum Albumin Nanoparticles for Effective Photodynamic Therapy

    Directory of Open Access Journals (Sweden)

    Hayoung Jeong, MyungSook Huh, So Jin Lee, Heebeom Koo, Ick Chan Kwon, Seo Young Jeong, Kwangmeyung Kim

    2011-01-01

    Full Text Available Photodynamic therapy (PDT is an emerging theranostic modality for various cancers and diseases. The focus of this study was the development of tumor-targeting albumin nanoparticles containing photosensitizers for efficient PDT. To produce tumor-targeting albumin nanoparticles, the hydrophobic photosensitizer, chlorin e6 (Ce6, was chemically conjugated to human serum albumin (HSA. The conjugates formed self-assembled nanoparticle structures with an average diameter of 88 nm under aqueous conditions. As expected, the Ce6-conjugated HSA nanoparticles (Ce6-HSA-NPs were nontoxic in their native state, but upon illumination with the appropriate wavelength of light, they produced singlet oxygen and damaged target tumor cells in a cell culture system. Importantly, when the nanoparticles were injected through the tail vein into tumor-bearing HT-29 mice, Ce6-HSA-NPs compared with free Ce6 revealed enhanced tumor-specific biodistribution and successful therapeutic results following laser irradiation. These results suggest that highly tumor-specific albumin nanoparticles have the potential to serve not only as efficient therapeutic agents, but also as photodynamic imaging (PDI reagents in cancer treatment.

  15. Short chain polyethylene glycols unusually assist thermal unfolding of human serum albumin.

    Science.gov (United States)

    Samanta, Nirnay; Mahanta, Debasish Das; Hazra, Soumitra; Kumar, Gopinatha Suresh; Mitra, Rajib Kumar

    2014-09-01

    In the present study we have investigated the thermal stability of the globular transport protein human serum albumin (HSA), in the presence of two small chain polyethylene glycols (namely PEG 200 and PEG 400). Both near- and far-UV circular dichroism (CD) study reveal that addition of PEG moderately increases the α-helical content of the protein without abruptly changing its tertiary structure. The hydration structure at the protein surface experiences a notable change at 30% PEG (v/v) concentration as evidenced from compressibility and dynamic light scattering (DLS) measurements. Thermal denaturation of HSA in the presence of PEG has been studied by CD and fluorescence spectroscopy using the intrinsic fluorophore tryptophan and it has been found that addition of PEG makes the protein more prone towards unfolding, which is in contrary to what has been observed in case of larger molecular weight polymers. The energetics of the thermal unfolding process has been obtained using differential scanning calorimetry (DSC) measurements. Our study concludes that both the indirect excluded volume principle as well as interaction of the polymer at the protein surface is responsible for the observed change of the unfolding process.

  16. Ethanol or/and captopril-induced precipitation and secondary conformational changes of human serum albumin

    Science.gov (United States)

    Lin, Shan-Yang; Li, Mei-Jane; Wei, Yen-Shan

    2004-11-01

    We determined the secondary structure of solid-state native human serum albumin (HSA) and its precipitates induced by ethanol, captopril, or a captopril/ethanol mixture. A transmission Fourier transform infrared (FT-IR) microspectroscopy equipped with a thermal analyzer was used. The secondary structural composition of solid-state native HSA was 54% α-helices (1655 cm -1), 22% β-turns (1679 cm -1), and 23% β-sheets (1633 cm -1). After ethanol treatment, a new peak was observed at 1690 cm -1, and the peak at 1633 cm -1 was more apparent in the HSA precipitates. The corresponding compositions consisted of 59% α-helices, 17% β-turns, and 24% β-sheets. After treatment with captopril with or without ethanol, the percentage of α-helices and β-turns decreased in both HSA precipitates, but the percentage of β-sheets increased. The temperature-dependent structural transformation from α-helices/random coils to β-sheets for the solid-state HSA samples occurred at markedly different onset temperatures. The onset temperature for native HSA was 85 °C, and that for HSA precipitates obtained from ethanol, captopril, or captopril/ethanol was 100, 48 or 57 °C, respectively. The thermal-induced structural transformation from α-helices/random coils to β-sheets implies a partial unfolding structure in these HSA samples.

  17. THE EFFECTS OF COPPER AND ZINC IONS DURING THEIR BINDING WITH HUMAN SERUM γ-GLOBULIN

    Directory of Open Access Journals (Sweden)

    S. B. Cheknev

    2006-01-01

    Full Text Available Abstract. Conformational changes of human serum γ-globulin were studied during and after its binding with copper and zinc ions, using molecular ultrafiltration and differential spectrophotometry. The contents of nonbound metals in the filtrate were evaluated, resp., with sodium diethyl thyocarbamate and o-phenanthroline. It has been shown that copper and zinc exhibited common biological properties during their interactions with protein, but the binding differed sufficiently under similar experimental conditions. E.g., it was confirmed that copper was more active at the external sites of γ-globulin molecule, whereas zinc demonstrated tropicity for the areas of protein intraglobular compartments. The metal-binding sites have been described that differ in their parameters of interactions with cations and their spatial location within globular domains. Approaches are suggested for dynamic analysis of saturation for these differently located sites by the metal ions. We discuss the issues of altered conformational state of the γ-globulin molecule during the binding of cations, as well as potential usage of these data in clinical immunology.

  18. Binding of doxyl stearic spin labels to human serum albumin: an EPR study.

    Science.gov (United States)

    Pavićević, Aleksandra A; Popović-Bijelić, Ana D; Mojović, Miloš D; Šušnjar, Snežana V; Bačić, Goran G

    2014-09-18

    The binding of spin-labeled fatty acids (SLFAs) to the human serum albumin (HSA) examined by electron paramagnetic resonance (EPR) spectroscopy was studied to evaluate the potential of the HSA/SLFA/EPR technique as a biomarking tool for cancer. A comparative study was performed on two spin labels with nitroxide groups attached at opposite ends of the fatty acid (FA) chain, 5-doxyl stearic (5-DS) and 16-doxyl stearic (16-DS) acid. The effects of incubation time, different [SLFA]/[HSA] molar ratios, ethanol, and temperature showed that the position of the nitroxide group produces certain differences in binding between the two SLFAs. Spectra for different [SLFA]/[HSA] molar ratios were decomposed into two spectral components, which correspond to the weakly and strongly bound SLFAs. The reduction of SLFA with ascorbate showed the existence of a two component process, fast and slow, confirming the decomposition results. Warfarin has no effect on the binding of the two SLFAs, whereas ibuprofen significantly decreases the binding of 5-DS and has no effect on 16-DS. Together, the results of this study indicate that both SLFAs, 5-DS and 16-DS, should be used for the study of HSA conformational changes in blood induced by various medical conditions.

  19. [Regularities of formation of chlorophyll-human serum albumin functionally active complexes in the aqueous medium].

    Science.gov (United States)

    Semichaevskiĭ, V D

    1975-01-01

    In the system with constant content of the chlorophyll a and increasing amounts of human serum albumin, dependence of pigment incorporation into the complex upon interaction of its aqueous associates with protein solutions was studied by applying the gel filtration on Sephadex G-75 and by measuring light scattering and rate of sensitized photoreduction of the methyl red by ascorbic-acid. The curves were obtained after extraction of the chlorophyll by acetone from dry pigment-protein films formed after desiccation of the aqueous systems. Sigmoid character of the above dependences, their linearization in Hill's coordinates and the value of cooperativity coefficient close to 2 testifies in favour of the cooperative character of the complex formation, two pigment molecules reacting with a single protein molecule. Measurement of adsorption isotherms and their treatment with use of the Brunauer-Emmett-Teller theory of polymolecular adsorption make it possible to evaluate the maximum molar ratio of the pigment to the protein in the complex (close to 2). The pigment-pigment interaction suggests that the chlorophyll molecules adsorbed on the protein are in the state of loosely packed dimers. Deaggregation of aqueus pigment associates by the protein in the course of complex formation results in a considerable increase of the protosensitizing chlorophyll activity.

  20. (99m)Tc-human serum albumin nanocolloids: particle sizing and radioactivity distribution.

