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Sample records for human plasma carboxypeptidase

  1. Cloning of cDNAs that encode human mast cell carboxypeptidase A, and comparison of the protein with mouse mast cell carboxypeptidase A and rat pancreatic carboxypeptidases

    SciTech Connect

    Reynolds, D.S.; Gurley, D.S.; Stevens, R.L.; Austen, K.F.; Serafin, W.E. Brigham and Women's Hospital, Boston, MA ); Sugarbaker, D.J. )

    1989-12-01

    Human skin and lung mast cells and rodent peritoneal cells contain a carboxypeptidase in their secretory granules. The authors have screened human lung cDNA libraries with a mouse mast cell carboxypeptidase A (MC-CPA) cDNA probe to isolate a near-full-length cDNA that encodes human MC-CPA. The 5{prime} end of the human MC-CPA transcript was defined by direct mRNA sequencing and by isolation and partial sequencing of the human MC-CPA gene. Human MC-CPA is predicted to be translated as a 417 amino acid preproenzyme which includes a 15 amino acid signal peptide and a 94-amino acid activation peptide. The mature human MC-CPA enzyme has a predicted size of 36.1 kDa, a net positive charge of 16 at neutral pH, and 86% amino acid sequence identity with mouse MC-CPA. DNA blot analyses showed that human MC-CPA mRNA is transcribed from a single locus in the human genome. Comparison of the human MC-CPA with mouse MC-CPA and with three rat pancreatic carboxypeptidases shows that these enzymes are encoded by distinct but homologous genes.

  2. Interactions between Human Glutamate Carboxypeptidase II and Urea-Based Inhibitors: Structural Characterization

    SciTech Connect

    Barinka, Cyril; Byun, Youngjoo; Dusich, Crystal L.; Banerjee, Sangeeta R.; Chen, Ying; Castanares, Mark; Kozikowski, Alan P.; Mease, Ronnie C.; Pomper, Martin G.; Lubkowski, Jacek

    2009-01-21

    Urea-based, low molecular weight ligands of glutamate carboxypeptidase II (GCPII) have demonstrated efficacy in various models of neurological disorders and can serve as imaging agents for prostate cancer. To enhance further development of such compounds, we determined X-ray structures of four complexes between human GCPII and urea-based inhibitors at high resolution. All ligands demonstrate an invariant glutarate moiety within the S1{prime} pocket of the enzyme. The ureido linkage between P1 and P1{prime} inhibitor sites interacts with the active-site Zn{sub 1}{sup 2+} ion and the side chains of Tyr552 and His553. Interactions within the S1 pocket are defined primarily by a network of hydrogen bonds between the P1 carboxylate group of the inhibitors and the side chains of Arg534, Arg536, and Asn519. Importantly, we have identified a hydrophobic pocket accessory to the S1 site that can be exploited for structure-based design of novel GCPII inhibitors with increased lipophilicity.

  3. Novel Substrate-Based Inhibitors of Human Glutamate Carboxypeptidase II with Enhanced Lipophilicity

    SciTech Connect

    Plechanovová, Anna; Byun, Youngjoo; Alquicer, Glenda; Škultétyová, L; ubica; Ml; #269; ochová, Petra; N; #283; mcová, Adriana; Kim, Hyung-Joon; Navrátil, Michal; Mease, Ronnie; Lubkowski, Jacek; Pomper, Martin; Konvalinka, Jan; Rulíšek, Lubomír; Ba; #345; inka, Cyril

    2012-10-09

    Virtually all low molecular weight inhibitors of human glutamate carboxypeptidase II (GCPII) are highly polar compounds that have limited use in settings where more lipophilic molecules are desired. Here we report the identification and characterization of GCPII inhibitors with enhanced liphophilicity that are derived from a series of newly identified dipeptidic GCPII substrates featuring nonpolar aliphatic side chains at the C-terminus. To analyze the interactions governing the substrate recognition by GCPII, we determined crystal structures of the inactive GCPII(E424A) mutant in complex with selected dipeptides and complemented the structural data with quantum mechanics/molecular mechanics calculations. Results reveal the importance of nonpolar interactions governing GCPII affinity toward novel substrates as well as formerly unnoticed plasticity of the S1' specificity pocket. On the basis of those data, we designed, synthesized, and evaluated a series of novel GCPII inhibitors with enhanced lipophilicity, with the best candidates having low nanomolar inhibition constants and clogD > -0.3. Our findings offer new insights into the design of more lipophilic inhibitors targeting GCPII.

  4. Effect of mutation of two critical glutamic acid residues on the activity and stability of human carboxypeptidase M and characterization of its signal for glycosylphosphatidylinositol anchoring.

    PubMed

    Tan, Fulong; Balsitis, Scott; Black, Judy K; Blöchl, Andrea; Mao, Ji-Fang; Becker, Robert P; Schacht, David; Skidgel, Randal A

    2003-03-01

    Human carboxypeptidase (CP) M was expressed in baculovirus-infected insect cells in a glycosylphosphatidylinositol-anchored form, whereas a truncated form, lacking the putative signal sequence for glycosylphosphatidylinositol anchoring, was secreted at high levels into the medium. Both forms had lower molecular masses (50 kDa) than native placental CPM (62 kDa), indicating minimal glycosylation. The predicted glycosylphosphatidylinositol-anchor attachment site was investigated by mutation of Ser(406) to Ala, Thr or Pro and expression in HEK-293 and COS-7 cells. The wild-type and S406A and S406T mutants were expressed on the plasma membrane in glycosylphosphatidylinositol-anchored form, but the S406P mutant was not and was retained in a perinuclear location. The roles of Glu(260) and Glu(264) in CPM were investigated by site-directed mutagenesis. Mutation of Glu(260) to Gln had minimal effects on kinetic parameters, but decreased heat stability, whereas mutation to Ala reduced the k(cat)/ K(m) by 104-fold and further decreased stability. In contrast, mutation of Glu(264) to Gln resulted in a 10000-fold decrease in activity, but the enzyme still bound to p-aminobenzoylarginine-Sepharose and was resistant to trypsin treatment, indicating that the protein was folded properly. These results show that Glu(264) is the critical catalytic glutamic acid and that Glu(260) probably stabilizes the conformation of the active site.

  5. Structural and biochemical characterization of the folyl-poly-γ-l-glutamate hydrolyzing activity of human glutamate carboxypeptidase II.

    PubMed

    Navrátil, Michal; Ptáček, Jakub; Šácha, Pavel; Starková, Jana; Lubkowski, Jacek; Bařinka, Cyril; Konvalinka, Jan

    2014-07-01

    In addition to its well-characterized role in the central nervous system, human glutamate carboxypeptidase II (GCPII; Uniprot ID Q04609) acts as a folate hydrolase in the small intestine, participating in the absorption of dietary polyglutamylated folates (folyl-n-γ-l-glutamic acid), which are the provitamin form of folic acid (also known as vitamin B9 ). Despite the role of GCPII as a folate hydrolase, nothing is known about the processing of polyglutamylated folates by GCPII at the structural or enzymological level. Moreover, many epidemiologic studies on the relationship of the naturally occurring His475Tyr polymorphism to folic acid status suggest that this polymorphism may be associated with several pathologies linked to impaired folate metabolism. In the present study, we report: (a) a series X-ray structures of complexes between a catalytically inactive GCPII mutant (Glu424Ala) and a panel of naturally occurring polyglutamylated folates; (b) the X-ray structure of the His475Tyr variant at a resolution of 1.83 Å; (c) the study of the recently identified arene-binding site of GCPII through mutagenesis (Arg463Leu, Arg511Leu and Trp541Ala), inhibitor binding and enzyme kinetics with polyglutamylated folates as substrates; and (d) a comparison of the thermal stabilities and folate-hydrolyzing activities of GCPII wild-type and His475Tyr variants. As a result, the crystallographic data reveal considerable details about the binding mode of polyglutamylated folates to GCPII, especially the engagement of the arene binding site in recognizing the folic acid moiety. Additionally, the combined structural and kinetic data suggest that GCPII wild-type and His475Tyr variant are functionally identical. PMID:24863754

  6. An N-terminal truncated carboxypeptidase E splice isoform induces tumor growth and is a biomarker for predicting future metastasis in human cancers

    PubMed Central

    Lee, Terence K.; Murthy, Saravana R.K.; Cawley, Niamh X.; Dhanvantari, Savita; Hewitt, Stephen M.; Lou, Hong; Lau, Tracy; Ma, Stephanie; Huynh, Thanh; Wesley, Robert A.; Ng, Irene O.; Pacak, Karel; Poon, Ronnie T.; Loh, Y. Peng

    2011-01-01

    Metastasis is a major cause of mortality in cancer patients. However, the mechanisms governing the metastatic process remain elusive, and few accurate biomarkers exist for predicting whether metastasis will occur, something that would be invaluable for guiding therapy. We report here that the carboxypeptidase E gene (CPE) is alternatively spliced in human tumors to yield an N-terminal truncated protein (CPE-ΔN) that drives metastasis. mRNA encoding CPE-ΔN was found to be elevated in human metastatic colon, breast, and hepatocellular carcinoma (HCC) cell lines. In HCC cells, cytosolic CPE-ΔN was translocated to the nucleus and interacted with histone deacetylase 1/2 to upregulate expression of the gene encoding neural precursor cell expressed, developmentally downregulated gene 9 (Nedd9) — which has been shown to promote melanoma metastasis. Nedd9 upregulation resulted in enhanced in vitro proliferation and invasion. Quantification of mRNA encoding CPE-ΔN in HCC patient samples predicted intrahepatic metastasis with high sensitivity and specificity, independent of cancer stage. Similarly, high CPE-ΔN mRNA copy numbers in resected pheochromocytomas/paragangliomas (PHEOs/PGLs), rare neuroendocrine tumors, accurately predicted future metastasis or recurrence. Thus, CPE-ΔN induces tumor metastasis and should be investigated as a potentially powerful biomarker for predicting future metastasis and recurrence in HCC and PHEO/PGL patients. PMID:21285511

  7. Efficient and versatile one-step affinity purification of in vivo biotinylated proteins: Expression, characterization and structure analysis of recombinant human glutamate carboxypeptidase II

    SciTech Connect

    Tykvart, J.; Sacha, P.; Barinka, C.; Knedlik, T.; Starkova, J.; Lubkowski, J.; Konvalinka, J.

    2012-02-07

    Affinity purification is a useful approach for purification of recombinant proteins. Eukaryotic expression systems have become more frequently used at the expense of prokaryotic systems since they afford recombinant eukaryotic proteins with post-translational modifications similar or identical to the native ones. Here, we present a one-step affinity purification set-up suitable for the purification of secreted proteins. The set-up is based on the interaction between biotin and mutated streptavidin. Drosophila Schneider 2 cells are chosen as the expression host, and a biotin acceptor peptide is used as an affinity tag. This tag is biotinylated by Escherichia coli biotin-protein ligase in vivo. We determined that localization of the ligase within the ER led to the most effective in vivo biotinylation of the secreted proteins. We optimized a protocol for large-scale expression and purification of AviTEV-tagged recombinant human glutamate carboxypeptidase II (Avi-GCPII) with milligram yields per liter of culture. We also determined the 3D structure of Avi-GCPII by X-ray crystallography and compared the enzymatic characteristics of the protein to those of its non-tagged variant. These experiments confirmed that AviTEV tag does not affect the biophysical properties of its fused partner. Purification approach, developed here, provides not only a sufficient amount of highly homogenous protein but also specifically and effectively biotinylates a target protein and thus enables its subsequent visualization or immobilization.

  8. Prolactin/Stat5 and androgen R1881 coactivate carboxypeptidase-D gene in breast cancer cells.

    PubMed

    Koirala, Samir; Thomas, Lynn N; Too, Catherine K L

    2014-03-01

    Plasma membrane-bound carboxypeptidase-D (CPD) cleaves C-terminal arginine from extracellular substrates. In the cell, arginine is converted to nitric oxide (NO). We have reported that up-regulation of CPD mRNA/protein levels by 17β-estradiol and prolactin (PRL) in breast cancer cells, and by testosterone in prostate cancer cells, increased NO production and cell survival. The CPD promoter contains a consensus γ-interferon-activated sequence (GAS) and 3 putative androgen response elements (ARE.1, ARE.2, ARE.3) that could potentially bind PRL-activated transcription factor Stat5 (signal transducer and activator of transcription 5) and the liganded androgen receptor (AR), respectively. This study showed that synthetic androgen R1881 and PRL elevated CPD mRNA/protein levels in human MCF-7 and T47D breast cancer cells in a time-/dose-dependent manner. PRL/R1881-elevated CPD expression was blocked by actinomycin-D, and a CPD promoter construct containing these GAS and AREs was stimulated by PRL or R1881, indicating transcriptional regulation by both hormones. Luciferase reporter assays showed that GAS and the adjacent ARE.1 only were active. Mutation of GAS in the ΔGAS-CPD construct (ARE.1 intact) abolished CPD promoter activity in response to PRL and, surprisingly, to R1881 as well. ΔGAS-CPD promoter activity was restored by PRL+R1881 in combination, and enhanced by ectopic Stat5, but abolished by Stat5 gene knockdown. Chromatin immunoprecipitation analysis confirmed binding of activated Stat5 and liganded AR to GAS and ARE.1, respectively. Activated Stat5 also induced binding of unliganded AR to ARE.1, and liganded AR induced binding of unactivated Stat5 to GAS. In summary, PRL and R1881, acting through Stat5 and AR, act cooperatively to stimulate CPD gene transcription in breast cancer cells. PMID:24433040

  9. Prolactin/Stat5 and androgen R1881 coactivate carboxypeptidase-D gene in breast cancer cells.

    PubMed

    Koirala, Samir; Thomas, Lynn N; Too, Catherine K L

    2014-03-01

    Plasma membrane-bound carboxypeptidase-D (CPD) cleaves C-terminal arginine from extracellular substrates. In the cell, arginine is converted to nitric oxide (NO). We have reported that up-regulation of CPD mRNA/protein levels by 17β-estradiol and prolactin (PRL) in breast cancer cells, and by testosterone in prostate cancer cells, increased NO production and cell survival. The CPD promoter contains a consensus γ-interferon-activated sequence (GAS) and 3 putative androgen response elements (ARE.1, ARE.2, ARE.3) that could potentially bind PRL-activated transcription factor Stat5 (signal transducer and activator of transcription 5) and the liganded androgen receptor (AR), respectively. This study showed that synthetic androgen R1881 and PRL elevated CPD mRNA/protein levels in human MCF-7 and T47D breast cancer cells in a time-/dose-dependent manner. PRL/R1881-elevated CPD expression was blocked by actinomycin-D, and a CPD promoter construct containing these GAS and AREs was stimulated by PRL or R1881, indicating transcriptional regulation by both hormones. Luciferase reporter assays showed that GAS and the adjacent ARE.1 only were active. Mutation of GAS in the ΔGAS-CPD construct (ARE.1 intact) abolished CPD promoter activity in response to PRL and, surprisingly, to R1881 as well. ΔGAS-CPD promoter activity was restored by PRL+R1881 in combination, and enhanced by ectopic Stat5, but abolished by Stat5 gene knockdown. Chromatin immunoprecipitation analysis confirmed binding of activated Stat5 and liganded AR to GAS and ARE.1, respectively. Activated Stat5 also induced binding of unliganded AR to ARE.1, and liganded AR induced binding of unactivated Stat5 to GAS. In summary, PRL and R1881, acting through Stat5 and AR, act cooperatively to stimulate CPD gene transcription in breast cancer cells.