    Science.gov (United States)

    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.

  1. Long term stability of paraoxonase-1 and high-density lipoprotein in human serum

    Directory of Open Access Journals (Sweden)

    Beekhof Piet K

    2012-05-01

    Full Text Available Abstract Background Paraoxonase-1 (PON1 is an enzyme with numerous functions and receives an increasing interest in clinical and epidemiological studies. Sometimes samples are stored for longer periods at a certain temperature. Therefore the stability of PON1 activity must be checked and retained upon storage for longer periods. Results In this study the stability of PON1 activity has been tested in human serum samples during storage up to 12 months at 3 commonly used temperatures, -20°C, -70°C and −196°C. It was found that the stability of the PON1 activity is constant during 12 months of storage at −70°C and −196°C. Storage at −20°C resulted in a small but statistically significant decrease after 6 months to about 94% of its original value. Nonetheless, the rank order between the samples at T = 0 and 12 months remained the same. The same temperature dependence was found for the associated high-density lipoprotein. Conclusions It can be concluded that −70°C is the right temperature for storage to maintain the PON1 activity for at least one year. Storage at a lower temperature in liquid nitrogen (−196°C is not necessary.

  2. Pollutant-induced modulation in conformation and β-lactamase activity of human serum albumin.

    Directory of Open Access Journals (Sweden)

    Ejaz Ahmad

    Full Text Available Structural changes in human serum albumin (HSA induced by the pollutants 1-naphthol, 2-naphthol and 8-quinolinol were analyzed by circular dichroism, fluorescence spectroscopy and dynamic light scattering. The alteration in protein conformational stability was determined by helical content induction (from 55 to 75% upon protein-pollutant interactions. Domain plasticity is responsible for the temperature-mediated unfolding of HSA. These findings were compared to HSA-hydrolase activity. We found that though HSA is a monomeric protein, it shows heterotropic allostericity for β-lactamase activity in the presence of pollutants, which act as K- and V-type non-essential activators. Pollutants cause conformational changes and catalytic modifications of the protein (increase in β-lactamase activity from 100 to 200%. HSA-pollutant interactions mediate other protein-ligand interactions, such as HSA-nitrocefin. Therefore, this protein can exist in different conformations with different catalytic properties depending on activator binding. This is the first report to demonstrate the catalytic allostericity of HSA through a mechanistic approach. We also show a correlation with non-microbial drug resistance as HSA is capable of self-hydrolysis of β-lactam drugs, which is further potentiated by pollutants due to conformational changes in HSA.

  3. Caffeine and sulfadiazine interact differently with human serum albumin: A combined fluorescence and molecular docking study

    Science.gov (United States)

    Islam, Mullah Muhaiminul; Sonu, Vikash K.; Gashnga, Pynsakhiat Miki; Moyon, N. Shaemningwar; Mitra, Sivaprasad

    2016-01-01

    The interaction and binding behavior of the well-known drug sulfadiazine (SDZ) and psychoactive stimulant caffeine (CAF) with human serum albumin (HSA) was monitored by in vitro fluorescence titration and molecular docking calculations under physiological condition. The quenching of protein fluorescence on addition of CAF is due to the formation of protein-drug complex in the ground state; whereas in case of SDZ, the experimental results were explained on the basis of sphere of action model. Although both these compounds bind preferentially in Sudlow's site 1 of the protein, the association constant is approximately two fold higher in case of SDZ (∼4.0 × 104 M-1) in comparison with CAF (∼9.3 × 102 M-1) and correlates well with physico-chemical properties like pKa and lipophilicity of the drugs. Temperature dependent fluorescence study reveals that both SDZ and CAF bind spontaneously with HSA. However, the binding of SDZ with the protein is mainly governed by the hydrophobic forces in contrast with that of CAF; where, the interaction is best explained in terms of electrostatic mechanism. Molecular docking calculation predicts the binding of these drugs in different location of sub-domain IIA in the protein structure.

  4. Stereoselective binding of mexiletine and ketoprofen enantiomers with human serum albumin domains

    Institute of Scientific and Technical Information of China (English)

    Da SHI; Yin-xiu JIN; Yi-hong TANG; Hai-hong HU; Si-yun XU; Lu-shanYU; Hui-di JIANG; Su ZENG

    2012-01-01

    To investigate the stereoselective binding of mexiletine or ketoprofen enantiomers with different recombinant domains of human serum albumin (HSA).Methods:Three domains (HSA DOM Ⅰ,Ⅱ and Ⅲ) were expressed in Pichia pastoris GS115 cells.Blue Sepharose 6 Fast Flow was employed to purify the recombinant HSA domains.The binding properties of the standard ligands,digitoxin,phenylbutazone and diazepam,and the chiral drugs to HSA domains were investigated using ultrafiltration.The concentrations of the standard ligands,ketoprofen and mexiletine were analyzed with HPLC.Results:The recombinant HSA domains were highly purified as shown by SDS-PAGE and Western blotting analyses,The standard HSA ligands digitoxin,phenylbutazone and diazepam selectively binds to DOM Ⅰ,DOM Ⅱ and DOM Ⅲ,respectively.For the chiral drugs,R-ketoprofen showed a higher binding affinity toward DOM Ⅲ than S-ketoprofen,whereas S-mexiletine bound to DOM Ⅱ with a greater affinity than R-mexiletine.Conclusion:The results demonstrate that HSA DOM Ⅲ possesses the chiral recognition ability for the ketoprofen enantiomers,whereas HSA DOM Ⅱ possesses that for the mexiletine enantiomers.

  5. Stabilization of Human Serum Albumin against Urea Denaturation by Diazepam and Ketoprofen.

    Science.gov (United States)

    Manoharan, Pralad; Wong, Yin H; Tayyab, Saad

    2015-01-01

    Stabilizing effect of diazepam and ketoprofen, Sudlow's site II markers on human serum albumin (HSA) against urea denaturation was studied using fluorescence spectroscopy. The two-step, three-state urea transition of HSA was transformed into a single-step, two-state transition with the abolishment of the intermediate state along with a shift of the transition curve towards higher urea concentrations in the presence of diazepam or ketoprofen. Interestingly, a greater shift in the transition curve of HSA was observed in the presence of ketoprofen compared to diazepam. A comparison of the intrinsic fluorescence and three-dimensional fluorescence spectra of HSA and partially-denatured HSAs, obtained in the absence and the presence of diazepam or ketoprofen suggested significant retention of native-like conformation in the partially-denatured states of HSA in the presence of Sudlow's site II markers. Taken together, all these results suggested stabilization of HSA in the presence of diazepam or ketoprofen, being greater in the presence of ketoprofen.

  6. Multidrug Delivery Systems Based on Human Serum Albumin for Combination Therapy with Three Anticancer Agents.

    Science.gov (United States)

    Qi, Jinxu; Zhang, Yao; Gou, Yi; Lee, Philbert; Wang, Jun; Chen, Shifang; Zhou, Zuping; Wu, Xiaoyang; Yang, Feng; Liang, Hong

    2016-09-06

    When administering several anticancer drugs within a single carrier, it is important to regulate their spatial distribution so as to avoid possible mutual interference and to thus enhance the drugs' selectivity and efficiency. To achieve this, we proposed to develop human serum albumin (HSA)-based multidrug delivery systems for combination anticancer therapy. We used three anticancer agents (an organic drug [5-fluorouracil, or 5FU], a metallic agent [2-benzoylpyridine thiosemicarbazide copper II, or BpT], and a gene agent [AS1411]) to treat liver cancer and confirm our hypothesis. The structure of the HSA-palmitic acid (PA)-5FU-BpT complex revealed that 5FU and BpT, respectively, bind to the IB and IIA subdomains of HSA. Our MALDI-TOF-MS spectral data show that one AS1411 molecule is conjugated to Cys-34 of the HSA-5FU-BpT complex via a linker. Compared with unregulated three-drug combination therapy, the HSA-5FU-BpT-AS1411 complex enhances cytotoxicity in Bel-7402 cells approximately 7-fold in vitro; however, in normal cells it does not raise cytotoxicity levels. Importantly, our in vivo results demonstrate that the HSA-5FU-BpT-AS1411 complex is superior to the unregulated three-drug combination in enhancing targeting ability, inhibiting liver tumor growth, and causing fewer side effects.