  10. Human Plasma Protein C

    PubMed Central

    Kisiel, Walter

    1979-01-01

    Protein C is a vitamin K-dependent protein, which exists in bovine plasma as a precursor of a serine protease. In this study, protein C was isolated to homogeneity from human plasma by barium citrate adsorption and elution, ammonium sulfate fractionation, DEAE-Sephadex chromatography, dextran sulfate agarose chromatography, and preparative polyacrylamide gel electrophoresis. Human protein C (Mr = 62,000) contains 23% carbohydrate and is composed of a light chain (Mr = 21,000) and a heavy chain (Mr = 41,000) held together by a disulfide bond(s). The light chain has an amino-terminal sequence of Ala-Asn-Ser-Phe-Leu- and the heavy chain has an aminoterminal sequence of Asp-Pro-Glu-Asp-Gln. The residues that are identical to bovine protein C are underlined. Incubation of human protein C with human α-thrombin at an enzyme to substrate weight ratio of 1:50 resulted in the formation of activated protein C, an enzyme with serine amidase activity. In the activation reaction, the apparent molecular weight of the heavy chain decreased from 41,000 to 40,000 as determined by gel electrophoresis in the presence of sodium dodecyl sulfate. No apparent change in the molecular weight of the light chain was observed in the activation process. The heavy chain of human activated protein C also contains the active-site serine residue as evidenced by its ability to react with radiolabeled diisopropyl fluorophosphate. Human activated protein C markedly prolongs the kaolin-cephalin clotting time of human plasma, but not that of bovine plasma. The amidolytic and anticoagulant activities of human activated protein C were completely obviated by prior incubation of the enzyme with diisopropyl fluorophosphate. These results indicate that human protein C, like its bovine counterpart, exists in plasma as a zymogen and is converted to a serine protease by limited proteolysis with attendant anticoagulant activity. Images PMID:468991

  11. Carboxypeptidase S- and carboxypeptidase Y-deficient mutants of Saccharomyces cerevisiae.

    PubMed

    Wolf, D H; Ehmann, C

    1981-08-01

    A new carboxypeptidase (carboxypeptidase S) was found in a Saccharomyces cerevisiae strain lacking carboxypeptidase Y (D. H. Wolf and U. Weiser, Eur. J. Biochem. 73:553-556, 1977). Mutants devoid of carboxypeptidase S activity were isolated from a mutant strain that was also deficient in carboxypeptidase Y. Four mutants were analyzed in detail and fell into one complementation group. The defect segregated 2:2 in meiotic tetrads. Gene dosage experiments indicated that the mutation might reside in the structural gene of carboxypeptidase S. The absence of both enzymes, carboxypeptidases Y and S, did not affect mitotic growth. Ascopore formation was only slightly affected by the absence of both carboxypeptidases. Protein degradation under conditions of nutrient deprivation and under sporulation conditions showed no obvious alteration in the absence of carboxypeptidases Y and S. When a proteinase B mutation, which led to the absence of proteinase B activity and resulted in the partial reduction of sporulation, was introduced into a mutant lacking both carboxypeptidases, the ability of diploid cells to sporulate was nearly completely lost. Mutants lacking both carboxypeptidases were unable to grow on the dipeptide benzyloxycarbonylglycyl-l-leucine as a sole nitrogen source, which indicates an additional function for carboxypeptidases Y and S in supplying nutrients from exogenous peptides. Catabolite inactivation of fructose-1,6-bisphosphatase, cytoplasmic malate dehydrogenase, and phosphoenolpyruvate carboxykinase and inactivation of nicotin-amide adenine dinucleotide phosphate-dependent, glutamate dehydrogenase, events which have been proposed to involve proteolysis in vivo, were not dependent on the presence of carboxypeptidase Y and S. In a mutant lacking both carboxypeptidases, four new proteolytic enzymes with carboxypeptidase activity were detected. PMID:7021530

  12. Design of highly potent urea-based, exosite-binding inhibitors selective for glutamate carboxypeptidase II.

    PubMed

    Tykvart, Jan; Schimer, Jiří; Jančařík, Andrej; Bařinková, Jitka; Navrátil, Václav; Starková, Jana; Šrámková, Karolína; Konvalinka, Jan; Majer, Pavel; Šácha, Pavel

    2015-05-28

    We present here a structure-aided design of inhibitors targeting the active site as well as exosites of glutamate carboxypeptidase II (GCPII), a prostate cancer marker, preparing potent and selective inhibitors that are more than 1000-fold more active toward GCPII than its closest human homologue, glutamate carboxypeptidase III (GCPIII). Additionally, we demonstrate that the prepared inhibitor conjugate can be used for sensitive and selective imaging of GCPII in mammalian cells. PMID:25923815

  13. Insect midgut carboxypeptidases with emphasis on S10 hemipteran and M14 lepidopteran carboxypeptidases.

    PubMed

    Ferreira, C; Rebola, K G O; Cardoso, C; Bragatto, I; Ribeiro, A F; Terra, W R

    2015-04-01

    We compared the whole complement of midgut carboxypeptidases from 10 insects pertaining to five orders based on transcriptomes obtained by deep sequencing and biochemical data. Most of the carboxypeptidases were metallocarboxypeptidases from family M14, with carboxypeptidase A (CPA) predominating over carboxypeptidase B (CPB). They were found in all of the insects studied except for the hemipterans and a bruchid beetle. M14 carboxypeptidases were expressed only in the midgut of Spodoptera frugiperda (Lepidoptera). The most expressed CPA from this insect (SfCPA) was cloned, sequenced and expressed as a recombinant enzyme. This enzyme was used to generate antibodies used to demonstrate that SfCPA is secreted by an exocytic route. Serine carboxypeptidases from family S10 were found in all of the insects studied here. In S. frugiperda, they are expressed in all tissues besides the midgut, in accordance with their presumed lysosomal role. In the hemipteran Dysdercus peruvianus, S10 carboxypeptidases are expressed only in midgut, suggesting that they are digestive enzymes. This was confirmed by enzyme assays of midgut contents. Furthermore, the substrate specificity of D. peruvianus S10 carboxypeptidases are predicted to be one CPC (preferring hydrophobic residues) and one CPD (preferring basic residues), thus able to hydrolyse the peptides formed by their digestive cathepsin D and cathepsin L, respectively. The role of S10 carboxypeptidases in bruchid beetles are suggested to be the same as in hemipterans.

  14. Limited mutagenesis increases the stability of human carboxypeptidase U (TAFIa) and demonstrates the importance of CPU stability over proCPU concentration in down-regulating fibrinolysis.

    PubMed

    Knecht, Wolfgang; Willemse, Johan; Stenhamre, Hanna; Andersson, Mats; Berntsson, Pia; Furebring, Christina; Harrysson, Anna; Hager, Ann-Christin Malmborg; Wissing, Britt-Marie; Hendriks, Dirk; Cronet, Philippe

    2006-02-01

    Procarboxypeptidase U [proCPU, thrombin-activatable fibrinolysis inhibitor (TAFI), EC 3.4.17.20] belongs to the metallocarboxypeptidase family and is a zymogen found in human plasma. ProCPU has been proposed to be a molecular link between coagulation and fibrinolysis. Upon activation of proCPU, the active enzyme (CPU) rapidly becomes inactive due to its intrinsic instability. The inherent instability of CPU is likely to be of major importance for the in vivo down-regulation of its activity, but the underlying structural mechanisms of this fast and spontaneous loss of activity of CPU have not yet been explained, and they severely inhibit the structural characterization of CPU. In this study, we screened for more thermostable versions of CPU to increase our understanding of the mechanism underlying the instability of CPU's activity. We have shown that single as well as a few 2-4 mutations in human CPU can prolong the half-life of CPU's activity at 37 degrees C from 0.2 h of wild-type CPU to 0.5-5.5 h for the mutants. We provide evidence that the gain in stable activity is accompanied by a gain in thermostability of the enzyme and increased resistance to proteolytic digest by trypsin. Using one of the stable mutants, we demonstrate the importance of CPU stability over proCPU concentration in down-regulating fibrinolysis.

  15. Pyrrolidon carboxypeptidase activities in the hypothalamus-pituitary-thyroid and hypothalamus-pituitary-ovary axes of rats with mammary gland cancer induced by N-methyl nitrosourea.

    PubMed

    Carrera, M P; Ramírez-Expósito, M J; Valenzuela, M T; García, M J; Mayas, M D; Arias de Saavedra, J M; Sánchez, R; Pérez, M C; Martínez-Martos, J M

    2005-02-01

    Pyrrolidon carboxypeptidase is an omega-peptidase that hydrolyses N-terminal pyroglutamyl residues from biologically active peptides such as gonadotropin-releasing and thyrotrophin-releasing hormones. We previously described a decrease in both rat and human pyrrolidon carboxypeptidase activity with breast cancer, suggesting that gonadotropin-releasing hormone may be an important local intracrine, autocrine and/or paracrine hormonal factor in the pathogenesis of breast cancer while playing a role in the tumoral process. However, the other susceptible substrate of pyrrolidon carboxypeptidase, thyrotrophin-releasing hormone, may also be modified with breast cancer, supporting an association between breast cancer and thyroid disorders. The present work analyses soluble and membrane-bound pyrrolidon carboxypeptidase activities in the hypothalamus-pituitary-thyroid and hypothalamus-pituitary-ovary axes in N-methyl nitrosourea-induced breast cancer in rats. Our aim was to determine the possible relationship between gonadotropin-releasing hormone and thyrotrophin-releasing hormone regulation through pyrrolidon carboxypeptidase activity. We propose that pyrrolidon carboxypeptidase activity dysregulation at various local and systemic levels may participate in the initiation, promotion and progression of breast cancer induced in rat by N-methyl nitrosourea through the increase in gonadotropin-releasing hormone. Since pyrrolidon carboxypeptidase activity also acts on thyrotrophin-releasing hormone, the dysregulation of this enzyme's activity could indirectly affect hypothalamus-pituitary-thyroid axis function, and thus potentially represent a link between the diseases of thyroid and breast cancer.

  16. New Roles of Carboxypeptidase E in Endocrine and Neural Function and Cancer

    PubMed Central

    Cawley, Niamh X.; Wetsel, William C.; Murthy, Saravana R. K.; Park, Joshua J.; Pacak, Karel

    2012-01-01

    Carboxypeptidase E (CPE) or carboxypeptidase H was first discovered in 1982 as an enkephalin-convertase that cleaved a C-terminal basic residue from enkephalin precursors to generate enkephalin. Since then, CPE has been shown to be a multifunctional protein that subserves many essential nonenzymatic roles in the endocrine and nervous systems. Here, we review the phylogeny, structure, and function of CPE in hormone and neuropeptide sorting and vesicle transport for secretion, alternative splicing of the CPE transcript, and single nucleotide polymorphisms in humans. With this and the analysis of mutant and knockout mice, the data collectively support important roles for CPE in the modulation of metabolic and glucose homeostasis, bone remodeling, obesity, fertility, neuroprotection, stress, sexual behavior, mood and emotional responses, learning, and memory. Recently, a splice variant form of CPE has been found to be an inducer of tumor growth and metastasis and a prognostic biomarker for metastasis in endocrine and nonendocrine tumors. PMID:22402194

  17. Expression and distribution of carboxypeptidase B in the hippocampal subregions of normal and Alzheimer's disease brain.

    PubMed

    Papp, Henrietta; Török, I; Matsumoto, A; Enomoto, T; Matsuyama, S; Kása, P

    2003-01-01

    Earlier neurochemical studies suggested that human brain carboxypeptidase B may play a significant role in the degradation of amyloid-beta1-42 in the brain. Using an immimohistochemical technique we report here on the neuronal expression and distribution of this enzyme in the segments (CA1a, CA1b and CA1c) of the CA1 subfield and in area CA4 of the hippocampus in normal and Alzheimer's disease brain samples. Its distribution was compared with the appearance of neurofibrillary tangles in the same brain sample. For immunohistochemical localization of carboxypeptidase B, a specific C14-module antibody was applied, together with the Gallyas silver impregnation technique for the demonstration of neurofibrillary tangles. The results revealed that, in the control samples, most of the immunoreactivity appeared in segment CA1a in the pyramidal cells, less in segment CA1b and least in segment CA1c. In the Alzheimer's disease samples, there was no particular immunostaining in the neurons, but, a large number of silver-impregnated degenerated neurons appeared. The results support the suggestion that carboxypeptidase B may play a significant role in elimination of the intracellular accumulation and toxicity of amyloid-beta in the human brain and thereby protect the neurons from degeneration. PMID:12705322

  18. Comparison of Substrate Specificity of Escherichia Coli p-Aminobenzoyl-Glutamate Hydrolase with Pseudomonas Carboxypeptidase G

    PubMed Central

    Larimer, Cassandra M.; Slavnic, Dejan; Pitstick, Lenore D.; Green, Jacalyn M.

    2016-01-01

    Reduced folic acid derivatives support biosynthesis of DNA, RNA and amino acids in bacteria as well as in eukaryotes, including humans. While the genes and steps for bacterial folic acid synthesis are known, those associated with folic acid catabolism are not well understood. A folate catabolite found in both humans and bacteria is p-aminobenzoyl-glutamate (PABA-GLU). The enzyme p-aminobenzoyl-glutamate hydrolase (PGH) breaks down PABA-GLU and is part of an apparent operon, the abg region, in E. coli. The subunits of PGH possess sequence and catalytic similarities to carboxypeptidase enzymes from Pseudomonas species. A comparison of the subunit sequences and activity of PGH, relative to carboxypeptidase enzymes, may lead to a better understanding of bacterial physiology and pathway evolution. We first compared the amino acid sequences of AbgA, AbgB and carboxypeptidase G2 from Pseudomonas sp. RS-16, which has been crystallized. Then we compared the enzyme activities of E. coli PGH and commercially available Pseudomonas carboxypeptidase G using spectrophotometric assays measuring cleavage of PABA-GLU, folate, aminopterin, methotrexate, 5-formyltetrahydrofolate, and 5-methyltetrahydrofolate. The Km and Vmax values for the folate and anti-folate substrates of PGH could not be determined, because the instrument reached its limit before the enzyme was saturated. Therefore, activity of PGH was compared to the activity of CPG, or normalized to PABA-GLU (nmole/min/µg). Relative to its activity with 10 µM PABA-GLU (100%), PGH cleaved glutamate from methotrexate (48%), aminopterin (45%) and folate (9%). Reduced folates leucovorin (5-formyltetrahydrofolate) and 5-methyltetrahydrofolate were not cleaved by PGH. Our data suggest that E. coli PGH is specific for PABA-GLU as its activity with natural folates (folate, 5-methyltetrahydrofolate, and leucovorin) was very poor. It does, however, have some ability to cleave anti-folates which may have clinical applications in

  19. Biochemical characterization of a novel carboxypeptidase inhibitor from a variety of Andean potatoes.

    PubMed

    Lufrano, Daniela; Cotabarren, Juliana; Garcia-Pardo, Javier; Fernandez-Alvarez, Roberto; Tort, Olivia; Tanco, Sebastián; Avilés, Francesc Xavier; Lorenzo, Julia; Obregón, Walter D

    2015-12-01

    Natural protease inhibitors of metallocarboxypeptidases are rarely reported. In this work, the cloning, expression and characterization of a proteinaceous inhibitor of the A/B-type metallocarboxypeptidases, naturally occurring in tubers of Solanum tuberosum, subsp. andigenum cv. Imilla morada, are described. The obtained cDNA encoded a polypeptide of 80 residues, which displayed the features of metallocarboxypeptidase inhibitor precursors from the Potato Carboxypeptidase Inhibitor (PCI) family. The mature polypeptide (39 residues) was named imaPCI and in comparison with the prototype molecule of the family (PCI from S. tuberosum subsp. tuberosum), its sequence showed one difference at its N-terminus and another three located at the secondary binding site, a region described to contribute to the stabilization of the complex inhibitor-target enzyme. In order to gain insights into the relevance of the secondary binding site in nature, a recombinant form of imaPCI (rimaPCI) having only differences at the secondary binding site with respect to recombinant PCI (rPCI) was cloned and expressed in Escherichia coli. The rimaPCI exhibited a molecular mass of 4234.8Da by MALDI-TOF/MS. It displayed potent inhibitory activity towards A/B-type carboxypeptidases (with a Ki in the nanomolar range), albeit 2-4-fold lower inhibitory capacity compared to its counterpart rPCI. This result is in agreement with our bioinformatic analysis, which showed that the main interaction established between the secondary binding site of rPCI and the bovine carboxypeptidase A is likely lost in the case of rimaPCI. These observations reinforce the importance of the secondary binding site of PCI-family members on inhibitory effects towards A/B-type metallocarboxypeptidases. Furthermore, as a simple proof of concept of its applicability in biotechnology and biomedicine, the ability of rimaPCI to protect human epidermal growth factor from C-terminal cleavage and inactivation by carboxypeptidases A and B

  20. Genome-Wide Identification and Characterization of Carboxypeptidase Genes in Silkworm (Bombyx mori).

    PubMed

    Ye, Junhong; Li, Yi; Liu, Hua-Wei; Li, Jifu; Dong, Zhaoming; Xia, Qingyou; Zhao, Ping

    2016-01-01

    The silkworm (Bombyx mori) is an economically-important insect that can secrete silk. Carboxypeptidases have been found in various metazoan species and play important roles in physiological and biochemical reactions. Here, we analyzed the silkworm genome database and characterized 48 carboxypeptidases, including 34 metal carboxypeptidases (BmMCP1-BmMCP34) and 14 serine carboxypeptidases (BmSCP1-BmSCP14), to better understand their diverse functions. Compared to other insects, our results indicated that carboxypeptidases from silkworm have more family members. These silkworm carboxypeptidases could be divided into four families: Peptidase_M2 carboxypeptidases, Peptidase_M14 carboxypeptidases, Peptidase_S10 carboxypeptidases and Peptidase_S28 carboxypeptidases. Microarray analysis showed that the carboxypeptidases had distinct expression patterns, whereas quantitative real-time PCR demonstrated that the expression level of 13 carboxypeptidases significantly decreased after starvation and restored after re-feeding. Overall, our study provides new insights into the functional and evolutionary features of silkworm carboxypeptidases.