  7. Structural characteristics of green tea catechins for formation of protein carbonyl in human serum albumin.

    Science.gov (United States)

    Ishii, Takeshi; Mori, Taiki; Ichikawa, Tatsuya; Kaku, Maiko; Kusaka, Koji; Uekusa, Yoshinori; Akagawa, Mitsugu; Aihara, Yoshiyuki; Furuta, Takumi; Wakimoto, Toshiyuki; Kan, Toshiyuki; Nakayama, Tsutomu

    2010-07-15

    Catechins are polyphenolic antioxidants found in green tea leaves. Recent studies have reported that various polyphenolic compounds, including catechins, cause protein carbonyl formation in proteins via their pro-oxidant actions. In this study, we evaluate the formation of protein carbonyl in human serum albumin (HSA) by tea catechins and investigate the relationship between catechin chemical structure and its pro-oxidant property. To assess the formation of protein carbonyl in HSA, HSA was incubated with four individual catechins under physiological conditions to generate biotin-LC-hydrazide labeled protein carbonyls. Comparison of catechins using Western blotting revealed that the formation of protein carbonyl in HSA was higher for pyrogallol-type catechins than the corresponding catechol-type catechins. In addition, the formation of protein carbonyl was also found to be higher for the catechins having a galloyl group than the corresponding catechins lacking a galloyl group. The importance of the pyrogallol structural motif in the B-ring and the galloyl group was confirmed using methylated catechins and phenolic acids. These results indicate that the most important structural element contributing to the formation of protein carbonyl in HSA by tea catechins is the pyrogallol structural motif in the B-ring, followed by the galloyl group. The oxidation stability and binding affinity of tea catechins with proteins are responsible for the formation of protein carbonyl, and consequently the difference in these properties of each catechin may contribute to the magnitude of their biological activities.

  8. Measurement of selected halogenated contaminants in human serum and milk using GCxGC-IDTOFMS

    Energy Technology Data Exchange (ETDEWEB)

    Focant, J. [Liege Univ. (Belgium); Sjoedin, A.; Turner, W.; Patterson, D. [Centers for Disease Control and Prevention, Atlanta, GA (United States)

    2004-09-15

    A new method using comprehensive two-dimensional gas chromatography and isotope dilution time-of-flight mass spectrometry (GCxGC-IDTOFMS) for the simultaneous measurement of selected polychlorinated biphenyls (PCBs), organochlorine pesticides (OCPs), and brominated flame retardants (BFRs) is presented. Conversely to reference methods based on classical GC-MS, a single injection of the extract containing all species of interest conducts to accurate identification and quantification. GCxGC ensures the chromatographic separation of most compounds and TOFMS allows mass spectral deconvolution of co-eluting compounds as well as the use of {sup 13}C-labeled internal standards for quantification. Isotope ratio measurements of the most intense ions for both natives and labels ensure the required specificity. Potentially interfering matrix compounds are usually kept away from the compounds to be measured in the chromatographic space. The use of this new method with automated sample preparation procedures developed at the Centers for Disease Control and Prevention (CDC) for the analysis of human serum and milk compared favorably to conventional isotope-dilution one-dimensional gas chromatography-high resolution mass spectrometry (GC-IDHRMS) for the different sample pools that were tested.

  9. In vitro stereoselective covalent binding of carprofen glucuronides to human serum albumin: characterization of the mechanism.

    Science.gov (United States)

    Greige-Georges, Hélène; Buronfosse, Thierry; Netter, Patrcik; Magdalou, Jacques; Lapicque, Françoise

    2003-01-01

    The reactivity, in terms of covalent binding, of R- and S-carprofen acylglucuronides with human serum albumin (HSA) has been investigated in vitro. The irreversible binding of these metabolites to the HSA 580 mM occurred at pH 7.4 and 37 degrees C instantaneously and stereoselectively in favour of the R-enentiomer glucuronide. The amount of carprofen adducts remained stable with time up to 48 hr, and increased with the glucuronide concentration. It was not modified by addiction of imine-trapping reagents, suggesting that the reaction is not mediated by a Schiff base mechanism. Moreover the extreme rapidity of the covalent binding supports a mechanism of nucleophilic attack. Competition studies with ligands known to bind to different sites of HSA, indicated that carprofen glucuronides interacted mainly with site II. The extent of the binding of R-carprofen glucuronide increased with pH, thus suggesting the participation of an alkaline group in the process. The modification of HSA by amino-acid directed chemicals led to the conclusion that Tyr, Lys or Arg residues in site II were mainly involved.

  10. Enhancing the copper(II) complexes cytotoxicity to cancer cells through bound to human serum albumin.

    Science.gov (United States)

    Gou, Yi; Zhang, Yao; Qi, Jinxu; Zhou, Zuping; Yang, Feng; Liang, Hong

    2015-03-01

    We use Schiff-base salicylaldehyde benzoylhydrazone (HL) as the ligand for copper(II), resulting in the complexes [CuCl(L)]·H2O (C1), [CuNO3(L)]·H2O (C2) and [CuBr(L)]2 (C3). We characterize the Cu(II) compounds' interactions with human serum albumin (HSA) using fluorescence spectroscopy and molecular docking. These studies revealed that Cu(II) compounds propensity bound to IIA subdomain of HSA possible by hydrophobic interactions and hydrogen bond. Cu(II) compounds produce intracellular reactive oxygen species (ROS) in cancer cells. Complexes of HSA and copper(II) compounds enhance about 2-fold cytotoxicity in cancer cells but do not raise cytotoxicity levels in normal cells in vitro. Compared with C3 alone, HSA-C3 complex promotes HepG2 cell apoptosis and has a stronger capacity to promote cell cycle arrest at the G2/M phase of HepG2.

  11. Comparison of Posttranslational Modification and the Functional Impairment of Human Serum Albumin in Commercial Preparations.

    Science.gov (United States)

    Miyamura, Shigeyuki; Imafuku, Tadashi; Anraku, Makoto; Taguchi, Kazuaki; Yamasaki, Keishi; Tominaga, Yuna; Maeda, Hitoshi; Ishima, Yu; Watanabe, Hiroshi; Otagiri, Masaki; Maruyama, Toru

    2016-03-01

    On account of its long circulating half-life, human serum albumin (HSA) is susceptible to posttranslational modifications that can alter its functions. Here, we comprehensively compared the degree of posttranslational modifications with the functional impairment of HSA derived from 5 different commercially available albumin preparations and clarified their relationships. We used electrospray ionization-time of flight mass spectrometry to evaluate the degree of posttranslational modification of the entire HSA molecule that was associated with disease development and found that the fraction of Cys34-cysteinylated HSA (Cys-Cys34-HSA), a major form of oxidative modification, varied substantially among the albumin preparations. Meanwhile, no remarkable difference was found in the degree of glycated or N-terminal truncated HSA among the preparations tested. The nonosmotic pressure maintenance functions of HSA, such as its antioxidative and ligand-binding activities significantly differed among the preparations. Interestingly, the alternations of these functions showed a significantly negative correlation only with the Cys-Cys34-HSA fraction. These findings suggest that the Cys-Cys34-HSA fraction, as estimated by electrospray ionization-time of flight mass spectrometry can be used as a predictive marker for the functional impairment of albumin preparations and that it would be preferable to use albumin preparations with higher contents of functionally effective albumin that correspond to a lower degree of cysteinylation of Cys34 in clinical practice.

  12. Characterization of Gallic Acid Interaction with Human Serum Albumin by Spectral and Molecular Modeling Methods

    Institute of Scientific and Technical Information of China (English)

    LIU Zuo-jia; LI Dan; NIU Feng-lan

    2012-01-01

    The binding of drugs with human serum albumin(HSA)is a crucial factor influencing the distribution and bioactivity of drugs in the body.To understand the action mechanisms between gallic acid(GA,3,4,5-trihydroxybenzoic acid)and HSA,the binding of GA with HSA was investigated by a combined experimental and computational approach.The fluorescence properties of HSA and the binding parameters of GA collectively indicate that the binding is characterized by static quenching mechanism at one high affinity binding site.According to the estimated molecular distance between the donor(HSA)and the acceptor(GA),the binding is related to the fluorescence resonance energy transfer.As indicated by the thermodynamic parameters,hydrophobic interaction plays a major role in the GA-HSA complex.Further,the experimental results reveal that GA is bound in the large hydrophobic cavity of subdomain ⅡA in the site Ⅰ of HSA,which is well approved by molecular docking.

  13. Free insulin-like growth factors (IGF-I and IGF-II) in human serum.

    Science.gov (United States)

    Frystyk, J; Skjaerbaek, C; Dinesen, B; Orskov, H

    1994-07-11

    Using ultrafiltration by centrifugation we have isolated the free, unbound fractions of insulin-like growth factor I and II (free IGF-I and IGF-II) in human serum. In this way near in vivo conditions could be maintained before and during isolation. The recovery was 80 to 100% in the ultrafiltrates, which contained no detectable amounts of IGF-binding proteins (IGFBPs) as measured by Western ligand blotting and IGFBP-1 and IGFBP-3 immunoassays. The concentration of free peptides was measured in two ultrasensitive non-competitive IGF-I and IGF-II time-resolved fluoroimmunoassays. We found that (i) equilibrium between free and protein-complexed IGF was strongly dependent on re-establishment of in vivo conditions (temperature, pH, ionic milieu and dilution); (ii) metabolic events (glucose load and fasting) caused significant changes in free IGF-I and IGF-II levels without concomitant changes in total circulating levels of IGFs; (iii) in 49 healthy adult subjects (20 to above 60 years) free IGF-I was inversely related to age and ranged from 950 +/- 150 ng/l (mean +/- S.E.M.) (20-30 years) to 410 +/- 70 ng/l (> 60 years). The relative percentage was, however, unchanged, being 0.38 +/- 0.02% of total IGF-I. In contrast, free IGF-II was independent of age, being 1,480 +/- 80 ng/l (approximately 0.20 +/- 0.01% of total IGF-II).

  14. Comparative Studies of Interactions between Fluorodihydroquinazolin Derivatives and Human Serum Albumin with Fluorescence Spectroscopy

    Directory of Open Access Journals (Sweden)

    Yi Wang

    2016-10-01

    Full Text Available In the present study, 3-(fluorobenzylideneamino-6-chloro-1-(3,3-dimethylbutanoyl-phenyl-2,3-dihydroquinazolin-4(1H-one (FDQL derivatives have been designed and synthesized to study the interaction between fluorine substituted dihydroquinazoline derivatives with human serum albumin (HSA using fluorescence, circular dichroism and Fourier transform infrared spectroscopy. The results indicated that the FDQL could bind to HSA, induce conformation and the secondary structure changes of HSA, and quench the intrinsic fluorescence of HSA through a static quenching mechanism. The thermodynamic parameters, ΔH, ΔS, and ΔG, calculated at different temperatures, revealed that the binding was through spontaneous and hydrophobic forces and thus played major roles in the association. Based on the number of binding sites, it was considered that one molecule of FDQL could bind to a single site of HSA. Site marker competition experiments indicated that the reactive site of HSA to FDQL mainly located in site II (subdomain IIIA. The substitution by fluorine in the benzene ring could increase the interactions between FDQL and HSA to some extent in the proper temperature range through hydrophobic effect, and the substitution at meta-position enhanced the affinity greater than that at para- and ortho-positions.

  15. Interaction of polyphenolic metabolites with human serum albumin: a circular dichroism study.

    Science.gov (United States)

    Nozaki, Akiko; Kimura, Toshikiro; Ito, Hideyuki; Hatano, Tsutomu

    2009-09-01

    Binding sites of polyphenolic compounds on human serum albumin (HSA) were investigated using induced Cotton effects on the circular dichroism (CD) spectra. Polyphenolic compounds used in this study are known to be metabolites from tannins and their related polyphenols in food and medicinal plants. The present investigation revealed that the structural differences markedly affected the binding of the compounds to HSA. Protocatechuic acid, together with its methylated compounds vanillic and isovanillic acids, were assigned to be bound to sites I and II of HSA, based on the competitive relationships with site-I-binding phenylbutazone (PB) and site-II-binding diazepam (DP). 4-O-Methylgallic acid, which is the metabolite from gallic acid, was bound to site I on HSA, while gallic acid did not affect the binding of PB and DP at the concentration examined. Neither ellagic acid nor its metabolite urolithin A was competitive with PB and DP on HSA. The addition of digitoxin did not affect the induced CD of the polyphenolic acids examined.

  16. The investigation of interaction between Thioguanine and human serum albumin by fluorescence and modeling

    Directory of Open Access Journals (Sweden)

    Xin An

    2017-05-01

    Full Text Available The interaction between Thioguanine (6-TG and human serum albumin (HSA under simulative physiological conditions was studied using fluorescence spectroscopy in combination with UV absorption and molecular modeling method. A strong fluorescence quenching reaction of 6-TG to HSA was observed and the quenching mechanism was suggested as static quenching according to the Stern–Volmer equation. The binding constants (K at different temperatures as well as thermodynamic parameters, enthalpy change (ΔH and entropy change (ΔS, were calculated according to relevant fluorescent data and thermodynamic equation. It was indicated that the hydrophobic interaction was a predominant intermolecular force in order to stabilize the copolymer, which was in agreement with the results of molecular modeling study. In addition, the binding distance between 6-TG and the tryptophan residue of HSA was studied according to Föster’s non-radiative energy transfer theory and the effects of common ions on the binding constant of 6-TG-HSA copolymer were also discussed at room temperature.

  17. A Rapid and Sensitive Method to Measure the Functional Activity of Shiga Toxins in Human Serum

    Directory of Open Access Journals (Sweden)

    Valentina Arfilli

    2015-11-01

    Full Text Available Shiga toxins (Stx have a definite role in the development of hemolytic uremic syndrome in children with hemorrhagic colitis caused by pathogenic Stx-producing Escherichia coli (STEC strains. The dramatic effects of these toxins on the microvasculature of different organs, particularly of the kidney, are well known, whereas there is no consensus on the mechanism by which Stx reach the endothelia of target organs and/or indirectly injure these body sites. We hereby describe a quick (4 h, radioactive, Raji cell-based method designed for the detection of Stx in human sera. The assay monitors the translation impairment induced by these powerful inhibitors of protein synthesis, which are identified properly by neutralizing their activity with specific monoclonal antibodies. By this method, we detected for the first time the functional activity of Stx in sera of STEC-infected patients during hemorrhagic colitis. Recent research has pointed to a dynamic process of Stx-induced renal intoxication in which concurrent and interactive steps are involved. Our rapid and specific method could be useful for studying the kinetics of Stx during the natural course of STEC infection and the interplay between Stx activity in serum and Stx presence in different blood fractions (neutrophils, monocytes, platelets, leukocyte-platelet aggregates, microvesicles, lipoproteins.