  1. Genome-Wide Identification and Characterization of Carboxypeptidase Genes in Silkworm (Bombyx mori)

    PubMed Central

    Ye, Junhong; Li, Yi; Liu, Hua-Wei; Li, Jifu; Dong, Zhaoming; Xia, Qingyou; Zhao, Ping

    2016-01-01

    The silkworm (Bombyx mori) is an economically-important insect that can secrete silk. Carboxypeptidases have been found in various metazoan species and play important roles in physiological and biochemical reactions. Here, we analyzed the silkworm genome database and characterized 48 carboxypeptidases, including 34 metal carboxypeptidases (BmMCP1–BmMCP34) and 14 serine carboxypeptidases (BmSCP1–BmSCP14), to better understand their diverse functions. Compared to other insects, our results indicated that carboxypeptidases from silkworm have more family members. These silkworm carboxypeptidases could be divided into four families: Peptidase_M2 carboxypeptidases, Peptidase_M14 carboxypeptidases, Peptidase_S10 carboxypeptidases and Peptidase_S28 carboxypeptidases. Microarray analysis showed that the carboxypeptidases had distinct expression patterns, whereas quantitative real-time PCR demonstrated that the expression level of 13 carboxypeptidases significantly decreased after starvation and restored after re-feeding. Overall, our study provides new insights into the functional and evolutionary features of silkworm carboxypeptidases. PMID:27483237

  2. Genome-Wide Identification and Characterization of Carboxypeptidase Genes in Silkworm (Bombyx mori).

    PubMed

    Ye, Junhong; Li, Yi; Liu, Hua-Wei; Li, Jifu; Dong, Zhaoming; Xia, Qingyou; Zhao, Ping

    2016-01-01

    The silkworm (Bombyx mori) is an economically-important insect that can secrete silk. Carboxypeptidases have been found in various metazoan species and play important roles in physiological and biochemical reactions. Here, we analyzed the silkworm genome database and characterized 48 carboxypeptidases, including 34 metal carboxypeptidases (BmMCP1-BmMCP34) and 14 serine carboxypeptidases (BmSCP1-BmSCP14), to better understand their diverse functions. Compared to other insects, our results indicated that carboxypeptidases from silkworm have more family members. These silkworm carboxypeptidases could be divided into four families: Peptidase_M2 carboxypeptidases, Peptidase_M14 carboxypeptidases, Peptidase_S10 carboxypeptidases and Peptidase_S28 carboxypeptidases. Microarray analysis showed that the carboxypeptidases had distinct expression patterns, whereas quantitative real-time PCR demonstrated that the expression level of 13 carboxypeptidases significantly decreased after starvation and restored after re-feeding. Overall, our study provides new insights into the functional and evolutionary features of silkworm carboxypeptidases. PMID:27483237

  3. Circulating Proteolytic Products of Carboxypeptidase N for Early Detection of Breast Cancer

    PubMed Central

    Li, Yaojun; Li, Yueguo; Chen, Tao; Kuklina, Anna; Bernard, Paul; Esteva, Francisco J.; Shen, Haifa; Ferrari, Mauro; Hu, Ye

    2014-01-01

    Background Carboxypeptidase N (CPN) plays an important role in regulating vasoactive peptide hormones, growth factors, and cytokines by specifically cleaving their C-terminal basic residues. Herein we demonstrated that the circulating peptides specifically cleaved by CPN in the tumor microenvironment can indeed be stage-specific indicators of breast cancer. Methods The activity of CPN was evaluated using an ex-vivo peptide cleavage assay, in which synthesized C3f peptide (His6-C3f_S1304-R1320-His6) is incubated in interstitial fluids of breast tumor and adjacent normal breast tissues in mice with orthotopic implantation of the human cell line MDA-MB-231. Fragment profiling, by an approach combining nanopore fractionation and mass spectrometry, revealed the nature and extent of cleavage by CPN. These results correlated with the level of CPN-catalyzed peptides in blood specimens taken from the tumor-bearing mice, healthy women and breast cancer patients. CPN expression in the same set of samples was further examined by immunohistochemistry and immunoblotting. Results We showed that generation of C3f_R1310-L1319 specifically correlated with the CPN expression level. In both the mouse and clinical patient samples, the amount of CPN was clearly increased in tumor tissues compared to that seen in normal breast tissue, while its counterpart in blood remained constant. The amount of 6 CPN-catalyzed peptides predominantly increased in sera taken from the mice (n=8) at 2 weeks after orthotropic implantation. Six homologous peptides displayed the significantly higher expression in the patients’ plasma as early as the first pathologic stage of breast cancer. Conclusions The amount of circulating CPN-catalyzed peptides reported here reflects the extent of this enzyme’s activity in tumors, and our results clearly indicate their strong potential as biomarkers for non-invasive and early diagnosis of breast cancer. PMID:24146311

  4. Human seminal plasma inhibition of complement.

    PubMed

    Petersen, B H; Lammel, C J; Stites, D P; Brooks, G F

    1980-10-01

    Recent studies have shown that human seminal plasma contains chemically and biologically distinct factors which inhibit lymphocyte functions and the serum bactericidal and opsonic activities associated with the killing of gram-negative organisms. Because of the direct association between complement action and serum bactericidal and opsonic activities, inhibition of complement may be one of the possible mechanisms of action of seminal plasma immunoinhibitory factors. Complement hemolytic activity was measured for C3 and C4 in serum Neisseria gonorrhoeae and Escherichia coli bactericidal reaction mixtures with and without addition of seminal plasma. In the presence of seminal plasma, where there was no bactericidal action, C3 and titers were reduced to approximately 50% of the titers in the reactions with complement donor serum. The C3 titers were lower than in the reaction mixtures with immune serum and complement donor serum, where N. gonorrhoeae bactericidal activity occurred. Individual human seminal plasma specimens depressed CH50 activity of pooled normal human sera up to 50% of normal levels. There were no differences in inhibition by seminal plasma specimens from normal or vasectomized men. Treatment with seminal plasma depressed the functional activity of complement components C1 and C3 by more than 50%. Seminal plasma also inhibited alternate pathway activity. Cleavage of factor B was demonstrated. The seminal plasma factor which inhibited complement was of low molecular weight. DPF blocked the seminal plasma complement-inhibitory factor. However, amidolytic activity for serine protease substrates could not be demonstrated. It is likely that the seminal plasma complement inhibitor is a protease inhibitor acting singly or in combination.

  5. Measurement of Human Blood and Plasma Volumes

    NASA Technical Reports Server (NTRS)

    Greenleaf, J. E.; Szalkay, H. G. H.

    1987-01-01

    Report reviews techniques for measuring blood-plasma volume in humans. Common technique of using radioactive iodine isotope to label plasma albumin involves unwarranted risks from low-level radiation. Report emphasizes techniques using Evans-blue-dye (T-1824) labeling of albumin, hematocrit or hemoglobin/hematocrit measurements, or blood densitometry. In Evans-blue-dye technique, plasma volume determined from decrease in dye concentration occurring after small amount of dye solution injected into circulatory system. Subjection of Evans blue dye to test for carcinogenicity gave negative results.

  6. Quantitation of phosphorothioate oligonucleotides in human plasma.

    PubMed

    Leeds, J M; Graham, M J; Truong, L; Cummins, L L

    1996-03-01

    Methods are presented for the extraction of phosphorothioate oligonucleotides from human plasma to permit quantitation by capillary gel electrophoresis. Extraction of the phosphorothioate oligonucleotides from plasma was accomplished using two solid-phase extraction columns, a strong anion-exchange column to remove plasma proteins and lipids, followed by a reverse-phase column to remove salts. A second desalting step, achieved by dialysis utilizing a membrane with a molecular weight cutoff of 2500 Da floating on distilled water, was required to remove residual ionic material from the extracted sample. This method should be generally applicable to the analysis and quantitation of phosphorothioate oligonucleotides. PMID:8850544

  7. Acid Carboxypeptidases in Grains and Leaves of Wheat, Triticum aestivum L.

    PubMed

    Mikola, L

    1986-07-01

    Extracts of resting and germinating (3 days at 20 degrees C) wheat (Triticum aestivum L. cv Ruso) grains rapidly hydrolyzed various benzyloxycarbonyldipeptides (Z-dipeptides) at pH 4 to 6. Similar activities were present in extracts of mature flag leaves. Fractionation by chromatography on CM-cellulose and on Sephadex G-200 showed that the activities in germinating grains were due to five acid carboxypeptidases with different and complementary substrate specificities. The wheat enzymes appeared to correspond to the five acid carboxypeptidases present in germinating barley (L Mikola 1983 Biochim Biophys Acta 747: 241-252). The enzymes were designated wheat carboxypeptidases I to V and their best or most characteristic substrates and approximate molecular weights were: I, Z-Phe-Ala, 120,000; II, Z-Ala-Arg, 120,000; III, Z-Ala-Phe, 40,000; IV, Z-Pro-Ala, 165,000; and V, Z-Pro-Ala, 150,000. Resting grains contained carboxypeptidase II as a series of three isoenzymes and low activities of carboxypeptidases IV and V. During germination the activity of carboxypeptidase II decreased, those of carboxypeptidases IV and V increased, and high activities of carboxypeptidases I and III appeared. The flag leaves contained high activity of carboxypeptidase I and lower activities of carboxypeptidases II, IV, and V, whereas carboxypeptidase III was absent.

  8. Acid Carboxypeptidases in Grains and Leaves of Wheat, Triticum aestivum L

    PubMed Central

    Mikola, Leena

    1986-01-01

    Extracts of resting and germinating (3 days at 20°C) wheat (Triticum aestivum L. cv Ruso) grains rapidly hydrolyzed various benzyloxycarbonyldipeptides (Z-dipeptides) at pH 4 to 6. Similar activities were present in extracts of mature flag leaves. Fractionation by chromatography on CM-cellulose and on Sephadex G-200 showed that the activities in germinating grains were due to five acid carboxypeptidases with different and complementary substrate specificities. The wheat enzymes appeared to correspond to the five acid carboxypeptidases present in germinating barley (L Mikola 1983 Biochim Biophys Acta 747: 241-252). The enzymes were designated wheat carboxypeptidases I to V and their best or most characteristic substrates and approximate molecular weights were: I, Z-Phe-Ala, 120,000; II, Z-Ala-Arg, 120,000; III, Z-Ala-Phe, 40,000; IV, Z-Pro-Ala, 165,000; and V, Z-Pro-Ala, 150,000. Resting grains contained carboxypeptidase II as a series of three isoenzymes and low activities of carboxypeptidases IV and V. During germination the activity of carboxypeptidase II decreased, those of carboxypeptidases IV and V increased, and high activities of carboxypeptidases I and III appeared. The flag leaves contained high activity of carboxypeptidase I and lower activities of carboxypeptidases II, IV, and V, whereas carboxypeptidase III was absent. PMID:16664910

  9. Production, purification, and properties of serine carboxypeptidase from Paecilomyces carneus.

    PubMed

    Umetsu, H; Hishinuma, K; Wake, H; Ichishima, E

    1996-07-01

    Seventeen strains of the genus Paecilomyces were examined for their ability to produce serine carboxypeptidase. Paecilomyces carneus IFO 7012 exhibited the highest potency for serine carboxypeptidase production. A maximum yield of serine carboxypeptidase was obtained by koji culture of the strain at 22 degrees C for 7 days. The serine carboxypeptidase was purified to homogeneity from an extract of the koji culture. The molecular weight of the enzyme was estimated to be 47,000 by HPLC. The isoelectric point of the enzyme was determined to be 4.0, and the optimum pH was 4.0 toward benzyloxycarbonyl-L-glutamyl-L-tyrosine (Z-Glu-Tyr) and benzyloxycarbonyl-L-phenylalanyl-L-alanine (Z-Phe-Ala), respectively. The enzyme was strongly inhibited by phenylmethylsulfonyl fluoride and p-chloromercurybenzoate. Relative hydrolysis rates of N-acylpeptides and kinetic studies indicated that the enzyme preferred substrates having bulky amino acids in the penultimate position from their carboxy-termini. PMID:8661688

  10. A digestive prolyl carboxypeptidase in Tenebrio molitor larvae

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Prolyl carboxypeptidase (PRCP) was purified from the anterior midgut of larvae of a stored product pest, Tenebrio molitor. The cDNA of PRCP was cloned, and the predicted protein was identical to the proteomic sequences of the purified enzyme. The substrate specificity of the enzyme was studied, and ...

  11. 21 CFR 640.90 - Plasma Protein Fraction (Human).

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 21 Food and Drugs 7 2010-04-01 2010-04-01 false Plasma Protein Fraction (Human). 640.90 Section...) BIOLOGICS ADDITIONAL STANDARDS FOR HUMAN BLOOD AND BLOOD PRODUCTS Plasma Protein Fraction (Human) § 640.90 Plasma Protein Fraction (Human). (a) Proper name and definition. The proper name of the product shall...

  12. 21 CFR 640.90 - Plasma Protein Fraction (Human).

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 21 Food and Drugs 7 2011-04-01 2010-04-01 true Plasma Protein Fraction (Human). 640.90 Section 640...) BIOLOGICS ADDITIONAL STANDARDS FOR HUMAN BLOOD AND BLOOD PRODUCTS Plasma Protein Fraction (Human) § 640.90 Plasma Protein Fraction (Human). (a) Proper name and definition. The proper name of the product shall...

  13. 21 CFR 640.90 - Plasma Protein Fraction (Human).

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 21 Food and Drugs 7 2012-04-01 2012-04-01 false Plasma Protein Fraction (Human). 640.90 Section...) BIOLOGICS ADDITIONAL STANDARDS FOR HUMAN BLOOD AND BLOOD PRODUCTS Plasma Protein Fraction (Human) § 640.90 Plasma Protein Fraction (Human). (a) Proper name and definition. The proper name of the product shall...

  14. 21 CFR 640.90 - Plasma Protein Fraction (Human).

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 21 Food and Drugs 7 2013-04-01 2013-04-01 false Plasma Protein Fraction (Human). 640.90 Section...) BIOLOGICS ADDITIONAL STANDARDS FOR HUMAN BLOOD AND BLOOD PRODUCTS Plasma Protein Fraction (Human) § 640.90 Plasma Protein Fraction (Human). (a) Proper name and definition. The proper name of the product shall...

  15. 21 CFR 640.90 - Plasma Protein Fraction (Human).

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 21 Food and Drugs 7 2014-04-01 2014-04-01 false Plasma Protein Fraction (Human). 640.90 Section...) BIOLOGICS ADDITIONAL STANDARDS FOR HUMAN BLOOD AND BLOOD PRODUCTS Plasma Protein Fraction (Human) § 640.90 Plasma Protein Fraction (Human). (a) Proper name and definition. The proper name of the product shall...

  16. Thermal diffusivity of human serum and plasma

    NASA Astrophysics Data System (ADS)

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

    2005-06-01

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

  17. Involvement of human plasma angiotensin I-converting enzyme in the degradation of the haemoregulatory peptide N-acetyl-seryl-aspartyl-lysyl-proline.

    PubMed Central

    Rieger, K J; Saez-Servent, N; Papet, M P; Wdzieczak-Bakala, J; Morgat, J L; Thierry, J; Voelter, W; Lenfant, M

    1993-01-01

    The degradation of N-Ac-Ser-Asp-Lys-Pro (AcSDKP), a negative regulator controlling the proliferation of the haematopoietic stem cell, by enzymes present in human plasma, has been investigated. Radiolabelled AcSD[4-3H]KP ([3H]AcSDKP, 1 mM) was completely metabolized in human plasma with a half-life of 80 min, leading exclusively to the formation of radiolabelled lysine. The cleavage of AcSDKP was insensitive to classical proteinase inhibitors including leupeptin, but sensitive to metalloprotease inhibitors. The degradation was completely blocked by specific inhibitors of angiotensin I-converting enzyme (ACE; kininase II; peptidyldipeptide hydrolase, EC 3.4.15.1), showing that the first step of the hydrolysis was indeed due to ACE. In dialysed plasma, the hydrolysis proceeded at only 17% of the maximal rate, whereas addition of 20 mM NaCl led to the recovery of the initial rate observed with normal plasma. Hydrolysis of AcSDKP by commercial rabbit lung ACE generated the C-terminal dipeptide Lys-Pro. Thus, ACE cleaves AcSDKP by a dipeptidyl carboxypeptidase activity. In fact the formation of Lys-Pro was observed when AcSDKP was incubated in human plasma in the presence of HgCl2. These results suggest that ACE is involved in the first limiting step of AcSDKP degradation in human plasma. The second step seems to be under the control of a leupeptin- and E-64-insensitive, HgCl2-sensitive plasmatic enzyme. PMID:8257427

  18. Human plasma lipocalins and serum albumin: Plasma alternative carriers?

    PubMed

    di Masi, Alessandra; Trezza, Viviana; Leboffe, Loris; Ascenzi, Paolo

    2016-04-28

    Lipocalins are an evolutionarily conserved family of proteins that bind and transport a variety of exogenous and endogenous ligands. Lipocalins share a conserved eight anti-parallel β-sheet structure. Among the different lipocalins identified in humans, α-1-acid glycoprotein (AGP), apolipoprotein D (apoD), apolipoprotein M (apoM), α1-microglobulin (α1-m) and retinol-binding protein (RBP) are plasma proteins. In particular, AGP is the most important transporter for basic and neutral drugs, apoD, apoM, and RBP mainly bind endogenous molecules such as progesterone, pregnenolone, bilirubin, sphingosine-1-phosphate, and retinol, while α1-m binds the heme. Human serum albumin (HSA) is a monomeric all-α protein that binds endogenous and exogenous molecules like fatty acids, heme, and acidic drugs. Changes in the plasmatic levels of lipocalins and HSA are responsible for the onset of pathological conditions associated with an altered drug transport and delivery. This, however, does not necessary result in potential adverse effects in patients because many drugs can bind both HSA and lipocalins, and therefore mutual compensatory binding mechanisms can be hypothesized. Here, molecular and clinical aspects of ligand transport by plasma lipocalins and HSA are reviewed, with special attention to their role as alterative carriers in health and disease. PMID:26951925

  19. Peptidoglycan LD-carboxypeptidase Pgp2 influences Campylobacter jejuni helical cell shape and pathogenic properties and provides the substrate for the DL-carboxypeptidase Pgp1.

    PubMed

    Frirdich, Emilisa; Vermeulen, Jenny; Biboy, Jacob; Soares, Fraser; Taveirne, Michael E; Johnson, Jeremiah G; DiRita, Victor J; Girardin, Stephen E; Vollmer, Waldemar; Gaynor, Erin C

    2014-03-21

    Despite the importance of Campylobacter jejuni as a pathogen, little is known about the fundamental aspects of its peptidoglycan (PG) structure and factors modulating its helical morphology. A PG dl-carboxypeptidase Pgp1 essential for maintenance of C. jejuni helical shape was recently identified. Bioinformatic analysis revealed the CJJ81176_0915 gene product as co-occurring with Pgp1 in several organisms. Deletion of cjj81176_0915 (renamed pgp2) resulted in straight morphology, representing the second C. jejuni gene affecting cell shape. The PG structure of a Δpgp2 mutant showed an increase in tetrapeptide-containing muropeptides and a complete absence of tripeptides, consistent with ld-carboxypeptidase activity, which was confirmed biochemically. PG analysis of a Δpgp1Δpgp2 double mutant demonstrated that Pgp2 activity is required to generate the tripeptide substrate for Pgp1. Loss of pgp2 affected several pathogenic properties; the deletion strain was defective for motility in semisolid agar, biofilm formation, and fluorescence on calcofluor white. Δpgp2 PG also caused decreased stimulation of the human nucleotide-binding oligomerization domain 1 (Nod1) proinflammatory mediator in comparison with wild type, as expected from the reduction in muropeptide tripeptides (the primary Nod1 agonist) in the mutant; however, these changes did not alter the ability of the Δpgp2 mutant strain to survive within human epithelial cells or to elicit secretion of IL-8 from epithelial cells after infection. The pgp2 mutant also showed significantly reduced fitness in a chick colonization model. Collectively, these analyses enhance our understanding of C. jejuni PG maturation and help to clarify how PG structure and cell shape impact pathogenic attributes.

  20. Inhibitors of Kallikrein in Human Plasma

    PubMed Central

    McConnell, David J.

    1972-01-01

    Human plasma was fractionated by ammonium sulfate precipitation, DEAE-cellulose chromatography, and Sephadex G-200 gel filtration to determine which method would give the greatest number of clearly separable kallikrein inhibitory peaks. With G-200 gel filtration three peaks could be separated which were demonstrated to contain α2-macroglobulin, C1̄ inactivator, and α1-antitrypsin. No other kallikrein inhibitors could be identified. The fractions containing C1̄ inactivator and α2-macroglobulin appeared to be more effective against kallikrein than that containing α1-antitrypsin. A patient with hereditary angioneurotic edema was shown to have an abnormal C1̄ inactivator protein capable of interfering with kallikrein's biologic, but not its esterolytic activity. Heat-treated human plasma, a commonly used source of kininogen for experiments with kallikrein, was shown to have kallikrein inhibitory activity. PMID:4113391

  1. Revision of the oligosaccharide structures of yeast carboxypeptidase Y

    SciTech Connect

    Ballou, L.; Hernandez, L.M.; Alvarado, E.; Ballou, C.E. )

    1990-05-01

    The N-linked oligosaccharides from baker's yeast carboxypeptidase Y were analyzed by {sup 1}H NMR and specific mannosidase digestion and found to be identical to those from the Saccharomyces cerevisiae mnn9 mutant bulk mannoprotein. The results support the view that the mnn mutants make oligosaccharides that are a true reflection of the normal biosynthetic pathway and confirm that a recently revised yeast oligosaccharide structure is applicable to wild-type mannoproteins.

  2. Revision of the oligosaccharide structures of yeast carboxypeptidase Y.

    PubMed Central

    Ballou, L; Hernandez, L M; Alvarado, E; Ballou, C E

    1990-01-01

    The N-linked oligosaccharides from baker's yeast carboxypeptidase Y were analyzed by 1H NMR and specific mannosidase digestion and found to be identical to those from the Saccharomyces cerevisiae mnn9 mutant bulk mannoprotein. The results support the view that the mnn mutants make oligosaccharides that are a true reflection of the normal biosynthetic pathway and confirm that a recently revised yeast oligosaccharide structure is applicable to wild-type mannoproteins. PMID:2185468

  3. Carboxypeptidase I from triticale grains and the hydrolysis of salt-soluble fractions of storage proteins.

    PubMed

    Drzymała, Adam; Prabucka, Beata; Bielawski, Wiesław

    2012-09-01

    Carboxypeptidase I was purified from triticale grains (×Triticosecale Wittm.) by a 5-step purification procedure including gel filtration, cation-exchange chromatography and affinity chromatography. The enzyme was purified 595.9 fold with a 1.58% recovery. Triticale carboxypeptidase I is a homodimer with a molecular weight of 124.2 kDa and a subunit weight of 55.2 kDa. Each subunit is composed of two polypeptide chains (33.4 and 21.3 kDa). Serine was found in the active site of triticale carboxypeptidase I; DFP (diisopropylflourophosphate) and other applied inhibitors of serine proteases inhibited the enzyme activity. Triticale carboxypeptidase I hydrolyzes N-CBZ-dipeptide (N-carbobenzoxy-dipeptide) substrates at low pH. N-CBZ-Phe-Ala, N-CBZ-Phe-Leu and N-CBZ-Ala-Met were hydrolyzed with the highest rates. The lowest K(m) value and the highest k(cat)/K(m) ratio were observed for hydrolysis of N-CBZ-Phe-Ala. Studies on the amino acid sequence revealed that the purified enzyme is homologous to carboxypeptidase I from barley. Analyses of conserved regions in the sequence of triticale carboxypeptidase I revealed the presence of Ser, Asp and His that compose the catalytic triad. Intact storage proteins were poor substrates for carboxypeptidases. Carboxypeptidase I together with carboxypeptidase III effectively degraded albumins proteolytically modified by endopeptidase EP8. Modified globulins were degraded at a slower rate, and all three carboxypeptidases were required for a significantly increased activity. Studies of the expression of the carboxypeptidase I gene revealed that the synthesis of the enzyme occurs mainly in the scutellum of the grain. The enzyme is also expressed in the aleurone layer of the grains, although its function in this tissue is unknown.

  4. Human plasma lecithin-cholesterol acyltransferase

    SciTech Connect

    Jauhiainen, M.; Stevenson, K.J.; Dolphin, P.J.

    1988-05-15

    Lecithin-cholesterol acyltransferase (LCAT) is a plasma enzyme which catalyzes the transacylation of the fatty acid at the sn-2 position of lecithin to cholesterol forming lysolecithin and cholesteryl ester. The substrates for and products of this reaction are present within the plasma lipoproteins upon which the enzyme acts to form the majority of cholesteryl ester in human plasma. The authors proposed a covalent catalytic mechanism of action for LCAT in which serine and histidine residues mediate lecithin cleavage and two cysteine residues cholesterol esterification. With the aid of sulfhydryl reactive trivalent organoarsenical compounds which are specific for vicinal thiols they have probed the geometry of the catalytic site. They conclude that the two catalytic cysteine residues of LCAT (Cys/sup 31/ and Cys /sup 184/) are vicinal with a calculated distance between their sulfur atoms of 3.50-3.62 A. The additional residue alkylated by teh bifunctional reagent is within the catalytic site and may represent a previously identified catalytic serine or histidine residue.

  5. Characterization of the human blood plasma proteome

    SciTech Connect

    Shen, Yufeng; Kim, Jeongkwon; Strittmatter, Eric F.; Jacobs, Jon M.; Camp, David G.; Fang, Ruihua; Tolic, Nikola; Moore, Ronald J.; Smith, Richard D.

    2005-10-15

    We describe methods for broad characterization of the human plasma proteome. The combination of stepwise IgG and albumin protein depletion by affinity chromatography and ultrahigh-efficiency capillary liquid chromatography separations coupled to ion trap-tandem mass spectrometry enabled identification of 2392 proteins from a single plasma sample with an estimated confidence level of >94%, and an additional 2198 proteins with an estimated confidence level of 80%. The relative abundances of the identified proteins span a range of over eight orders of magnitude in concentration (<30 pg/mL to {approx}30 mg/mL), facilitated by the attomole-level sensitivity of the analysis methods. More than 80% of the observed proteins demonstrate interactions with IgG and/or albumin. The results from this study provide a basis for a wide range of plasma proteomics studies, including broad quantitation of relative abundances in comparative studies for the identification of novel protein disease markers, as well as further studies of protein-protein interactions.

  6. Effects of orchidectomy and testosterone replacement on mouse pyrrolidone carboxypeptidase activity in the HPA axis.

    PubMed

    García-López, M J; Martínez-Martos, J M; Mayas, M D; Carrera, M P; Ramírez-Expósito, M J

    2004-03-01

    Pyrrolidone carboxypeptidase, also known as pyroglutamyl aminopeptidase, removes pyroglutamyl terminal residues from biologically active peptides such as thyrotropin-releasing hormone. The aim of the present work was to study the influence of orchidectomy and testosterone replacement on soluble (pyrrolidone carboxypeptidase type I) and membrane-bound (pyrrolidone carboxypeptidase type II) activities in the hypothalamus-pituitary-adrenal axis. Forty male mice (Balb/C) were distributed into five groups: sham-operated controls, orchidectomized, and orchidectomized treated with increasing doses of testosterone in each group (3, 6 and 12 mg/kg). In the hypothalamus, orchidectomy increased pyrrolidone carboxypeptidase type I, whereas the highest dose of testosterone returned this activity to control levels. In the pituitary, neither pyrrolidone carboxypeptidase type I nor type II activities changed after orchidectomy, although both activities increased after administration of testosterone in both cases. On the other hand, orchidectomy increased pyrrolidone carboxypeptidase type I and type II activities in adrenal glands, while testosterone replacement returned it to control levels. These results suggest that testosterone differentially modulates pyrrolidone carboxypeptidase type I and type II activities, and therefore also their endogenous substrate regulation. Thus, the influence of sex hormones in the physiology of the HPA axis through the modulation of the Pyrrolidone carboxypeptidase type I and type II activities is of great importance on stress and neuropathology associated with HPA dysfunction

  7. Structural characterization of P1'-diversified urea-based inhibitors of glutamate carboxypeptidase II.

    PubMed

    Pavlicek, Jiri; Ptacek, Jakub; Cerny, Jiri; Byun, Youngjoo; Skultetyova, Lubica; Pomper, Martin G; Lubkowski, Jacek; Barinka, Cyril

    2014-05-15

    Urea-based inhibitors of human glutamate carboxypeptidase II (GCPII) have advanced into clinical trials for imaging metastatic prostate cancer. In parallel efforts, agents with increased lipophilicity have been designed and evaluated for targeting GCPII residing within the neuraxis. Here we report the structural and computational characterization of six complexes between GCPII and P1'-diversified urea-based inhibitors that have the C-terminal glutamate replaced by more hydrophobic moieties. The X-ray structures are complemented by quantum mechanics calculations that provide a quantitative insight into the GCPII/inhibitor interactions. These data can be used for the rational design of novel glutamate-free GCPII inhibitors with tailored physicochemical properties.

  8. Purification of glycocalicin from human plasma.

    PubMed

    HadjKacem, Basma; Mkaouar, Héla; Ben Amor, Ikram; Gargouri, Jalel; Gargouri, Ali

    2016-01-01

    Glycocalicin (GC) is a large extracellular proteolytic fragment of glycoprotein Ib, a membrane platelet component playing an essential role in the physiological processes of platelet adhesion and aggregation. GC contains the binding sites for thrombin and von Willebrand factor. GC circulates normally in vivo in significant concentrations and the plasma level of this protein reflects a complex function of factors including platelet count or platelet turnover. It can therefore serve as a good indicator for many diseases like hypoplastic thrombocytopenia and idiopathic thrombocytopenic purpura. For this reason, several purification assays have been previously described. In this work, we describe a novel analytical method for GC purification from human platelets based on preparative HPLC gel filtration followed by immuno-affinity chromatography on NHS activated column conjugated with specific antibody. Pure GC was obtained from tiny amount of starting material. Our protocol of GC purification is simple, fast and provides a pure end product. PMID:26606109

  9. Purification of glycocalicin from human plasma.

    PubMed

    HadjKacem, Basma; Mkaouar, Héla; Ben Amor, Ikram; Gargouri, Jalel; Gargouri, Ali

    2016-01-01

    Glycocalicin (GC) is a large extracellular proteolytic fragment of glycoprotein Ib, a membrane platelet component playing an essential role in the physiological processes of platelet adhesion and aggregation. GC contains the binding sites for thrombin and von Willebrand factor. GC circulates normally in vivo in significant concentrations and the plasma level of this protein reflects a complex function of factors including platelet count or platelet turnover. It can therefore serve as a good indicator for many diseases like hypoplastic thrombocytopenia and idiopathic thrombocytopenic purpura. For this reason, several purification assays have been previously described. In this work, we describe a novel analytical method for GC purification from human platelets based on preparative HPLC gel filtration followed by immuno-affinity chromatography on NHS activated column conjugated with specific antibody. Pure GC was obtained from tiny amount of starting material. Our protocol of GC purification is simple, fast and provides a pure end product.

  10. Carboxypeptidase X-1 (CPX-1) is a secreted collagen-binding glycoprotein.

    PubMed

    Kim, Yu-Hee; O'Neill, Hayley M; Whitehead, Jonathan P

    2015-12-25

    Carboxypeptidase X-1 (CPX-1) is an atypical member of the carboxypeptidase (CP) family of proteins involved in a variety of physiological and pathological processes. However, unlike most other family members CPX-1 lacks catalytic activity making its biological function unclear. CPX-1 contains a 160 amino acid discoidin domain (DSD) that serves as a binding domain in other proteins prompting us to investigate a putative functional role for this domain in CPX-1. Sequence alignment confirmed the overarching homology between the DSD of CPX-1 and other DSDs whilst more detailed analysis revealed conservation of the residues known to form the collagen-binding trench within the DSD of the discoidin domain receptors (DDRs) 1 and 2. Biochemical characterisation of transiently expressed human CPX-1 revealed that CPX-1 was secreted in an N-glycosylation-dependent manner as treatment with the N-glycosylation inhibitor tunicamycin inhibited secretion concomitant with a reduction in CPX-1 mobility on Western blot. Using a collagen pull-down assay we found that secreted CPX-1 bound collagen and this appeared independent of N-glycosylation as treatment with PNGaseF did not affect binding. Further analysis under non-reducing and reducing (+DTT) conditions revealed that CPX-1 was secreted in both monomeric and dimeric forms and only the former bound collagen. Finally, mutation of a key residue situated within the putative collagen-binding trench within the DSD of CPX-1 (R192A) significantly reduced secretion and collagen-binding by 40% and 60%, respectively. Collectively these results demonstrate that CPX-1 is a secreted collagen-binding glycoprotein and provide a foundation for future studies investigating the function of CPX-1.

  11. Structural insight into the evolutionary and pharmacologic homology of glutamate carboxypeptidases II and III

    SciTech Connect

    Hlouchova, Klara; Barinka, Cyril; Konvalinka, Jan; Lubkowski, Jacek

    2009-10-23

    Glutamate carboxypeptidase III (GCPIII) is a metalloenzyme that belongs to the transferrin receptor/glutamate carboxypeptidase II (GCPII; EC 3.4.17.21) superfamily. GCPIII has been studied mainly because of its evolutionary relationship to GCPII, an enzyme involved in a variety of neuropathologies and malignancies, such as glutamatergic neurotoxicity and prostate cancer. Given the potential functional and pharmacological overlap between GCPIII and GCPII, studies addressing the structural and physiological properties of GCPIII are crucial for obtaining a deeper understanding of the GCPII/GCPIII system. In the present study, we report high-resolution crystal structures of the human GCPIII ectodomain in a 'pseudo-unliganded' state and in a complex with: (a) L-glutamate (a product of hydrolysis); (b) a phosphapeptide transition state mimetic, namely (2S,3'S)-{l_brace}[(3'-amino-3'-carboxy-propyl)-hydroxyphosphinoyl]methyl{r_brace}-pentanedioic acid; and (c) quisqualic acid, a glutamate biostere. Our data reveal the overall fold and quaternary arrangement of the GCPIII molecule, define the architecture of the GCPIII substrate-binding cavity, and offer an experimental evidence for the presence of Zn{sup 2+} ions in the bimetallic active site. Furthermore, the structures allow us to detail interactions between the enzyme and its ligands and to characterize the functional flexibility of GCPIII, which is essential for substrate recognition. A comparison of these GCPIII structures with the equivalent GCPII complexes reveals differences in the organization of specificity pockets, in surface charge distribution, and in the occupancy of the co-catalytic zinc sites. The data presented here provide information that should prove to be essential for the structurally-aided design of GCPIII-specific inhibitors and might comprise guidelines for future comparative GCPII/GCPIII studies.

  12. Diverse human extracellular RNAs are widely detected in human plasma

    PubMed Central

    Freedman, Jane E.; Gerstein, Mark; Mick, Eric; Rozowsky, Joel; Levy, Daniel; Kitchen, Robert; Das, Saumya; Shah, Ravi; Danielson, Kirsty; Beaulieu, Lea; Navarro, Fabio C. P.; Wang, Yaoyu; Galeev, Timur R.; Holman, Alex; Kwong, Raymond Y.; Murthy, Venkatesh; Tanriverdi, Selim E.; Koupenova, Milka; Mikhalev, Ekaterina; Tanriverdi, Kahraman

    2016-01-01

    There is growing appreciation for the importance of non-protein-coding genes in development and disease. Although much is known about microRNAs, limitations in bioinformatic analyses of RNA sequencing have precluded broad assessment of other forms of small-RNAs in humans. By analysing sequencing data from plasma-derived RNA from 40 individuals, here we identified over a thousand human extracellular RNAs including microRNAs, piwi-interacting RNA (piRNA), and small nucleolar RNAs. Using a targeted quantitative PCR with reverse transcription approach in an additional 2,763 individuals, we characterized almost 500 of the most abundant extracellular transcripts including microRNAs, piRNAs and small nucleolar RNAs. The presence in plasma of many non-microRNA small-RNAs was confirmed in an independent cohort. We present comprehensive data to demonstrate the broad and consistent detection of diverse classes of circulating non-cellular small-RNAs from a large population. PMID:27112789

  13. Plasma cadmium and zinc in human hypertension.

    PubMed

    Thind, G S; Fischer, G M

    1976-11-01

    1. Plasma cadmium and zinc were determined by atomic absorption spectrophotometry in inferior venal caval or peripheral venous blood in thrity hypertensive patients and fifteen normal subjects. 2. The mean plasma cadium in hypertensive patients was significantly higher than in normal control subjects. 3. The plasma cadmium/zinc ratio was significantly greater in hypertensive patients. 4. There was a significant positive correlation between the plasma cadmium/zinc ratio and the mean arterial blood pressure.