  18. A Rapid and Sensitive Method to Measure the Functional Activity of Shiga Toxins in Human Serum

    Science.gov (United States)

    Arfilli, Valentina; Carnicelli, Domenica; Ardissino, Gianluigi; Torresani, Erminio; Scavia, Gaia; Brigotti, Maurizio

    2015-01-01

    Shiga toxins (Stx) have a definite role in the development of hemolytic uremic syndrome in children with hemorrhagic colitis caused by pathogenic Stx-producing Escherichia coli (STEC) strains. The dramatic effects of these toxins on the microvasculature of different organs, particularly of the kidney, are well known, whereas there is no consensus on the mechanism by which Stx reach the endothelia of target organs and/or indirectly injure these body sites. We hereby describe a quick (4 h), radioactive, Raji cell-based method designed for the detection of Stx in human sera. The assay monitors the translation impairment induced by these powerful inhibitors of protein synthesis, which are identified properly by neutralizing their activity with specific monoclonal antibodies. By this method, we detected for the first time the functional activity of Stx in sera of STEC-infected patients during hemorrhagic colitis. Recent research has pointed to a dynamic process of Stx-induced renal intoxication in which concurrent and interactive steps are involved. Our rapid and specific method could be useful for studying the kinetics of Stx during the natural course of STEC infection and the interplay between Stx activity in serum and Stx presence in different blood fractions (neutrophils, monocytes, platelets, leukocyte-platelet aggregates, microvesicles, lipoproteins). PMID:26556372

  19. Quantitation of the residual DNA from rice-derived recombinant human serum albumin.

    Science.gov (United States)

    Chen, Zhen; Dai, Huixia; Liu, Zhenwei; Zhang, Liping; Pang, Jianlei; Ou, Jiquan; Yang, Daichang

    2014-04-01

    Residual DNA in recombinant protein pharmaceuticals can potentially cause safety issues in clinical applications; thus, maximum residual limit has been established by drug safety authorities. Assays for residual DNA in Escherichia coli, yeast, and Chinese hamster ovary (CHO) cell expression systems have been established, but no rice residual DNA assay for rice expression systems has been designed. To develop an assay for the quantification of residual DNA that is produced from rice seed, we established a sensitive assay using quantitative real-time polymerase chain reaction (qPCR) based on the 5S ribosomal RNA (rRNA) genes. We found that a 40-cycle qPCR exhibited a linear response when the template concentration was in the range of 2×10(4) to 0.2pg of DNA per reaction in TaqMan and SYBR Green I assays. The amplification efficiency was 103 to 104%, and the amount of residual DNA from recombinant human serum albumin from Oryza sativa (OsrHSA) was less than 3.8ng per dosage, which was lower than that recommended by the World Health Organization (WHO). Our results indicate that the current purification protocol could efficiently remove residual DNA during manufacturing and processing. Furthermore, this protocol could be viable in other cereal crop endosperm expression systems for developing a residual DNA quantitation assay using the highly conserved 5S rRNA gene of the crops.

  20. Monte carlo method-based QSAR modeling of penicillins binding to human serum proteins.

    Science.gov (United States)

    Veselinović, Jovana B; Toropov, Andrey A; Toropova, Alla P; Nikolić, Goran M; Veselinović, Aleksandar M

    2015-01-01

    The binding of penicillins to human serum proteins was modeled with optimal descriptors based on the Simplified Molecular Input-Line Entry System (SMILES). The concentrations of protein-bound drug for 87 penicillins expressed as percentage of the total plasma concentration were used as experimental data. The Monte Carlo method was used as a computational tool to build up the quantitative structure-activity relationship (QSAR) model for penicillins binding to plasma proteins. One random data split into training, test and validation set was examined. The calculated QSAR model had the following statistical parameters: r(2)  = 0.8760, q(2)  = 0.8665, s = 8.94 for the training set and r(2)  = 0.9812, q(2)  = 0.9753, s = 7.31 for the test set. For the validation set, the statistical parameters were r(2)  = 0.727 and s = 12.52, but after removing the three worst outliers, the statistical parameters improved to r(2)  = 0.921 and s = 7.18. SMILES-based molecular fragments (structural indicators) responsible for the increase and decrease of penicillins binding to plasma proteins were identified. The possibility of using these results for the computer-aided design of new penicillins with desired binding properties is presented. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  1. Electrochemical oxidation of amphetamine-like drugs and application to electroanalysis of ecstasy in human serum.

    Science.gov (United States)

    Garrido, E M P J; Garrido, J M P J; Milhazes, N; Borges, F; Oliveira-Brett, A M

    2010-08-01

    Amphetamine and amphetamine-like drugs are popular recreational drugs of abuse because they are powerful stimulants of the central nervous system. Due to a dramatic increase in the abuse of methylenedioxylated derivatives, individually and/or in a mixture, and to the incoherent and contradictory interpretation of the electrochemical data available on this subject, a comprehensive study of the redox properties of amphetamine-like drugs was accomplished. The oxidative behaviour of amphetamine (A), methamphetamine (MA), methylenedioxyamphetamine (MDA) and methylenedioxymethamphetamine (MDMA) was studied in different buffer systems by cyclic, differential pulse and square-wave voltammetry using a glassy carbon electrode. A quantitative electroanalytical method was developed and successfully applied to the determination of MDMA in seized samples and in human serum. Validation parameters, such as sensitivity, precision and accuracy, were evaluated. The results found using the developed electroanalytical methodology enabled to gather some information about the content and amount of MDMA present in ecstasy tablets found in Portugal. Moreover, the data found in this study outlook the possibility of using the voltammetric methods to investigate the potential harmful effects of interaction between drugs such as MDMA and methamphetamine and other substances often used together in ecstasy tablets.

  2. Noncovalent interactions between a trinuclear monofunctional platinum complex and human serum albumin.

    Science.gov (United States)

    Wang, Yanqing; Wang, Xiaoyong; Wang, Jing; Zhao, Yongmei; He, Weijiang; Guo, Zijian

    2011-12-19

    Interactions between platinum complexes and human serum albumin (HSA) play crucial roles in the metabolism, distribution, and efficacy of platinum-based anticancer drugs. Polynuclear monofunctional platinum(II) complexes represent a new class of anticancer agents that display distinct molecular characters of pharmacological action from those of cisplatin. In this study, the interaction between a trinuclear monofunctional platinum(II) complex, [Pt(3)LCl(3)](ClO(4))(3) (L = N,N,N',N',N",N"-hexakis(2-pyridylmethyl)-1,3,5-tris(aminomethyl)benzene) (1), and HSA was investigated using ultraviolet-visible spectroscopy, Fourier transform infrared spectroscopy, circular dichroism spectroscopy, fluorescence spectroscopy, molecular docking, and inductively coupled plasma mass spectrometry. The spectroscopic and thermodynamic data show that the interaction is a spontaneous process with the estimated enthalpy and entropy changes being 14.6 kJ mol(-1) and 145.5 J mol(-1) K(-1), respectively. The reactive sites of HSA to complex 1 mainly locate within its hydrophobic cavity in domain II. Noncovalent actions such as π-π stacking and hydrophobic bonding are the primary contributors to the interaction between HSA and complex 1, which is different from the scenario for cisplatin in similar conditions. The results suggest that the connection between complex 1 and HSA is reversible, and therefore the cytotoxic activity of the complex could be preserved during blood circulation.

  3. Simple, fast preparation of gallium-68-labelled human serum albumin microspheres.

    Science.gov (United States)

    Yvert, J P; Mazière, B; Verhas, M; Comar, D

    1979-04-01

    Following a study of the main factors involved in the 68-Ga labelling of human serum albumin microspheres (H.S.A.M.), especially methods of production and preparation of active solution and conditions of radioelement fixation on the protein support, the practical details of a fast technique (60 min) based on the process described by Hnatowich are presented. This method gives high labelling yields (93 +/- 3%), and after washing of the microspheres leads to a radiopharmaceutical product almost without free 68Ga (less than 2%). The spheres ready for use carry a total radioactivity corresponding to about 35%, including decay, of the activity originally recovered in the generator eluate and to more than 98% of that, found in the final suspension. The labelled product is sterile, non-pyrogenic and non-toxic. When it is injected in animals by left ventrical catheterization the uptake rates in the heart, lungs, spleen, left kidney and right kidney are similar to those observed with reference 85Sr-labelled carbonized microspheres. This radiopharmaceutical, easy to prepare and having excellent biological and nuclear properties, seems ideally suited for the scanning of organs by position emission tomoscintigraphy.