  14. The Expression of Hepatic Carboxypeptidase E is Decreased in Patients with Cholesterol Gallstone

    PubMed Central

    Dai, Shu-Long; Zhou, Jin; Yang, Kun-Xing; Yang, Shi-Yong

    2015-01-01

    Background/Aims: Decreased carboxypeptidase E (CPE) expression is associated with numerous pathophysiological conditions. This study aimed to investigate the potential function of hepatic CPE in cholesterol gallstone formation. Patients and Methods: Patients with cholesterol gallstone (CGS group) and patients without cholesterol gallstones (non-CGS group) were enrolled. The serum total cholesterol, triglyceride, and biliary composition were analyzed. Eight liver samples from two patients without CGS and six patients with CGS were subjected to cDNA microarray analysis. Hepatic CPE expression was detected by quantitative real-time polymerase chain reaction (qRT-PCR), Western blot, and immunohistochemical analysis. Plasma CCK level was measured by ELISA. Results: cDNA microarray identified CPE as a gene downregulated in the CGS group. RT-PCR showed that CPE mRNA level was lower in CGS group than in control (P < 0.05, t-test). Moreover, Western blot and immunohistochemistry analysis showed that CPE protein level was significantly lower in CGS group than in the control group. In addition, plasma CCK level was lower in CGS group than in the control group. A positive correlation was found between serum CCK level and hepatic CPE mRNA level (r2 = 0.713, P = 0.003). Conclusions: Down-expression of liver CPE may reduce the secretion of serum CCK and contribute to the formation of cholesterol gallstone. PMID:26228366

  15. Paradoxical role of C1561T glutamate carboxypeptidase II (GCPII) genetic polymorphism in altering disease susceptibility.

    PubMed

    Divyya, Shree; Naushad, Shaik Mohammad; Addlagatta, Anthony; Murthy, P V L N; Reddy, Ch Ram; Digumarti, Raghunadha Rao; Gottumukkala, Suryanarayana Raju; Kumar, Ajit; Rammurti, S; Kutala, Vijay Kumar

    2012-04-15

    Glutamate carboxypeptidase II (GCPII) is predominantly expressed in brain, intestinal mucosa and prostate cancer in the form of three splice variants i.e. N-acetylated-α-linked acidic dipeptidase (NAALADase), folyl poly-γ-glutamate carboxypeptidase (FGCP) and prostate specific membrane antigen (PSMA) respectively. Its inhibition was found to confer protection against certain neurological disorders and cancer. Despite the pivotal role of this enzyme, the most common polymorphism i.e. H475Y has not been explored comprehensively in all its splice variants. In this study, we have determined the role of this variant in different disease conditions such as breast and prostate cancers, autism, coronary artery disease (CAD) and miscarriages (N=1561). Genotyping was done by PCR-RFLP and dideoxy sequencing. Plasma folate levels were estimated by Axysm folate kit. GCPII expression was studied by semi-quantitative RT-PCR. In silico model was developed using PYMOL. We observed the protective role of H475Y variant in cancers [breast cancer; OR (95% CI): 0.81 (0.55-1.19), prostate cancer: OR (95% CI): 0.00 (0.00-0.66)], and in autism (OR (95% CI): 0.47 (0.21-1.03), whereas inflated risk was observed in CAD (OR (95% CI): 1.69 (1.20-2.37) and miscarriages [Maternal OR (95% CI): 3.26 (2.11-5.04); Paternal OR(95% CI): 1.99 (1.23-3.21)]. Further, this variant was found to impair the intestinal folate absorption in subjects with dietary folate intake in the lowest tertile (CC vs. CT in lowest tertile; 7.56±0.85ng/ml vs. 2.73±045ng/ml, p=0.005). In silico model of GCPII showed steric hindrance with H475Y resulting in stereochemical alteration of catalytic site, thus interfering with ligand binding. Statistically significant association was not observed between dietary folate levels and GCPII expression. However, a positive correlation was seen between plasma folate levels and GCPII expression (r=0.70, p<0.05). To conclude, our data suggests that GCPII H475Y variant shows inverse

  16. Purified human plasma kallikrein aggregates human blood neutrophils.

    PubMed Central

    Schapira, M; Despland, E; Scott, C F; Boxer, L A; Colman, R W

    1982-01-01

    Exposure of human blood polymorphonuclear leukocytes (PMN) to purified active plasma kallikrein resulted in PMN aggregation when kallikrein was present at concentrations ranging from 0.4 to 0.6 U/ml (0.18-0.27 microM). Kallikrein-induced PMN aggregation was not mediated through C5-derived peptides, because identical responses were observed whether or not kallikrein had been preincubated with an antibody to C5. Moreover, kallikrein was specific for aggregating PMN, because no aggregation was observed with Factor XII active fragments (23 nM), Factor XIa (0.6 U/ml or 15nM), thrombin (1.6 microM), plasmin (2 microM), porcine pancreatic elastase (2 microM), bovine pancreatic chymotrypsin (2 microM), or bradykinin (1 microM). Bovine pancreatic trypsin (2 microM) aggregated PMN, but to a lesser extent than kallikrein (0.18 microM). Kallikrein was a potent aggregant agent for PMN because similar responses were observed with kallikrein (0.5 U/ml or 0.23 microM) and an optimal dose (0.2 microM) of N-formyl-methionyl-leucyl-phenylalanine. In addition, PMN incubation with kallikrein resulted in stimulation of their oxidative metabolism as assessed by an increased oxygen uptake. Neutropenia and leukostasis observed in diseases associated with activation of the contact phase system may be the result of PMN aggregation by plasma kallikrein. PMID:6917855

  17. Primary structure and tissue distribution of anglerfish carboxypeptidase H.

    PubMed

    Roth, W W; Mackin, R B; Spiess, J; Goodman, R H; Noe, B D

    1991-07-01

    Most peptide hormones are synthesized as part of larger precursor proteins which must be processed after translation to generate bioactive peptides. This usually involves cleavage of the precursor by an endopeptidase at sites marked by basic amino acids, followed by removal of N- or C-terminal basic residues by the action of an aminopeptidase or carboxypeptidase. These processing events have been observed in a variety of species, from yeast to mammals. As part of an effort to characterize prohormone processing enzymes in the anglerfish, Lophius americanus, we have cloned and sequenced a cDNA for the fish prohormone processing carboxypeptidase H (CPH). Polyadenylated RNA from anglerfish (AF) islet organs was used to construct a cDNA library in phage lambda gt11. The library was screened with a probe derived from the cDNA for rat CPH. A 2400 base pair AF cDNA clone was isolated. This cDNA encodes a polypeptide which is similar in size and composition to mammalian CPH. The sequence data indicate that the AF CPH precursor is a 454 amino acid polypeptide. The derived amino acid sequence of the putative fish CPH is 81% homologous to the rat and bovine CPH enzymes. Significantly, all of the amino acid residues thought to be important for metal ion and substrate binding, glycosylation, and catalytic activity of mammalian CPH are conserved in the fish enzyme. Northern hybridization using RNA from AF tissues indicates that a 2.5 kb fish CPH mRNA is expressed in brain, pituitary and islet organs, but not in other tissues which do not secrete peptide hormones. PMID:1778303

  18. Structures of Potent Selective Peptide Mimetics Bound to Carboxypeptidase B

    SciTech Connect

    Adler, M.; Buckman, B.; Bryant, J.; Chang, Z.; Chu, K.; Emayan, K.; Hrvatin, P.; Islam, I.; Morser, J.; Sukovich, D.; West, C.; Yuan, S.; Whitlow, M.

    2009-05-11

    This article reports the crystal structures of inhibitors of the functional form of thrombin-activatable fibrinolysis inhibitor (TAFIa). In vivo experiments indicate that selective inhibitors of TAFIa would be useful in the treatment of heart attacks. Since TAFIa rapidly degrades in solution, the homologous protein porcine pancreatic carboxypeptidase B (pp-CpB) was used in these crystallography studies. Both TAFIa and pp-CpB are zinc-based exopeptidases that are specific for basic residues. The final development candidate, BX 528, is a potent inhibitor of TAFIa (2 nM) and has almost no measurable effect on the major selectivity target, carboxypeptidase N. BX 528 was designed to mimic the tripeptide Phe-Val-Lys. A sulfonamide replaces the Phe-Val amide bond and a phosphinate connects the Val and Lys groups. The phosphinate also chelates the active-site zinc. The electrostatic interactions with the protein mimic those of the natural substrate. The primary amine in BX 528 forms a salt bridge to Asp255 at the base of the S1 pocket. The carboxylic acid interacts with Arg145 and the sulfonamide is hydrogen bonded to Arg71. Isopropyl and phenyl groups replace the side chains of Val and Phe, respectively. A series of structures are presented here that illustrate the evolution of BX 528 from thiol-based inhibitors that mimic a free C-terminal arginine. The first step in development was the replacement of the thiol with a phosphinate. This caused a precipitous drop in binding affinity. Potency was reclaimed by extending the inhibitors into the downstream binding sites for the natural substrate.

  19. Preparation, crystallization, and preliminary X-ray diffraction study of mutant carboxypeptidase T containing the primary specificity pocket of carboxypeptidase B

    NASA Astrophysics Data System (ADS)

    Akparov, V. Kh.; Grishin, A. M.; Timofeev, V. I.; Kuranova, I. P.

    2010-09-01

    Recombinant G215S, A251G, T257A, D260G, T262D mutant carboxypeptidase T from Thermoactinomyces vulgaris containing mutations in the primary specificity pocket was prepared and crystallized. Single crystals with a size of up to 0.3 mm were grown and investigated by X-ray diffraction. Recombinant mutant carboxypeptidase T containing the primary specificity subsite compositionally identical to that of pancreatic carboxypeptidase B crystallizes in the same space group as the natural enzyme. The crystals belong to sp. gr. P6322; the unit-cell parameters are a = b = 157.867 Å, c = 104.304 Å, α = β = 90°, γ = 120°. X-ray diffraction data suitable for determining the three-dimensional structure at atomic resolution were collected from one crystal.

  20. The p11 subunit of annexin II heterotetramer is regulated by basic carboxypeptidase.

    PubMed

    Fogg, Darin K; Bridges, David E; Cheung, Kitty Kit-Ting; Kassam, Geetha; Filipenko, Nolan R; Choi, Kyu-Sil; Fitzpatrick, Sandra L; Nesheim, Michael; Waisman, David M

    2002-04-16

    The Ca(2+)-dependent phospholipid-binding protein annexin II heterotetramer (AIIt) is composed of two copies of annexin II and a p11 dimer. The interaction of the carboxyl-terminal lysine residues of the p11 subunit of AIIt with the lysine-binding kringle domains of plasminogen is believed to play a key role in plasminogen binding and stimulation of the tPA-catalyzed cleavage of plasminogen to plasmin. In the current report, we show that AIIt-stimulated plasminogen activation is regulated by basic carboxypeptidases, in vitro. The incubation of AIIt with a 1/400 molar ratio of carboxypeptidase B for periods as short as 2 min resulted in a significant loss in AIIt-stimulated plasminogen activation. Carboxypeptidase B (CpB) as well as thrombin-activated fibrinolysis inhibitor (TAFIa) and carboxypeptidase N (CpN) rapidly reduced AIIt-stimulated plasminogen activation by 80%. The molar ratio of carboxypeptidase/AIIt for half-maximal inhibition of AIIt was 1/4700, 1/700, and 1/500 for CpB, TAFIa, and CpN, respectively. Treatment of AIIt with carboxypeptidase resulted in loss of both carboxyl-terminal lysine residues from the p11 subunit, which correlated with a decrease in the k(cat) and an increase in the K(m) for plasminogen activation. The data reveal a novel mechanism for the regulation of AIIt-stimulated plasminogen activation. PMID:11939791

  1. Helical Shape of Helicobacter pylori Requires an Atypical Glutamine as a Zinc Ligand in the Carboxypeptidase Csd4*

    PubMed Central

    Chan, Anson C. K.; Blair, Kris M.; Liu, Yanjie; Frirdich, Emilisa; Gaynor, Erin C.; Tanner, Martin E.; Salama, Nina R.; Murphy, Michael E. P.

    2015-01-01

    Peptidoglycan modifying carboxypeptidases (CPs) are important determinants of bacterial cell shape. Here, we report crystal structures of Csd4, a three-domain protein from the human gastric pathogen Helicobacter pylori. The catalytic zinc in Csd4 is coordinated by a rare His-Glu-Gln configuration that is conserved among most Csd4 homologs, which form a distinct subfamily of CPs. Substitution of the glutamine to histidine, the residue found in prototypical zinc carboxypeptidases, resulted in decreased enzyme activity and inhibition by phosphate. Expression of the histidine variant at the native locus in a H. pylori csd4 deletion strain did not restore the wild-type helical morphology. Biochemical assays show that Csd4 can cleave a tripeptide peptidoglycan substrate analog to release m-DAP. Structures of Csd4 with this substrate analog or product bound at the active site reveal determinants of peptidoglycan specificity and the mechanism to cleave an isopeptide bond to release m-DAP. Our data suggest that Csd4 is the archetype of a new CP subfamily with a domain scheme that differs from this large family of peptide-cleaving enzymes. PMID:25505267

  2. Pathogen reduction in human plasma using an ultrashort pulsed laser.

    PubMed

    Tsen, Shaw-Wei D; Kingsley, David H; Kibler, Karen; Jacobs, Bert; Sizemore, Sara; Vaiana, Sara M; Anderson, Jeanne; Tsen, Kong-Thon; Achilefu, Samuel

    2014-01-01

    Pathogen reduction is a viable approach to ensure the continued safety of the blood supply against emerging pathogens. However, the currently licensed pathogen reduction techniques are ineffective against non-enveloped viruses such as hepatitis A virus, and they introduce chemicals with concerns of side effects which prevent their widespread use. In this report, we demonstrate the inactivation of both enveloped and non-enveloped viruses in human plasma using a novel chemical-free method, a visible ultrashort pulsed laser. We found that laser treatment resulted in 2-log, 1-log, and 3-log reductions in human immunodeficiency virus, hepatitis A virus, and murine cytomegalovirus in human plasma, respectively. Laser-treated plasma showed ≥70% retention for most coagulation factors tested. Furthermore, laser treatment did not alter the structure of a model coagulation factor, fibrinogen. Ultrashort pulsed lasers are a promising new method for chemical-free, broad-spectrum pathogen reduction in human plasma.

  3. Potent cough suppression by physiologically active substance in human plasma.

    PubMed

    Akaike, Norio; Ito, Yushi; Ogawa, Sachie K; Maeda, Megumi; Wakita, Masahito; Takahama, Kazuo; Noguchi, Tetsuro; Kamei, Shintaro; Hamamoto, Takayoshi; Umehashi, Misako; Maeda, Hiroaki

    2014-01-01

    Human plasma contains wide variety of bioactive proteins that have proved essential in therapeutic discovery. However many human plasma proteins remain orphans with unknown biological functions. Evidences suggest that some plasma components target the respiratory system. In the present study we adapted heparin affinity chromatography to fractionate human plasma for functional bioassay. Fractions from pooled human plasma yielded particular plasma fractions with strong cough suppressing effects. Purification yielded a fraction that was finally identified as an activated blood coagulation factor fXIa using liquid chromatography-tandem mass spectrometry (LC-MS/MS) and matrix-assisted laser desorption ionization/time-of-flight mass spectrometry (MALDI/TOF-MS). The fraction almost completely suppressed coughs induced by either chemical or mechanical stimulation applied to larynx or bifurcation of guinea-pig trachea. Cough suppressing effect of the fraction and commercially available fXIa were one million times stronger than codeine and codeine only partially suppressed the mechanically triggered coughing in animal model. Recent reviews highlighted prominent shortcomings of current available antitussives, including narcotic opioids such as codeine and their unpleasant or intolerable side effects. Therefore, safer and more effective cough suppressants would be welcome, and present findings indicate that fXIa in human plasma as a very promising, new therapeutic candidate for effective antitussive action.

  4. Photothermal Measurements on Human Serum and Plasma

    NASA Astrophysics Data System (ADS)

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

    2003-09-01

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

  5. Accumulation of Paprika Carotenoids in Human Plasma and Erythrocytes.

    PubMed

    Nishino, Azusa; Ichihara, Takashi; Takaha, Takeshi; Kuriki, Takashi; Nihei, Hideko; Kawamoto, Kazuhisa; Yasui, Hiroyuki; Maoka, Takashi

    2015-01-01

    The accumulation (incorporation) of paprika carotenoid in human plasma and erythrocytes was investigated. A paprika carotenoid supplement (14 mg/day) was ingested for 4 weeks by 5 young healthy volunteers (3 men and 2 women). After 2 weeks of carotenoid ingestion, the carotenoid levels in plasma and erythrocytes increased by 1.2-fold and 2.2-fold, respectively. Characteristic carotenoids found in paprika (capsanthin, cucurbitaxanthin A, and cryptocapsin) were detected in both plasma and erythrocytes. An oxidative metabolite of capsanthin (capsanthone) was also found in both plasma and erythrocytes.