  4. Neo-epitopes on methylglyoxal modified human serum albumin lead to aggressive autoimmune response in diabetes.

    Science.gov (United States)

    Jyoti; Mir, Abdul Rouf; Habib, Safia; Siddiqui, Sheelu Shafiq; Ali, Asif; Moinuddin

    2016-05-01

    Glyco-oxidation of proteins has implications in the progression of diabetes type 2. Human serum albumin is prone to glyco-oxidative attack by sugars and methylglyoxal being a strong glycating agent may have severe impact on its structure and consequent role in diabetes. This study has probed the methylglyoxal mediated modifications of HSA, the alterations in its immunological characteristics and possible role in autoantibody induction. We observed an exposure of chromophoric groups, loss in the fluorescence intensity, generation of AGEs, formation of cross-linked products, decrease in α-helical content, increase in hydrophobic clusters, FTIR band shift, attachment of methylglyoxal to HSA and the formation of N(ε)-(carboxyethyl) lysine in the modified HSA, when compared to the native albumin. MG-HSA was found to be highly immunogenic with additional immunogenicity invoking a highly specific immune response than its native counterpart. The binding characteristics of circulating autoantibodies in type 2 diabetes mellitus (DM) patients showed the generation of anti-MG-HSA auto-antibodies in the these patients, that are preferentially recognized by the modified albumin. We propose that MG induced structural perturbations in HSA, result in the generation of neo-epitopes leading to an aggressive auto-immune response and may contribute to the immunopathogenesis of diabetes type 2 associated complications.

  5. Spectroscopic and calorimetric studies of interaction of methimazole with human serum albumin

    Energy Technology Data Exchange (ETDEWEB)

    Afrin, Sadaf [Department of Chemistry, Aligarh Muslim University, Aligarh 202002, Uttar Pradesh (India); Riyazuddeen, E-mail: rz1@rediffmail.com [Department of Chemistry, Aligarh Muslim University, Aligarh 202002, Uttar Pradesh (India); Rabbani, Gulam; Khan, Rizwan Hasan [Interdisciplinary Biotechnology Unit, Aligarh Muslim University, Aligarh 202002, Uttar Pradesh (India)

    2014-07-01

    The interaction of the anti-thyroid drug, 2-mercapto 1-methylimidazole (methimazole) with human serum albumin (HSA) has been examined by fluorescence and isothermal titration calorimetry (ITC) techniques. Fluorescence results indicate that in case of HSA–drug complex the quenching of fluorescence intensity is at 340 nm. The methimazole has an ability to quench the intrinsic fluorescence of HSA tryptophan through a static quenching procedure. The binding constant has been determined using Stern–Volmer modified equation and energy transfer mechanisms of quenching are discussed. The ΔG°, ΔH°, and ΔS° values are also calculated by ITC measurements. The experimental spectroscopic and thermodynamic parameters have been used for understanding the binding mechanism of anti-thyroid drug with HSA. - Highlights: • The binding of methimazole to HSA quenches the intrinsic fluorescence of tryptophan. • The negative ΔG° value suggests the binding of methimazole with HSA is spontaneous. • The main contribution to ΔG° arises from the ΔS° rather than from ΔH°, so hydrophobic forces most likely play a major role in the binding of methimazole to HSA.

  6. Effects of titania nanotubes with or without bovine serum albumin loaded on human gingival fibroblasts.

    Science.gov (United States)

    Liu, Xiangning; Zhou, Xiaosong; Li, Shaobing; Lai, Renfa; Zhou, Zhiying; Zhang, Ye; Zhou, Lei

    2014-01-01

    Modifying the surface of the transmucosal area is a key research area because this process positively affects the three functions of implants: attachment to soft tissue, inhibiting bacterial biofilm adhesion, and the preservation of the crestal bone. To exploit the potential of titania nanotube arrays (TNTs) with or without using bovine serum albumin (BSA) to modify the surface of a dental implant in contact with the transmucosal area, BSA was loaded into TNTs that were fabricated by anodizing Ti sheets; the physical characteristics of these arrays, including their morphology, chemical composition, surface roughness, contact angle, and surface free energy (SFE), were assessed. The effect of Ti surfaces with TNTs or TNTs-BSA on human gingival fibroblasts (HGFs) was determined by analyzing cell morphology, early adhesion, proliferation, type I collagen (COL-1) gene expression, and the extracellular secretion of COL-1. The results indicate that early HGF adhesion and spreading behavior is positively correlated with surface characteristics, including hydrophilicity, SFE, and surface roughness. Additionally, TNT surfaces not only promoted early HGF adhesion, but also promoted COL-1 secretion. BSA-loaded TNT surfaces promoted early HGF adhesion, while suppressing late proliferation and COL-1 secretion. Therefore, TNT-modified smooth surfaces are expected to be applicable for uses involving the transmucosal area. Further study is required to determine whether BSA-loaded TNT surfaces actually affect closed loop formation of connective tissue because BSA coating actions in vivo are very rapid.

  7. Thermostabilisation of human serum butyrylcholinesterase for detection of its inhibitors in water and biological fluids

    Directory of Open Access Journals (Sweden)

    Lakshmanan Jaganathan

    1999-01-01

    Full Text Available The ability of gelatine-trehalose to convert the normally fragile, dry human serum BChE into a thermostable enzyme and its use in the detection of cholinesterase inhibitors in water and biological fluids is described. Gelatine or trehalose alone is unable to protect the dry enzyme against exposure to high temperature, while a combination of gelatine and trehalose were able to protect the enzyme activity against prolonged exposure to temperature as high as +50°C. A method for rapid, simple and inexpensive means of screening for cholinesterase inhibitors such as carbamates and organophosphates in water, vegetables and human blood has been developed.A capacidade da gelatina-trehalose em converter a frágil BChE do soro humano em uma enzima termoestável e seu uso na descoberta de inibidores de colinesterase em água e fluidos biológicos é apresentado. A Gelatina ou trehalose são incapazes de proteger a enzima seca BchE do soro humano contra exposição a elevadas temperaturas, enquanto que uma combinação de gelatina e trehalose são capazes de proteger a atividade de enzima contra exposição prolongada a temperaturas elevadas e da ordem de 50° C. Um método barato, simples e rápido de screening para inibidores de colinesterase tal como carbamatos e organofosfatos em água, verduras e sangue humano foi desenvolvido.

  8. Interaction of biocompatible natural rosin-based surfactants with human serum albumin: A biophysical study

    Energy Technology Data Exchange (ETDEWEB)

    Ishtikhar, Mohd [Protein Biophysics Laboratory, Interdisciplinary Biotechnology Unit, Aligarh Muslim University, Aligarh 202002 (India); Ali, Mohd Sajid [Surfactant Research Chair, Department of Chemistry, King Saud University, P.O. Box-2455, Riyadh 11451 (Saudi Arabia); Atta, Ayman M. [Surfactant Research Chair, Department of Chemistry, King Saud University, P.O. Box-2455, Riyadh 11451 (Saudi Arabia); Petroleum Application department, Egyptian Petroleum Research Institute, Ahmad Elzomor St., Nasr city, Cairo-11727 (Egypt); Al-Lohedan, H.A. [Surfactant Research Chair, Department of Chemistry, King Saud University, P.O. Box-2455, Riyadh 11451 (Saudi Arabia); Nigam, Lokesh; Subbarao, Naidu [Centre for Computational Biology and Bioinformatics, School of Computational and Integrative Sciences, Jawaharlal Nehru University, New Delhi 110067 (India); Hasan Khan, Rizwan, E-mail: rizwanhkhan@hotmail.com [Protein Biophysics Laboratory, Interdisciplinary Biotechnology Unit, Aligarh Muslim University, Aligarh 202002 (India)

    2015-11-15

    Biophysical insight into interaction of biocompatible rosin-based surfactants with human serum albumin (HSA) was studied at physiological conditions using various spectroscopic, calorimetric and molecular docking approaches. The binding constant (K{sub b}), enthalpy (ΔH{sup 0}), entropy (ΔS{sup 0}) and Gibbs free energy change (ΔG{sup 0}) were calculated by spectroscopic and calorimetric method. We have also calculated the probability of energy transfer by FRET analysis. The circular dichroism study showed that the cationic surfactant QRMAE significantly altered the secondary structure of HSA as compared to the nonionic rosin surfactants. The thermodynamic study was performed by ITC to determine binding constant as well as change in enthalpy of HSA in presence of rosin surfactants. It clearly showed that hydrogen binding and hydrophobic interaction play an important role in the binding of HSA to rosin surfactants. We have also performed molecular docking studies to locate the binding site on HSA and to visualize the mode of interaction. The present study provides a significant insight into HSA–rosin surfactants interaction, which also improves our understanding of the possible effect of rosin surfactants on human health. - Highlights: • RMPEG 750 has the highest Kb, Kq and Ksv value as compared to other rosin surfactants. • The probability of energy transfer from HSA to rosin surfactants was maximum in the case of RMPEG 750. • Cationic surfactant QRMAE significantly altered the secondary structure of the HSA as compared to other rosin surfactants. • Molecular docking and ITC experiment studies, to locate the binding site on HSA and to investigate the mode of interaction.