  6. Catalytic activity of an in vivo tumor targeted anti-CEA scFv::carboxypeptidase G2 fusion protein.

    PubMed

    Bhatia, J; Sharma, S K; Chester, K A; Pedley, R B; Boden, R W; Read, D A; Boxer, G M; Michael, N P; Begent, R H

    2000-02-15

    Antibody-directed enzyme prodrug therapy (ADEPT) targets an enzyme selectively to a tumor where it converts a relatively non-toxic prodrug to a potent cytotoxic drug. Previous clinical work using antibody-enzyme chemical conjugates has been limited by the moderate efficiency of tumor targeting of these molecules. To address this a recombinant fusion protein composed of MFE-23, an anti-carcinoembryonic antigen (CEA) single chain Fv (scFv) antibody, fused to the amino-terminus of the enzyme carboxypeptidase G2 (CPG2) has been constructed to achieve ADEPT in CEA-producing tumors. MFE-23::CPG2 fusion protein was overexpressed in Escherichia coli and purified using CEA affinity chromatography. Efficacy of MFE-23::CPG2 delivery to tumors in vivo was assessed by measuring catalytic activity after intravenous injection of purified MFE-23::CPG2 into nude mice bearing CEA-positive LS174T human colon adenocarcinoma xenografts. Recombinant MFE-23::CPG2 cleared rapidly from circulation and catalytic activity in extracted tissues showed tumor to plasma ratios of 1.5:1 (6 hr), 10:1 (24 hr), 19:1 (48 hr) and 12:1 (72 hr). (125)I-MFE-23::CPG2 was retained in kidney, liver and spleen but MFE-23::CPG2 catalytic activity was not, resulting in excellent tumor to normal tissue enzyme ratios 48 hr after injection. These were 371:1 (tumor to liver), 450:1 (tumor to lung), 562:1 (tumor to kidney), 1,477:1 (tumor to colon) and 1,618:1 (tumor to spleen). Favorable tumor : normal tissue ratios occurred at early time points when there was still 21% (24 hr) and 9.5% (48 hr) of the injected activity present per gram of tumor tissue. The high tumor concentrations and selective tumor retention of active enzyme delivered by MFE-23::CPG2 establish that this recombinant fusion protein has potential to give improved clinical efficiency for ADEPT.

  7. Molecular cloning, nucleotide sequence, and expression of a carboxypeptidase-encoding gene from the archaebacterium Sulfolobus solfataricus.

    PubMed

    Colombo, S; Toietta, G; Zecca, L; Vanoni, M; Tortora, P

    1995-10-01

    Mammalian metallocarboxypeptidases play key roles in major biological processes, such as digestive-protein degradation and specific proteolytic processing. A Sulfolobus solfataricus gene (cpsA) encoding a recently described zinc carboxypeptidase with an unusually broad substrate specificity was cloned, sequenced, and expressed in Escherichia coli. Despite the lack of overall sequence homology with known carboxypeptidases, seven homology blocks, including the Zn-coordinating and catalytic residues, were identified by multiple alignment with carboxypeptidases A, B, and T. S. solfataricus carboxypeptidase expressed in E. coli was found to be enzymatically active, and both its substrate specificity and thermostability were comparable to those of the purified S. solfataricus enzyme. PMID:7559343

  8. Molecular cloning, nucleotide sequence, and expression of a carboxypeptidase-encoding gene from the archaebacterium Sulfolobus solfataricus.

    PubMed Central

    Colombo, S; Toietta, G; Zecca, L; Vanoni, M; Tortora, P

    1995-01-01

    Mammalian metallocarboxypeptidases play key roles in major biological processes, such as digestive-protein degradation and specific proteolytic processing. A Sulfolobus solfataricus gene (cpsA) encoding a recently described zinc carboxypeptidase with an unusually broad substrate specificity was cloned, sequenced, and expressed in Escherichia coli. Despite the lack of overall sequence homology with known carboxypeptidases, seven homology blocks, including the Zn-coordinating and catalytic residues, were identified by multiple alignment with carboxypeptidases A, B, and T. S. solfataricus carboxypeptidase expressed in E. coli was found to be enzymatically active, and both its substrate specificity and thermostability were comparable to those of the purified S. solfataricus enzyme. PMID:7559343

  9. Molecular cloning, nucleotide sequence, and expression of a carboxypeptidase-encoding gene from the archaebacterium Sulfolobus solfataricus.

    PubMed

    Colombo, S; Toietta, G; Zecca, L; Vanoni, M; Tortora, P

    1995-10-01

    Mammalian metallocarboxypeptidases play key roles in major biological processes, such as digestive-protein degradation and specific proteolytic processing. A Sulfolobus solfataricus gene (cpsA) encoding a recently described zinc carboxypeptidase with an unusually broad substrate specificity was cloned, sequenced, and expressed in Escherichia coli. Despite the lack of overall sequence homology with known carboxypeptidases, seven homology blocks, including the Zn-coordinating and catalytic residues, were identified by multiple alignment with carboxypeptidases A, B, and T. S. solfataricus carboxypeptidase expressed in E. coli was found to be enzymatically active, and both its substrate specificity and thermostability were comparable to those of the purified S. solfataricus enzyme.

  10. Rheology of human blood plasma: viscoelastic versus Newtonian behavior.

    PubMed

    Brust, M; Schaefer, C; Doerr, R; Pan, L; Garcia, M; Arratia, P E; Wagner, C

    2013-02-15

    We investigate the rheological characteristics of human blood plasma in shear and elongational flows. While we can confirm a Newtonian behavior in shear flow within experimental resolution, we find a viscoelastic behavior of blood plasma in the pure extensional flow of a capillary breakup rheometer. The influence of the viscoelasticity of blood plasma on capillary blood flow is tested in a microfluidic device with a contraction-expansion geometry. Differential pressure measurements revealed that the plasma has a pronounced flow resistance compared to that of pure water. Supplementary measurements indicate that the viscoelasticity of the plasma might even lead to viscoelastic instabilities under certain conditions. Our findings show that the viscoelastic properties of plasma should not be ignored in future studies on blood flow.

  11. Purification of selenoprotein P from human plasma using immunoaffinity chromatography

    SciTech Connect

    Aakesson, B.; Bellew, T.; Burk, R.F. )

    1991-03-11

    Selenoprotein P was purified from rat plasma using immunoaffinity chromatography. The same approach was used with human plasma. HepG2 cells were labeled with {sup 75}Se. The labeled medium, containing proteins secreted by the cells, was added to human plasma and the {sup 75}Se was used as a marker for {gt}1,000-fold purification of the major {sup 75}Se-containing protein. This material was used to produce 2 monoclonal antibodies. In a competitive assay, human plasma, but not plasma from 5 other species, inhibited binding of {sup 75}Se by these 2 antibodies. The antibodies were coupled to agarose and columns were made. Human plasma was processed in 2 steps. Step 1 was an antibody column and step 2 was a heparin-agarose column. SDS-PAGE demonstrated bands at 61 and 55 kDa. Both bands stained with PAS. Amino acid analysis of carboxymethylated material indicated that selenocysteine was {gt}1% of the total amino acids. N-terminal sequencing revealed a strong similarity to rat selenoprotein P. Immunodepleted human plasma and control plasma were chromatographed on Sephacryl S200 and selenium was measured in the eluted fractions. Immunodepletion removed one-third of the selenium. The elution pattern of control plasma revealed a broad peak of selenium just ahead of and including the albumin peak. Most of this peak was absent from the immunodepleted serum and a graph of the difference between the 2 chromatograms was a single peak of selenium well separated from the albumin peak.

  12. Plasma expansion does not precipitate the fall in plasma vasopressin in humans drinking isotonic fluids.

    PubMed Central

    Cotter, T P; Gebruers, E M; Hall, W J; O'Sullivan, M F

    1986-01-01

    In a group of healthy humans, plasma vasopressin (AVP) levels fell on drinking either Tyrode or mannitol solutions isosmotic with plasma. Both the timing and magnitude of the fall were appropriate to account for the transient diuresis which followed the drinking. Although plasma expansion follows drinking Tyrode solution it occurred too late to account for the fall in plasma AVP. It was also too small to inhibit AVP secretion. Even though plasma volume tended to contract on drinking isosmotic mannitol solution a fall in plasma AVP and a diuresis occurred, similar to those found after drinking Tyrode solution. These findings appear to eliminate plasma volume expansion as the stimulus for the fall in plasma AVP and the associated diuresis on drinking isotonic fluids. In a further group of human subjects, bypassing the oropharynx by intragastric infusion resulted in a slower onset of diuresis after a water load. We suggest that receptors, as yet undefined, in the upper gastrointestinal tract contribute to the early stages of a water diuresis and account for the apparently inappropriate transient diuresis which follows the drinking of isotonic fluids. PMID:3098967

  13. Recall performance, plasma cortisol and plasma norepinephrine in normal human subjects.

    PubMed

    Bemelmans, Karel J; Goekoop, Jaap G; de Rijk, Roel; van Kempen, Godfried M J

    2003-01-01

    This study investigated hypothalamic-pituitary-adrenal (HPA)-axis and sympathetic nervous system (SNS) correlates of recall performance in normal human subjects. Twenty-two normal human subjects were given one memory task: short-term recall of unrelated non-organizable lists of neutral words, in immediate recall conditions. Two types of memory were individualized: measures reflecting effortful processing and measures reflecting automatic processing, which were related to 3 daytime plasma cortisol (CORT) and plasma NE values, and assessed after venipuncture. It was hypothesized that plasma CORT is positively related and plasma norepinephrine (NE) is negatively related to effortful processing. Pearson correlation was computed and regression analysis was performed. Positive correlation appeared between plasma CORT values and negative correlation appeared between plasma NE values and measures reflecting effortful processing. However, stepwise multiple regression analysis showed that only morning plasma CORT values are functionally positively and afternoon plasma NE values are functionally negatively related to effortful processing. This suggests that morning HPA-axis activities enhance and afternoon SNS activities inhibit effortful processing.

  14. Comparison of a spectrophotometric, a fluorometric, and a novel radiometric assay for carboxypeptidase E and other carboxypeptidase B-like enzymes

    SciTech Connect

    Fricker, L.D.; Devi, L. )

    1990-01-01

    Carboxypeptidase E (CPE) is a carboxypeptidase B-like enzyme involved in the biosynthesis of numerous peptide hormones and neurotransmitters. A sensitive assay for CPE and other carboxypeptidase B-like enzymes has been developed using 125I-acetyl-Tyr-Ala-Arg (125I-AcYAR) as the substrate. This peptide is poorly soluble in ethyl acetate whereas the product of carboxypeptidase B-like enzymatic activity (125I-AcYA) can be quantitatively extracted with this solvent, allowing the rapid separation of product from substrate. This radiometric assay can detect less than 1 pg of either CPE or carboxypeptidase B. For CPE, the assay with 125I-AcYAR is approximately 1000 times more sensitive than a fluorescent assay using dansyl-Phe-Ala-Arg (dans-FAR), and 6000 times more sensitive than a spectrophotometric assay using hippuryl-Arg (hipp-R). CPE hydrolyzes the three substrates with Kcat values of 16 s-1 for AcYAR, 13 s-1 for dans-FAR, and 8.5 s-1 for hipp-R. The Km values for CPE with AcYAR (28 microM) and dans-FAR (34 microM) are similar, and are much lower than the Km with hipp-R (400 microM). Thus, the primary reason for the increased sensitivity of the 125I-AcYAR assay over the fluorescent assay is not a result of kinetic differences but is due to the detection limit of iodinated product (10(-15) mol), compared to the fluorescent product (5 x 10(-11) mol). Applications of this rapid and sensitive radiometric assay to detect CPE in cultured cells and in subcellular fractions of the pituitary are described.

  15. Characterization of an islet carboxypeptidase B involved in prohormone processing.

    PubMed

    Mackin, R B; Noe, B D

    1987-02-01

    An islet carboxypeptidase B-like enzyme (CP B) has been identified and characterized in secretory granules of anglerfish islets. By employing several different column chromatography methods (gel filtration, ion exchange, and hydroxylapatite), it was determined that the islet secretory granules contained only one detectable CP B. This enzyme is present in both secretory granule- and microsome-enriched subcellular fractions and is membrane associated at pH 5.2. The specific activity of the islet CP B was approximately 4-fold higher in the secretory granule- and microsome-enriched subcellular fractions than in the lysosome-enriched fraction. It is a metallo-enzyme that is stimulated by Co++, and has a pH optimum in the range of 5.2-6.2. The isoelectric point of the islet CP B is at pH 4.9. The enzyme is a glycoprotein and has an approximate molecular size of Mr 30,000 by gel filtration. The substrate analogs guanidinoethylmercaptosuccinic acid, guanidinopropylsuccinic acid, and aminopropylmercaptosuccinic acid competitively inhibited the islet CP B with inhibition constant (Ki) values of 23, 21, and 230 nM, respectively. In experiments employing purified prohormone substrates it was demonstrated that the action of a CP B-like enzyme was required for the complete processing of anglerfish proinsulin and prosomatostatin-II. These results indicate that the anglerfish islet CP B is involved in prohormone processing and has properties which are very similar to those of enkephalin convertase. PMID:3542502

  16. A digestive prolyl carboxypeptidase in Tenebrio molitor larvae.

    PubMed

    Goptar, Irina A; Shagin, Dmitry A; Shagina, Irina A; Mudrik, Elena S; Smirnova, Yulia A; Zhuzhikov, Dmitry P; Belozersky, Mikhail A; Dunaevsky, Yakov E; Oppert, Brenda; Filippova, Irina Yu; Elpidina, Elena N

    2013-06-01

    Prolyl carboxypeptidase (PRCP) is a lysosomal proline specific serine peptidase that also plays a vital role in the regulation of physiological processes in mammals. In this report, we isolate and characterize the first PRCP in an insect. PRCP was purified from the anterior midgut of larvae of a stored product pest, Tenebrio molitor, using a three-step chromatography strategy, and it was determined that the purified enzyme was a dimer. The cDNA of PRCP was cloned and sequenced, and the predicted protein was identical to the proteomic sequences of the purified enzyme. The substrate specificity and kinetic parameters of the enzyme were determined. The T. molitor PRCP participates in the hydrolysis of the insect's major dietary proteins, gliadins, and is the first PRCP to be ascribed a digestive function. Our collective data suggest that the evolutionary enrichment of the digestive peptidase complex in insects with an area of acidic to neutral pH in the midgut is a result of the incorporation of lysosomal peptidases, including PRCP.

  17. Radioimmunoassay and characterization of atrial natriuretic peptide in human plasma

    SciTech Connect

    Yandle, T.G.; Espiner, E.A.; Nicholls, M.G.; Duff, H.

    1986-07-01

    A RIA for alpha-human atrial natriuretic peptide (alpha hANP) in plasma was developed and used to study the immunoreactive components secreted by the heart and circulating in peripheral venous plasma. The assay used (125I)diiodotyrosyl-alpha hANP, purified by high pressure liquid chromatography (HPLC), and a C-terminal-specific antiserum purchased from Peninsula Laboratories. Serial dilution curves of coronary sinus plasma samples were parallel with the standard curve, but significant nonparallelism was found in peripheral plasma samples of low immunoreactivity. When plasma was extracted using C-18 Sep-Pak cartridges, serial dilution curves from both coronary sinus and peripheral plasma samples were parallel to the standard curve. Although values for plasma samples assayed before and after extraction agreed closely (r = 0.99; n = 76), immunoreactive ANP in unextracted plasma was consistently greater (70-79 pmol/liter) than in extracts of plasma, suggesting non-specific interference by a component in plasma when assayed without extraction. Mean plasma immunoreactive ANP in 19 normal subjects consuming a normal salt intake was 14 +/- 1 (+/- SE) pmol/liter. In 5 normal men, increasing dietary sodium intake from 10 to 200 mmol sodium/day was associated with a 2-fold increment in ANP levels, and similar changes accompanied acute sodium loading using iv saline. Elevated values were found in patients with congestive heart failure (mean, 58 pmol/liter; range, 0-200; n = 9), chronic renal failure (mean, 118 pmol/liter; range, 30-290; n = 8), and primary aldosteronism (range, 32-90 pmol/liter; n = 3). HPLC and gel chromatographic analysis of the immunoreactive material found in coronary sinus plasma extracts showed that a large amount of the material eluted in the position of alpha hANP.

  18. Cytosolic Carboxypeptidase 1 Is Involved in Processing α- and β-Tubulin*

    PubMed Central

    Berezniuk, Iryna; Vu, Hang T.; Lyons, Peter J.; Sironi, Juan J.; Xiao, Hui; Burd, Berta; Setou, Mitsutoshi; Angeletti, Ruth H.; Ikegami, Koji; Fricker, Lloyd D.

    2012-01-01

    The Purkinje cell degeneration (pcd) mouse has a disruption in the gene encoding cytosolic carboxypeptidase 1 (CCP1). This study tested two proposed functions of CCP1: degradation of intracellular peptides and processing of tubulin. Overexpression (2–3-fold) or knockdown (80–90%) of CCP1 in human embryonic kidney 293T cells (HEK293T) did not affect the levels of most intracellular peptides but altered the levels of α-tubulin lacking two C-terminal amino acids (delta2-tubulin) ≥5-fold, suggesting that tubulin processing is the primary function of CCP1, not peptide degradation. Purified CCP1 produced delta2-tubulin from purified porcine brain α-tubulin or polymerized HEK293T microtubules. In addition, CCP1 removed Glu residues from the polyglutamyl side chains of porcine brain α- and β-tubulin and also generated a form of α-tubulin with two C-terminal Glu residues removed (delta3-tubulin). Consistent with this, pcd mouse brain showed hyperglutamylation of both α- and β-tubulin. The hyperglutamylation of α- and β-tubulin and subsequent death of Purkinje cells in pcd mice was counteracted by the knock-out of the gene encoding tubulin tyrosine ligase-like-1, indicating that this enzyme hyperglutamylates α- and β-tubulin. Taken together, these results demonstrate a role for CCP1 in the processing of Glu residues from β- as well as α-tubulin in vitro and in vivo. PMID:22170066

  19. Serum pyrrolidone carboxypeptidase activity in N-methyl-nitrosourea induced rat breast cancer.

    PubMed

    Carrera, M P; Ramírez-Expósito, M J; Valenzuela, M T; García, M J; Mayas, M D; Martínez-Martos, J M

    2003-08-01

    Pyrrolidone carboxypeptidase (Pcp) (E.C. 3.4.19.3) is an omega peptidase widely distributed in animal fluids and tissues and hydrolyses N-terminal pyroglutamic residues from biologically active peptides such as gonadotropin releasing hormone (GnRH). Previous results obtained by us showed a decrease in human breast cancer Pcp activity, suggesting that this enzyme activity or its putative substrates may play a major role in breast cancer pathogenesis. The aim of the present work is to analyse serum Pcp activity in N-methyl-nitrosourea (NMU) induced rat mammary tumours using pyroglutamyl-beta-naphthylamide as substrate. Serum Pcp activity was significantly lower in NMU-treated rats than in controls. Moreover, multiple regression analysis showed a significant correlation between Pcp activity and the number and size of tumours and the body weight of the animals. Since NMU-induced carcinomas are mainly oestrogen-dependent, the decrease observed in Pcp activity may reflect an increase in circulating levels of GnRH that lead to an increase in gonadal steroid hormones production responsible, at least in part, for the initiation and promotion of the disease.

  20. Determination of Carboxypeptidase Activity in Clinical Pathogens by Gas Chromatography–Mass Spectrometry

    PubMed Central

    Lough, Fraser; Perry, John D.; Stanforth, Stephen P.; Dean, John R.

    2016-01-01

    ABSTRACT A novel method for the determination of benzoic acid has been employed to identify carboxypeptidase activities in clinically relevant pathogens. Benzoic acid was determined after chemical derivatization by gas chromatography–mass spectrometry (GC–MS). N-Benzoyl amino acid substrates were evaluated for the detection of carboxypeptidase activities in a number of clinical pathogens. Upon enzymatic hydrolysis of these substrates, benzoic acid was produced which was detected by extraction from the liquid culture supernatant, derivatization as the trimethylsilyl ester, with subsequent analysis by GC–MS. Enzymatic hydrolysis of N-benzoyl glycine was observed for S. agalactiae, M. morganii, and A. baumannii. In addition, P. fluorescens was found to hydrolyze N-benzoyl-L-glutamic acid. Although the method provides an alternative approach for determining carboxypeptidase activity, ultimately it would not be a suitable method in a clinical setting. However, the method is well-suited for identifying carboxypeptidase activities that have not been previously described or to corroborate a carboxypeptidase assay with the ninhydrin reagent. PMID:27226648

  1. A radioimmunoassay for bone Gla protein (BGP) in human plasma.

    PubMed

    Johansen, J S; Mølholm Hansen, J E; Christiansen, C

    1987-03-01

    To study the value of bone Gla protein (BGP) as a biochemical marker of normal bone physiology and metabolic bone disorders, we have developed a radioimmunoassay (RIA) for the detection of BGP in human plasma. Antibodies were generated in rabbits immunized with purified calf BGP conjugated to thyroglobulin. Human plasma BGP reacted identically with the calf BGP standard, thus demonstrating the suitability of the assay to measure plasma BGP levels in man. The RIA is sensitive, accurate, and technically simple. Plasma BGP levels were determined in normal subjects (N = 35) and in patients with hypothyroidism (N = 10), hyperthyroidism (N = 22) and chronic renal failure (N = 35). The mean (+/- 1 SEM) concentration of plasma BGP in normal subjects was 1.27 +/- 0.07 nmol/l. Plasma BGP was significantly increased in patients with hyperthyroidism, 4.04 +/- 0.78 nmol/l (P less than 0.001) and chronic renal failure, 10.17 +/- 2.47 nmol/l (P less than 0.001). Low concentrations were found in patients with hypothyroidism, 0.74 +/- 0.11 nmol/l (P less than 0.01). Our studies indicate that plasma BGP provides a useful technique in the diagnosis of patients with bone disease.

  2. Human plasma protein N-glycosylation.

    PubMed

    Clerc, Florent; Reiding, Karli R; Jansen, Bas C; Kammeijer, Guinevere S M; Bondt, Albert; Wuhrer, Manfred

    2016-06-01

    Glycosylation is the most abundant and complex protein modification, and can have a profound structural and functional effect on the conjugate. The oligosaccharide fraction is recognized to be involved in multiple biological processes, and to affect proteins physical properties, and has consequentially been labeled a critical quality attribute of biopharmaceuticals. Additionally, due to recent advances in analytical methods and analysis software, glycosylation is targeted in the search for disease biomarkers for early diagnosis and patient stratification. Biofluids such as saliva, serum or plasma are of great use in this regard, as they are easily accessible and can provide relevant glycosylation information. Thus, as the assessment of protein glycosylation is becoming a major element in clinical and biopharmaceutical research, this review aims to convey the current state of knowledge on the N-glycosylation of the major plasma glycoproteins alpha-1-acid glycoprotein, alpha-1-antitrypsin, alpha-1B-glycoprotein, alpha-2-HS-glycoprotein, alpha-2-macroglobulin, antithrombin-III, apolipoprotein B-100, apolipoprotein D, apolipoprotein F, beta-2-glycoprotein 1, ceruloplasmin, fibrinogen, immunoglobulin (Ig) A, IgG, IgM, haptoglobin, hemopexin, histidine-rich glycoprotein, kininogen-1, serotransferrin, vitronectin, and zinc-alpha-2-glycoprotein. In addition, the less abundant immunoglobulins D and E are included because of their major relevance in immunology and biopharmaceutical research. Where available, the glycosylation is described in a site-specific manner. In the discussion, we put the glycosylation of individual proteins into perspective and speculate how the individual proteins may contribute to a total plasma N-glycosylation profile determined at the released glycan level. PMID:26555091

  3. Statistical Analysis of Variation in the Human Plasma Proteome

    DOE PAGES

    Corzett, Todd H.; Fodor, Imola K.; Choi, Megan W.; Walsworth, Vicki L.; Turteltaub, Kenneth W.; McCutchen-Maloney, Sandra L.; Chromy, Brett A.

    2010-01-01

    Quantifying the variation in the human plasma proteome is an essential prerequisite for disease-specific biomarker detection. We report here on the longitudinal and individual variation in human plasma characterized by two-dimensional difference gel electrophoresis (2-D DIGE) using plasma samples from eleven healthy subjects collected three times over a two week period. Fixed-effects modeling was used to remove dye and gel variability. Mixed-effects modeling was then used to quantitate the sources of proteomic variation. The subject-to-subject variation represented the largest variance component, while the time-within-subject variation was comparable to the experimental variation found in a previous technical variability study where onemore » human plasma sample was processed eight times in parallel and each was then analyzed by 2-D DIGE in triplicate. Here, 21 protein spots had larger than 50% CV, suggesting that these proteins may not be appropriate as biomarkers and should be carefully scrutinized in future studies. Seventy-eight protein spots showing differential protein levels between different individuals or individual collections were identified by mass spectrometry and further characterized using hierarchical clustering. The results present a first step toward understanding the complexity of longitudinal and individual variation in the human plasma proteome, and provide a baseline for improved biomarker discovery.« less

  4. Purification and primary structure determination of human lysosomal dipeptidase.

    PubMed

    Dolenc, Iztok; Mihelic, Marko

    2003-02-01

    The lysosomal metallopeptidase is an enzyme that acts preferentially on dipeptides with unsubstituted N- and C-termini. Its activity is highest in slightly acidic pH. Here we describe the isolation and characterization of lysosomal dipeptidase from human kidney. The isolated enzyme has the amino-terminal sequence DVAKAIINLAVY and is a homodimer with a molecular mass of 100 kDa. So far no amino acid sequence has been determined for this metallopeptidase. The complete primary structure as deduced from the nucleotide sequence revealed that the isolated dipeptidase is similar to blood plasma glutamate carboxypeptidase.

  5. Characterization of human plasma proteome dynamics using deuterium oxide

    PubMed Central

    Wang, Ding; Liem, David A; Lau, Edward; Ng, Dominic CM; Bleakley, Brian J; Cadeiras, Martin; Deng, Mario C; Lam, Maggie PY; Ping, Peipei

    2016-01-01

    Purpose High-throughput quantification of human protein turnover via in vivo administration of deuterium oxide (2H2O) is a powerful new approach to examine potential disease mechanisms. Its immediate clinical translation is contingent upon characterizations of the safety and hemodynamic effects of in vivo administration of 2H2O to human subjects. Experimental design We recruited 10 healthy human subjects with a broad demographic variety to evaluate the safety, feasibility, efficacy, and reproducibility of 2H2O intake for studying protein dynamics. We designed a protocol where each subject orally consumed weight-adjusted doses of 70% 2H2O daily for 14 days to enrich body water and proteins with deuterium. Plasma proteome dynamics was measured using a high-resolution MS method we recently developed. Results This protocol was successfully applied in 10 human subjects to characterize the endogenous turnover rates of 542 human plasma proteins, the largest such human dataset to-date. Throughout the study, we did not detect physiological effects or signs of discomfort from 2H2O consumption. Conclusions and clinical relevance Our investigation supports the utility of a 2H2O intake protocol that is safe, accessible, and effective for clinical investigations of large-scale human protein turnover dynamics. This workflow shows promising clinical translational value for examining plasma protein dynamics in human diseases. PMID:24946186

  6. Human plasma kallikrein releases neutrophil elastase during blood coagulation.

    PubMed Central

    Wachtfogel, Y T; Kucich, U; James, H L; Scott, C F; Schapira, M; Zimmerman, M; Cohen, A B; Colman, R W

    1983-01-01

    Elastase is released from human neutrophils during the early events of blood coagulation. Human plasma kallikrein has been shown to stimulate neutrophil chemotaxis, aggregation, and oxygen consumption. Therefore, the ability of kallikrein to release neutrophil elastase was investigated. Neutrophils were isolated by dextran sedimentation, and elastase release was measured by both an enzyme-linked immunosorbent assay, and an enzymatic assay using t-butoxy-carbonyl-Ala-Ala-Pro-Val-amino methyl coumarin as the substrate. Kallikrein, 0.1-1.0 U/ml, (0.045-0.45 microM), was incubated with neutrophils that were preincubated with cytochalasin B (5 micrograms/ml). The release of elastase was found to be proportional to the kallikrein concentration. Kallikrein released a maximum of 34% of the total elastase content, as measured by solubilizing the neutrophils in the nonionic detergent Triton X-100. A series of experiments was carried out to determine if kallikrein was a major enzyme involved in neutrophil elastase release during blood coagulation. When 10 million neutrophils were incubated in 1 ml of normal plasma in the presence of 30 mM CaCl2 for 90 min, 2.75 micrograms of elastase was released. In contrast, neutrophils incubated in prekallikrein-deficient or Factor XII-deficient plasma released less than half of the elastase, as compared with normal plasma. The addition of purified prekallikrein to prekallikrein-deficient plasma restored neutrophil elastase release to normal levels. Moreover, release of elastase was enhanced in plasma deficient in C1-inhibitor, the major plasma inhibitor of kallikrein. This release was not dependent upon further steps in the coagulation pathway, or on C5a, since levels of elastase, released in Factor XI- or C5-deficient plasma, were similar to that in normal plasma, and an antibody to C5 failed to inhibit elastase release. These data suggest that kallikrein may be a major enzyme responsible for the release of elastase during blood

  7. Detection of RSV Antibodies in Human Plasma by Enzyme Immunoassays.

    PubMed

    Jadhao, Samadhan J; Anderson, Larry J

    2016-01-01

    Enzyme immunoassays (EIAs) to detect and quantify antibodies against respiratory syncytial virus (RSV) and RSV proteins in human plasma or sera are described. The first EIA uses RSV lysate antigens produced in HEp-2 cell line. The second EIA uses RSV F or G gene-expressed antigen in HEp-2 cells. The third EIA uses 30-amino acid synthetic peptides from central conserved region of G protein of RSV A2 or RSV B1 virus and a peptide from the SARS CoV nucleoprotein as a negative control peptide. All three EIAs have been evaluated for detecting and quantifying the respective antibodies in human sera or plasma. PMID:27464686

  8. Sensitive radiometric assay for enkephalin convertase and other carboxypeptidase B-like enzymes

    SciTech Connect

    Stack, G.; Fricker, L.D.; Snyder, S.H.

    1984-01-09

    A sensitive radiometric assay for carboxypeptidase B-like enzymes has been developed using enkephalin convertase, an enkephalin synthesizing carboxypeptidase. The assay is based on the differential solubility of /sup 3/H-labeled substrate and product in chloroform. The substrates /sup 3/H-benzoyl-Phe-Ala-Arg or /sup 3/H-benzoyl-Phe-Leu-Arg are poorly soluble in chloroform due to the charged arginine. The products of carboxypeptidase B-like activity on these substrates, /sup 3/H-benzoyl-Phe-Ala or /sup 3/H-benzoyl Phe-Leu partition quantitatively into chloroform, allowing rapid separation of product from substrate. This assay is approximately 100 times more sensitive than a similar fluorometric assay utilizing dansyl-Phe-Ala-Arg as a substrate.

  9. An extraovarian protein accumulated in mosquito oocytes is a carboxypeptidase activated in embryos

    SciTech Connect

    Wenlong Cho; Deitsch, K.W.; Raikhel, A.S. )

    1991-12-01

    The authors report a phenomenon previously unknown for oviparous animals; in Aedes aegypti mosquitoes a serine carboxypeptidase is synthesized extraovarially and then internalized by oocytes. The cDNA encoding mosquito vitellogenic carboxypeptidase (VCP) was cloned and sequenced. The VCP cDNA hybridizes to a 1.5-kilobase mRNA present only in the fat body of vitellogenic females. The deduced amino acid sequence of VCP shares significant homology with members of the serine carboxypeptidase family. Binding assays using a serine protease inhibitor, ({sup 3}H)diisopropyl fluorophosphate, showed that VCP is activated in eggs at the onset of embryonic development. Activation of VCP is associated with the reduction in its size from 53 kDa (inactive proenzyme) to 48 kDa (active enzyme). The active, 48-kDa, form of VCP is maximally present at the middle of embryonic development and disappears by the end.

  10. Variability in pK'1 of human plasma.

    PubMed

    Tibi, L; Bhattacharya, S S; Flear, C T

    1982-05-01

    (1) Results were presented of an investigation of the relationship between ionic strength and PK'1 (negative logarithmic form of the apparent overall first dissociation constant of carbonic acid in plasma) in separated human plasma at constant PCO2. (2) Ionic strength was varied by adding dry NaCl to diluted aliquots of plasma from six healthy people and dry NaCl plus dry NaHCO3 to diluted aliquots of plasma from six other healthy people. pK'1 was determined from simultaneous measurements of pH and PCO2, and measurements of TCO2. Values for solubility factor for CO2 (s) were corrected for differences in plasma water and in Na concentration. All plasmas were equilibrated at 37 degrees C in a tonometer with a constant gas mixture (5% CO2, 12% O2, 83% N2). Precision of pK'1 determinations averaged 0.003. pK'1 was also determined on fresh undiluted healthy plasma, similarly tonometered. (3) We report (a) considerable variability in pK'1 of fresh undiluted healthy plasma (from 6.0197 to 6.1217); and also in extrapolated values for pK'1 at a notional zero ionic strength (6.179 to 6.325); (b) that variation in plasma ionic strength alters pK'1; (c) that change in plasma bicarbonate [HCO3]p can also change pK'1; (d) that change in pK'1, changes measured pH. (4) Implications are discussed. PMID:6805986

  11. Dysregulation of glutamate carboxypeptidase II in psychiatric disease.

    PubMed

    Guilarte, Tomás R; Hammoud, Dima A; McGlothan, Jennifer L; Caffo, Brian S; Foss, Catherine A; Kozikowski, Alan P; Pomper, Martin G

    2008-02-01

    Experimental evidence is beginning to converge on an important role for dysregulation of glutamate carboxypeptidase II (GCPII) in schizophrenia. The goal of this study was to determine GCPII levels in postmortem brain specimens of patients with schizophrenia, bipolar disorder or unipolar depression and age-matched control subjects. We used N-[N-(S)-1,3-dicarboxypropyl]carbamoyl]-S-3-[(125)I]iodo-l-tyrosine ([(125)I]DCIT), a high-affinity radioligand for GCPII, to probe for GCPII expression in prefrontal cortex (PFC) and mesial temporal lobe, two brain regions implicated in the pathophysiology of schizophrenia. We found that GCPII levels measured by [(125)I]DCIT quantitative autoradiography were significantly lower in the PFC and entorhinal cortex in patients with schizophrenia compared to age-matched controls. Patients with bipolar disorder also expressed significantly lower GCPII levels in PFC than controls. The decrease in [(125)I]DCIT binding in schizophrenia and bipolar disorder remained significant after adjusting for drug abuse. A significant difference in GCPII levels was also observed between schizophrenia relative to bipolar disorder and depressed subjects in the hippocampus-stratum lucidum and between schizophrenia and bipolar in the CA2 region of the hippocampus, with bipolar and depressed subjects expressing higher levels of GCPII than subjects with schizophrenia. These differences in hippocampal GCPII levels may implicate differences in the etiologies of these mental disorders. In summary, this study demonstrates a regional dysregulation of GCPII expression in the brain of patients with schizophrenia and other psychiatric disorders and supports a hypoglutamatergic state of the former illness. GCPII may represent a viable therapeutic target for intervention in psychiatric disease. PMID:18191545