  9. Physiologically relevant plasma d,l-homocysteine concentrations mobilize Cd from human serum albumin.

    Science.gov (United States)

    Sagmeister, Peter; Gibson, Matthew A; McDade, Kyle H; Gailer, Jürgen

    2016-08-01

    Although low-level chronic exposure of humans to cadmium (Cd(2+)) can result in a variety of adverse health effects, little is known about the role that its interactions with plasma proteins and small molecular weight (SMW) ligands in the bloodstream may play in delivering this metal to its target organs. To gain insight, a Cd-human serum albumin (HSA) 1:1 (molar ratio) complex was analyzed by size exclusion chromatography (SEC) coupled on-line to a flame atomic absorption spectrometer (FAAS). Using a phosphate buffered saline (PBS)-buffer mobile phase, the stability of the Cd-HSA complex was investigated in the presence of 2.0mM of SMW ligands, including taurine, acetaminophen, l-methionine, l-cysteine (Cys), d,l-homocysteine (hCys) or l-cysteine methyl-ester (Cys-Me). While taurine, acetaminophen and l-methionine did not affect its integrity, Cys, hCys and Cys-Me completely abstracted Cd from HSA. Subsequent investigations into the effect of 1.5, 1.0 and 0.5mM Cys and hCys on the integrity of the Cd-HSA complex revealed clear differences with regard to the nature of the eluting SMW-Cd species between these structurally related endogenous thiols. Interestingly, the Cd-specific chromatograms that were obtained for 0.5mM hCys revealed the elution of an apparent mixture of the parent Cd-HSA complex with a significant contribution of a structurally uncharacterized CdxhCysy species. Since this hCys concentration is encountered in blood plasma of hyperhomocysteinemia patients and since previous studies by others have revealed that a SH-containing carrier mediates the uptake of Cd into hepatocytes, our results suggest that plasma hCys may play a role in the toxicologically relevant translocation of Cd from the bloodstream to mammalian target organs.

  10. Pharmacokinetics and effects on serum cholinesterase activities of organophosphorus pesticides acephate and chlorpyrifos in chimeric mice transplanted with human hepatocytes.

    Science.gov (United States)

    Suemizu, Hiroshi; Sota, Shigeto; Kuronuma, Miyuki; Shimizu, Makiko; Yamazaki, Hiroshi

    2014-11-01

    Organophosphorus pesticides acephate and chlorpyrifos in foods have potential to impact human health. The aim of the current study was to investigate the pharmacokinetics of acephate and chlorpyrifos orally administered at lowest-observed-adverse-effect-level doses in chimeric mice transplanted with human hepatocytes. Absorbed acephate and its metabolite methamidophos were detected in serum from wild type mice and chimeric mice orally administered 150mg/kg. Approximately 70% inhibition of cholinesterase was evident in plasma of chimeric mice with humanized liver (which have higher serum cholinesterase activities than wild type mice) 1day after oral administrations of acephate. Adjusted animal biomonitoring equivalents from chimeric mice studies were scaled to human biomonitoring equivalents using known species allometric scaling factors and in vitro metabolic clearance data with a simple physiologically based pharmacokinetic (PBPK) model. Estimated plasma concentrations of acephate and chlorpyrifos in humans were consistent with reported concentrations. Acephate cleared similarly in humans and chimeric mice but accidental/incidental overdose levels of chlorpyrifos cleared (dependent on liver metabolism) more slowly from plasma in humans than it did in mice. The data presented here illustrate how chimeric mice transplanted with human hepatocytes in combination with a simple PBPK model can assist evaluations of toxicological potential of organophosphorus pesticides.

  11. Adsorption of biopolymers human serum albumin and human gamma globulin to well-defined surfaces of self-assembled monolayers

    Science.gov (United States)

    Cregger, Tricia Ann

    The tenacity with which the blood proteins Human Serum Albumin (HSA) and Human Gamma Globulin (HGG) adsorb to a surface modified with a monomolecular coating varies with the packing of the alkyl chains in the coating. The adsorption of proteins onto well-defined surfaces of self-assembled monolayers (SAMs) was studied with X-ray reflectometry (XR), neutron reflectometry (NR), optical reflectometry, and total internal reflection fluorescence (TIRF). NR and XR was used to study adsorption in the absence of flow, while optical reflectometry and TIRF were used to probe the adsorption under flow conditions. In particular, competitive adsorption measurements of binary solutions of HSA, HGG and Fibrinogen (FIB) were performed with TIRE The properties of the surface were varied by altering the alkyl chains' packing density and the chain end functionality of the SAMs. The depth profiles of protein concentration near the adsorbing surface measured by NR were dependent upon the chain packing density in the case of HSA. The concentration depth profile of HGG was unaltered by varying chain packing density. Measurements performed under flow using optical reflectometry showed a different behavior: the surface excess of adsorbed HSA was relatively independent of the surface packing, while the surface excess of HGG depended on the packing density of the SAM. The tenacity with which the proteins adsorbed to different functionalized surfaces was determined by attempting to remove the protein using a strong surfactant, sodium dodecyl sulfate (SDS). Ex situ XR measurements suggested that both HSA and HGG adsorb more tenaciously to a less densely-packed monolayer, almost independent of surface functionality. Two exceptions were a less densely-packed vinyl-terminated monolayer and a less densely-packed bromine-terminated monolayer, from which HSA could not be removed at all.

  12. Differential binding of thyroxine and triiodothyronine to acidic isoforms of thyroid hormone binding globulin in human serum

    Energy Technology Data Exchange (ETDEWEB)

    Terasaki, T.; Pardridge, W.M.

    1988-05-17

    The differential availability of thyroxine (T/sub 4/) and 3,5,3'-triiodothyronine (T/sub 3/) to liver from the circulating thyroid hormone binding globulin (TBG)-bound pool suggests that the two thyroid hormones may bind to different TBG isoforms in human serum. In the present study, the binding of (/sup 125/I)T/sub 4/ and (/sup 125/I)T/sub 3/ to human serum proteins was investigated by using slab gel isoelectric focusing and chromatofocusing. In normal human male serum, (/sup 125/I)T/sub 4/ was localized to four isoforms of TBG called TBG-I, -II, -III, and -IV, with isoelectric points (pI's) of 4.30, 4.35, 4.45, and 4.55, respectively. (/sup 125/I)T/sub 3/ was localized to only two isoforms of TBG, TBG-III, and -IV, with pI's that were identical with those for (/sup 125/I)T/sub 4/. In normal female serum, (/sup 125/I)T/sub 4/ was localized to the same four isoforms of TBG as those of normal male serum, while (/sup 125/I)T/sub 3/ was localized to TBG-II, -III, -IV, and -V (pI = 4.65). In pregnant female serum, (/sup 125/I)T/sub 4/ was localized to five isoforms, whereas (/sup 125/I)T/sub 3/ was localized to four. IEF was also performed with male serum loaded with various concentrations of unlabeled T/sub 3/. The K/sub i/ values of T/sub 3/ binding to TBG-I, -II, -III, and -IV were 5.0, 2.4, 0.86, and 0.46 nM, respectively. The TBG isoforms in normal male serum were also separated by sequential concanavalin A-Sepharose affinity chromatography and the chromatofocusing (pH range of 3.5-5.0). T/sub 4/ preferentially bound to the most acidic isoforms of TBG in the pI range of 3.8-4.0, whereas the less acidic fractions (pH 4.0-4.2) bound both T/sub 4/ and T/sub 3/. In conclusion, this study shows that T/sub 4/ and T/sub 3/ do not bind to a single competitive binding site on TBG. Instead, T/sub 4/ is preferentially bound by the most acidic TBG isoforms owing to a 10-fold lower affinity of T/sub 3/ for these proteins.

  13. Maternal serum human placental growth hormone at 11 to 13 weeks in trisomy 21 and trisomy 18 pregnancies.

    Science.gov (United States)

    Sifakis, Stavros; Akolekar, Ranjit; Syngelaki, Argyro; De Cruz, Jader; Nicolaides, Kypros H

    2010-03-01

    To investigate the maternal serum concentration of human placental growth hormone (hPGH) in trisomy 21 and trisomy 18 pregnancies at 11 to 13 weeks of gestation and to examine the possible association between fetal nuchal translucency (NT) thickness and maternal serum free beta-human chorionic gonadotrophin (beta-hCG) and pregnancy-associated plasma protein-A (PAPP-A). The maternal serum concentration of hPGH at 11 to 13 weeks was measured in a case-control study from 28 pregnancies with fetal trisomy 21, 28 with trisomy 18 and 112 pregnancies with euploid fetuses. The median hPGH multiple of the median (MoM) in trisomy 21 and trisomy 18 pregnancies were compared with euploid pregnancies. Serum hPGH was significantly lower in trisomy 21 (0.93 MoM) and trisomy 18 (0.62 MoM) compared to euploid pregnancies (1.02 MoM). There was a significant association between serum hPGH and PAPP-A in both the euploid (r = 0.258, p = 0.006) and trisomy 21 pregnancies (r = 0.410, p = 0.030) but not in trisomy 18 pregnancies (p = 0.445). In the first trimester, serum hPGH in trisomy 21 and trisomy 18 pregnancies is reduced. This is the opposite of findings in previous studies reporting that in the second trimester, trisomy 21 and 18 pregnancies have increased hPGH. Copyright (c) 2010 John Wiley & Sons, Ltd.

  14. Effect of dietary cis and trans fatty acids on serum lipoprotein(a) levels in humans.

    NARCIS (Netherlands)

    Mensink, R.P.; Zock, P.L.; Katan, M.B.; Hornstra, G.

    1992-01-01

    Serum lipoprotein[a] (Lp[a]) is a strong risk factor for coronary heart disease. We therefore examined the effect of dietary fatty acid composition on serum Lp[a] levels in three strictly controlled experiments with healthy normocholesterolemic men and women. In Expt. I, 58 subjects consumed a contr

  15. Serum From Advanced Heart Failure Patients Promotes Angiogenic Sprouting and Affects the Notch Pathway in Human Endothelial Cells.

    Science.gov (United States)

    Pannella, Micaela; Caliceti, Cristiana; Fortini, Francesca; Aquila, Giorgio; Vieceli Dalla Sega, Francesco; Pannuti, Antonio; Fortini, Cinzia; Morelli, Marco Bruno; Fucili, Alessandro; Francolini, Gloria; Voltan, Rebecca; Secchiero, Paola; Dinelli, Giovanni; Leoncini, Emanuela; Ferracin, Manuela; Hrelia, Silvana; Miele, Lucio; Rizzo, Paola

    2016-12-01

    It is unknown whether components present in heart failure (HF) patients' serum provide an angiogenic stimulus. We sought to determine whether serum from HF patients affects angiogenesis and its major modulator, the Notch pathway, in human umbilical vein endothelial cells (HUVECs). In cells treated with serum from healthy subjects or from patients at different HF stage we determined: (1) Sprouting angiogenesis, by measuring cells network (closed tubes) in collagen gel. (2) Protein levels of Notch receptors 1, 2, 4, and ligands Jagged1, Delta-like4. We found a higher number of closed tubes in HUVECs treated with advanced HF patients serum in comparison with cells treated with serum from mild HF patients or controls. Furthermore, as indicated by the reduction of the active form of Notch4 (N4IC) and of Jagged1, advanced HF patients serum inhibited Notch signalling in HUVECs in comparison with mild HF patients' serum and controls. The circulating levels of NT-proBNP (N-terminal of the pro-hormone brain natriuretic peptide), a marker for the detection and evalutation of HF, were positively correlated with the number of closed tubes (r = 0.485) and negatively with Notch4IC and Jagged1 levels in sera-treated cells (r = -0.526 and r = -0.604, respectively). In conclusion, we found that sera from advanced HF patients promote sprouting angiogenesis and dysregulate Notch signaling in HUVECs. Our study provides in vitro evidence of an angiogenic stimulus arising during HF progression and suggests a role for the Notch pathway in it. J. Cell. Physiol. 231: 2700-2710, 2016. © 2016 Wiley Periodicals, Inc.

  16. Quantitative comparison of lipoprotein fractions derived from human plasma and serum by liquid chromatography-tandem mass spectrometry

    Directory of Open Access Journals (Sweden)

    Collins Lisamarie A

    2010-07-01

    Full Text Available Abstract Background Lipoproteins are complex, globular molecules which play essential roles in the transport and metabolism of cholesterol. Their implication in the development of cardiovascular diseases, such as atherosclerosis, has sustained a great deal of interest in these particles. Their various functions, and their contribution to the development of atherosclerosis, are often attributed to their protein constituents, which vary greatly among the different lipoprotein classes. Recent advances in the field of mass spectrometry have provided insight into the array of proteins associated with low-density lipoproteins (LDLs and, even more so, with high-density lipoproteins (HDLs. Plasma and serum are the most commonly used samples for the analysis of lipoproteins. Although these lipoprotein sources are unique, it was our goal to determine whether or not their inherent differences would ultimately affect a quantitative analysis of the LDL and HDL proteomes. To this end, we isolated LDL and HDL fractions with fast protein liquid chromatography-size exclusion chromatography (FPLC-SEC from both human plasma and serum samples from the same individuals. After delipidating these samples, we performed a quantitative proteomic analysis to compare the lipoprotein-associated proteins derived from both plasma and serum. Results Although the primary differences between the samples are found in fibrinogen proteins which are removed from serum, it of interest to note that, with respect to LDL-associated proteins, apolipoproteinB-100 was found at significantly higher levels in serum samples. Complement component 3 was found at significantly higher levels in serum-derived HDL fractions. Both of these proteins are known LDL- and HDL-associated proteins, respectively. Conclusion Overall, the results from our study indicate that both plasma and serum samples are equally suited for proteomic studies, and provide similar results. These findings are particularly

  17. A Preliminary Observation of Weight Loss Following Left Gastric Artery Embolization in Humans

    Directory of Open Access Journals (Sweden)

    Andrew J. Gunn

    2014-01-01

    Full Text Available Background/Objectives. Embolization of the left gastric artery (LGA, which preferentially supplies the gastric fundus, has been shown to produce weight loss in animal models. However, weight loss after LGA embolization in humans has not been previously established. The aim of this study was to evaluate postprocedural weight loss in patients following LGA embolization. Subjects/Methods. A retrospective analysis of the medical records of patients who underwent LGA embolization for upper gastrointestinal (GI bleeding was performed. Postprocedural weight loss in this group was compared to a control group of patients who had undergone embolization of other arteries for upper GI bleeding. Results. The experimental group (N=19 lost an average of 7.3% of their initial body weight within three months of LGA embolization, which was significantly greater than the 2% weight loss observed in the control group (N=28 (P=0.006. No significant differences were seen between the groups in preprocedural body mass index (BMI, age, postprocedural care in the intensive care unit, history of malignancy, serum creatinine, or left ventricular ejection fraction. Conclusions. The current data suggest that body weight in humans may be modulated via LGA embolization. Continued research is warranted with prospective studies to further investigate this phenomenon.

  18. In vito Effect of Dithiocarbamate Pesticides and of CaNa2 EDTA on Human Serum Dopamine-β-hydroxylase

    Institute of Scientific and Technical Information of China (English)

    1995-01-01

    Serum dopamine-β-hydroxylase(DBH)inhibition has been reported in lead workers treated with CaNa2EDTA and in alcoholic patients repatedly treated with the alcohol aversive drug Disulfiram.The mechanism of inhibition involves Cu++ c