  12. Carbohydrate chains on yeast carboxypeptidase Y are phosphorylated.

    PubMed Central

    Hashimoto, C; Cohen, R E; Zhang, W J; Ballou, C E

    1981-01-01

    Carboxypeptidase Y, a vacuolar enzyme from Saccharomyces cerevisiae, was digested with endo-beta-N-acetyl-D-glucosaminidase H to release the four oligosaccharide chains that are linked to asparagine in the glycoprotein. The oligosaccharides were fractionated into a neutral and acidic component, and the latter proved to phosphorylated. From its gel filtration pattern, the neutral fraction was shown to be a mixture of at least four homologs, the smallest of which had a proton NMR spectrum almost identical to that given by an IgM oligosaccharide with eight mannoses and one N-acetylglucosamine [Cohen, R. E. & Ballou, C. E. (1980) Biochemistry 19, 4345--4358]. The yeast oligosaccharide has one additional mannose unit in an alpha 1 leads to 3 or alpha 1 leads to 6 linkage, whereas the larger homologs appear to have two, three, and four more mannose units. One phosphorylated oligosaccharides with a mannose/phosphate ratio of 12.5 was reduced with NaB3H4 and then subjected to mild acid hydrolysis. This released mannose and mannobiose that were glycosidically linked to the phosphate group, whereas complete acid hydrolysis yielded D-mannose 6-phosphate. The recovered oligosaccharide phosphomonoester, which contained 11 or 12 mannose units, was digested exhaustively with alpha-mannosidase, and the product of this reaction was treated with alkaline phosphatase, which yielded radioactive Man3GlcNAcH2. These results suggest that the mannosidase-resistant phosphorylated oligosaccharide has the structure Man leads to P leads to 6 alpha Man leads to alpha Man leads to 6 beta Man leads to 4GlcNAcH2, in which some of the phosphate groups are substituted with mannobiose instead of mannose. A second phosphorylated oligosaccharide with a mannose/phosphate ratio of 6.5 probably contains two phosphodiester groups, but its structure has not been investigated in detail. Images PMID:7017728

  13. Recombinant human LCAT normalizes plasma lipoprotein profile in LCAT deficiency.

    PubMed

    Simonelli, Sara; Tinti, Cristina; Salvini, Laura; Tinti, Laura; Ossoli, Alice; Vitali, Cecilia; Sousa, Vitor; Orsini, Gaetano; Nolli, Maria Luisa; Franceschini, Guido; Calabresi, Laura

    2013-11-01

    Lecithin:cholesterol acyltransferase (LCAT) is the enzyme responsible for cholesterol esterification in plasma. Mutations in the LCAT gene leads to two rare disorders, familial LCAT deficiency and fish-eye disease, both characterized by severe hypoalphalipoproteinemia associated with several lipoprotein abnormalities. No specific treatment is presently available for genetic LCAT deficiency. In the present study, recombinant human LCAT was expressed and tested for its ability to correct the lipoprotein profile in LCAT deficient plasma. The results show that rhLCAT efficiently reduces the amount of unesterified cholesterol (-30%) and promotes the production of plasma cholesteryl esters (+210%) in LCAT deficient plasma. rhLCAT induces a marked increase in HDL-C levels (+89%) and induces the maturation of small preβ-HDL into alpha-migrating particles. Moreover, the abnormal phospholipid-rich particles migrating in the LDL region were converted in normally sized LDL.

  14. Human parvovirus PARV4 in plasma pools of Chinese origin.

    PubMed

    Ma, Y-Y; Guo, Y; Zhao, X; Wang, Z; Lv, M-M; Yan, Q-P; Zhang, J-G

    2012-10-01

    Human parvovirus 4 (PARV4) is present in blood and blood products. As the presence and levels of PARV4 in Chinese source plasma pools have never been determined, we implemented real-time quantitative PCR to investigate the presence of PARV4 in source plasma pools in China. Results showed that 26·15% (51/195) of lots tested positive for PARV4. The amounts of DNA ranged from 2·83 × 10(3) copies/ml to 2·35×10(7) copies/ml plasma. The high level of PARV4 in plasma pools may pose a potential risk to recipients. Further studies on the pathogenesis of PARV4 are urgently required.

  15. Recombinant human LCAT normalizes plasma lipoprotein profile in LCAT deficiency.

    PubMed

    Simonelli, Sara; Tinti, Cristina; Salvini, Laura; Tinti, Laura; Ossoli, Alice; Vitali, Cecilia; Sousa, Vitor; Orsini, Gaetano; Nolli, Maria Luisa; Franceschini, Guido; Calabresi, Laura

    2013-11-01

    Lecithin:cholesterol acyltransferase (LCAT) is the enzyme responsible for cholesterol esterification in plasma. Mutations in the LCAT gene leads to two rare disorders, familial LCAT deficiency and fish-eye disease, both characterized by severe hypoalphalipoproteinemia associated with several lipoprotein abnormalities. No specific treatment is presently available for genetic LCAT deficiency. In the present study, recombinant human LCAT was expressed and tested for its ability to correct the lipoprotein profile in LCAT deficient plasma. The results show that rhLCAT efficiently reduces the amount of unesterified cholesterol (-30%) and promotes the production of plasma cholesteryl esters (+210%) in LCAT deficient plasma. rhLCAT induces a marked increase in HDL-C levels (+89%) and induces the maturation of small preβ-HDL into alpha-migrating particles. Moreover, the abnormal phospholipid-rich particles migrating in the LDL region were converted in normally sized LDL. PMID:24140107

  16. Coffee polyphenols protect human plasma from postprandial carbonyl modifications.

    PubMed

    Sirota, Roman; Gorelik, Shlomit; Harris, Raviv; Kohen, Ron; Kanner, Joseph

    2013-05-01

    The antioxidant capability of coffee polyphenols to inhibit red-meat lipid peroxidation in stomach medium and absorption into blood of malondialdehyde (MDA) in humans was studied. Roasted-ground coffee polyphenols that were found to inhibit lipid peroxidation in stomach medium are 2- to 5-fold more efficient antioxidant than those found in instant coffee. Human plasma from ten volunteers analyzed after a meal of red-meat cutlets (250 g) revealed a rapid accumulation of MDA. The accumulation of MDA in human plasma modified low-density lipoprotein is known to trigger atherogenesis. Consumption of 200 mL roasted coffee by ten volunteers during a meal of red-meat cutlets, resulted after 2 and 4 h in the inhibition by 80 and 50%, respectively, of postprandial plasma MDA absorption. The results obtained in vitro simulated stomach model on MDA accumulation were predictive for the amount of MDA absorbed into circulating human plasma, in vivo. Timing the consumption of coffee during the meals may make it a very active functional food.

  17. The Cell Shape-determining Csd6 Protein from Helicobacter pylori Constitutes a New Family of l,d-Carboxypeptidase*

    PubMed Central

    Kim, Hyoun Sook; Im, Ha Na; An, Doo Ri; Yoon, Ji Young; Jang, Jun Young; Mobashery, Shahriar; Hesek, Dusan; Lee, Mijoon; Yoo, Jakyung; Cui, Minghua; Choi, Sun; Kim, Cheolhee; Lee, Nam Ki; Kim, Soon-Jong; Kim, Jin Young; Bang, Geul; Han, Byung Woo; Lee, Byung Il; Yoon, Hye Jin; Suh, Se Won

    2015-01-01

    Helicobacter pylori causes gastrointestinal diseases, including gastric cancer. Its high motility in the viscous gastric mucosa facilitates colonization of the human stomach and depends on the helical cell shape and the flagella. In H. pylori, Csd6 is one of the cell shape-determining proteins that play key roles in alteration of cross-linking or by trimming of peptidoglycan muropeptides. Csd6 is also involved in deglycosylation of the flagellar protein FlaA. To better understand its function, biochemical, biophysical, and structural characterizations were carried out. We show that Csd6 has a three-domain architecture and exists as a dimer in solution. The N-terminal domain plays a key role in dimerization. The middle catalytic domain resembles those of l,d-transpeptidases, but its pocket-shaped active site is uniquely defined by the four loops I to IV, among which loops I and III show the most distinct variations from the known l,d-transpeptidases. Mass analyses confirm that Csd6 functions only as an l,d-carboxypeptidase and not as an l,d-transpeptidase. The d-Ala-complexed structure suggests possible binding modes of both the substrate and product to the catalytic domain. The C-terminal nuclear transport factor 2-like domain possesses a deep pocket for possible binding of pseudaminic acid, and in silico docking supports its role in deglycosylation of flagellin. On the basis of these findings, it is proposed that H. pylori Csd6 and its homologs constitute a new family of l,d-carboxypeptidase. This work provides insights into the function of Csd6 in regulating the helical cell shape and motility of H. pylori. PMID:26306031

  18. Molecular interactions of graphene oxide with human blood plasma proteins

    NASA Astrophysics Data System (ADS)

    Kenry, Affa Affb Affc; Loh, Kian Ping; Lim, Chwee Teck

    2016-04-01

    We investigate the molecular interactions between graphene oxide (GO) and human blood plasma proteins. To gain an insight into the bio-physico-chemical activity of GO in biological and biomedical applications, we performed a series of biophysical assays to quantify the molecular interactions between GO with different lateral size distributions and the three essential human blood plasma proteins. We elucidate the various aspects of the GO-protein interactions, particularly, the adsorption, binding kinetics and equilibrium, and conformational stability, through determination of quantitative parameters, such as GO-protein association constants, binding cooperativity, and the binding-driven protein structural changes. We demonstrate that the molecular interactions between GO and plasma proteins are significantly dependent on the lateral size distribution and mean lateral sizes of the GO nanosheets and their subtle variations may markedly influence the GO-protein interactions. Consequently, we propose the existence of size-dependent molecular interactions between GO nanosheets and plasma proteins, and importantly, the presence of specific critical mean lateral sizes of GO nanosheets in achieving very high association and fluorescence quenching efficiency of the plasma proteins. We anticipate that this work will provide a basis for the design of graphene-based and other related nanomaterials for a plethora of biological and biomedical applications.

  19. Survey of Argentinean human plasma for ochratoxin A.

    PubMed

    Pacin, A M; Ciancio Bovier, E V; Motta, E; Resnik, S L; Villa, D; Olsen, M

    2008-05-01

    The presence of ochratoxin A (OTA) in human blood has been reported for many countries, especially in Europe. However, so far no report exists concerning such a presence in Argentina. The aim of this study was to assess OTA concentration in human plasma in two different areas of Buenos Aires province. OTA was determined by high-performance liquid chromatography (HPLC) in 199 plasma samples from blood donors in Mar del Plata and 236 from General Rodriguez. Solid-phase extraction with Bakerbond C-18 cartridge and a final purification with Ochraprep immunoaffinity columns was employed. The limit of quantification of ochratoxin A was 0.019ngml(-1) and the confirmation of OTA was by formation of ochratoxin A methyl ester. The results showed that 63.8% of human plasma samples from Mar del Plata and 62.3% from General Rodriguez were positive for OTA, with Winsorized means of 0.15 and 0.43ngml(-1), respectively. It is important to continue the research to detect the foods responsible of the presence of OTA in plasma. PMID:18473217

  20. Cardiovascular-related proteins identified in human plasma by the HUPO Plasma Proteome Project pilot phase.

    PubMed

    Berhane, Beniam T; Zong, Chenggong; Liem, David A; Huang, Aaron; Le, Steven; Edmondson, Ricky D; Jones, Richard C; Qiao, Xin; Whitelegge, Julian P; Ping, Peipei; Vondriska, Thomas M

    2005-08-01

    Proteomic profiling of accessible bodily fluids, such as plasma, has the potential to accelerate biomarker/biosignature development for human diseases. The HUPO Plasma Proteome Project pilot phase examined human plasma with distinct proteomic approaches across multiple laboratories worldwide. Through this effort, we confidently identified 3020 proteins, each requiring a minimum of two high-scoring MS/MS spectra. A critical step subsequent to protein identification is functional annotation, in particular with regard to organ systems and disease. Performing exhaustive literature searches, we have manually annotated a subset of these 3020 proteins that have cardiovascular-related functions on the basis of an existing body of published information. These cardiovascular-related proteins can be organized into eight groups: markers of inflammation and/or cardiovascular disease, vascular and coagulation, signaling, growth and differentiation, cytoskeletal, transcription factors, channels/receptors and heart failure and remodeling. In addition, analysis of the peptide per protein ratio for MS/MS identification reveals group-specific trends. These findings serve as a resource to interrogate the functions of plasma proteins, and moreover, the list of cardiovascular-related proteins in plasma constitutes a baseline proteomic blueprint for the future development of biosignatures for diseases such as myocardial ischemia and atherosclerosis. PMID:16052623

  1. Human red blood cells' physiological water exchange with the plasma.

    PubMed

    Kargol, M; Kargol, A; Przestalski, M; Siedlecki, J; Karpińska, M; Rogowski, M

    2005-01-01

    In the present paper, fundamental issues related to the mechanisms of human red blood cells' physiological water exchange with the plasma (for the stationary conditions) have been discussed. It has been demonstrated, on the basis of mechanistic transport equations for membrane transport that red blood cells are capable of exchanging considerable amounts of water with the plasma. Water absorption is osmosis-driven, and its removal occurs according to the hydromechanics principle, i.e. is driven by the turgor pressure of red blood cells. This newly-acquired knowledge of these issues may appear highly useful for clinical diagnosis of blood diseases and blood circulation failures. PMID:16358974

  2. Murein structure and lack of DD- and LD-carboxypeptidase activities in Caulobacter crescentus.

    PubMed Central

    Markiewicz, Z; Glauner, B; Schwarz, U

    1983-01-01

    High-pressure liquid chromatography of a muramidase digest of murein sacculi from Caulobacter crescentus showed that the absence of D-alanine carboxypeptidase activity in the cells was reflected by a very high content of pentapeptide in the murein. Approximately half of the pentapeptide side chains were shown to contain glycine, which replaced D-alanine as the terminal amino acid. PMID:6630150

  3. Carboxypeptidase D is the only enzyme responsible for antibody C-terminal lysine cleavage in Chinese hamster ovary (CHO) cells.

    PubMed

    Hu, Zhilan; Zhang, Henry; Haley, Benjamin; Macchi, Frank; Yang, Feng; Misaghi, Shahram; Elich, Joseph; Yang, Renee; Tang, Yun; Joly, John C; Snedecor, Bradley R; Shen, Amy

    2016-10-01

    Heterogeneity of C-terminal lysine levels often observed in therapeutic monoclonal antibodies is believed to result from the proteolysis by endogenous carboxypeptidase(s) during cell culture production. Identifying the responsible carboxypeptidase(s) for C-terminal lysine cleavage in CHO cells would provide valuable insights for antibody production cell culture processes development and optimization. In this study, five carboxypeptidases, CpD, CpM, CpN, CpB, and CpE, were studied for message RNA (mRNA) expression by qRT-PCR analysis in two most commonly used blank hosts (DUXB-11 derived DHFR-deficient DP12 host and DHFR-positive CHOK1 host), used for therapeutic antibody production, as well an antibody-expressing cell line derived from each host. Our results showed that CpD had the highest mRNA expression. When CpD mRNA levels were reduced by RNAi (RNA interference) technology, C-terminal lysine levels increased, whereas there was no obvious change in C-terminal lysine levels when a different carboxypeptidase mRNA level was knocked down suggesting that carboxypeptidase D is the main contributor for C-terminal lysine processing. Most importantly, when CpD expression was knocked out by CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) technology, C-terminal lysine cleavage was completely abolished in CpD knockout cells based on mass spectrometry analysis, demonstrating that CpD is the only endogenous carboxypeptidase that cleaves antibody heavy chain C-terminal lysine in CHO cells. Hence, our work showed for the first time that the cleavage of antibody heavy chain C-terminal lysine is solely mediated by the carboxypeptidase D in CHO cells and our finding provides one solution to eliminating C-terminal lysine heterogeneity for therapeutic antibody production by knocking out CpD gene expression. Biotechnol. Bioeng. 2016;113: 2100-2106. © 2016 Wiley Periodicals, Inc.

  4. Abrogation of immune complex glomerulonephritis by native carboxypeptidase and pharmacological antagonism of the C5a receptor

    PubMed Central

    Alexander, Jessy J.; Chaves, Lee D.; Chang, Anthony; Dighe, Shruti; Jacob, Alexander; Quigg, Richard J.

    2016-01-01

    Activation of complement generates C5a which leads to signaling through C5aR1. This is tightly controlled, including by the plasma proteins factor H (FH) and carboxypeptidase N. Here we studied a chronic serum sickness (CSS) model of glomerulonephritis (GN) in which there is an active humoral immune response, formation of glomerular immune complexes (ICs), and resulting glomerular inflammation. The antibody response, glomerular IC deposition, the degree of GN, and consequent renal functional insufficiency in CSS were all worse in FH−/− mice compared to wild-type FH+/+ animals. This was ameliorated in the former by giving a C5aR1 antagonist for the final 3 weeks of the 5-week protocol. In contrast, blocking CP-mediated inactivation of C5a increased these disease measures. Thus, complement regulation by both plasma FH and CP to limit the quantity of active C5a is important in conditions where the humoral immune response is directed to a continuously present foreign antigen. Signaling through C5aR1 enhances the humoral immune response as well as the inflammatory response to ICs that have formed in glomeruli. Both effects are relevant even after disease has begun. Thus, pharmacological targeting of C5a in IC-mediated GN has potential clinical relevance. PMID:26166765

  5. Carboxypeptidase E protects hippocampal neurons during stress in male mice by up-regulating prosurvival BCL2 protein expression.

    PubMed

    Murthy, S R K; Thouennon, E; Li, W-S; Cheng, Y; Bhupatkar, J; Cawley, N X; Lane, M; Merchenthaler, I; Loh, Y P

    2013-09-01

    Prolonged chronic stress causing elevated plasma glucocorticoids leads to neurodegeneration. Adaptation to stress (allostasis) through neuroprotective mechanisms can delay this process. Studies on hippocampal neurons have identified carboxypeptidase E (CPE) as a novel neuroprotective protein that acts extracellularly, independent of its enzymatic activity, although the mechanism of action is unclear. Here, we aim to determine if CPE plays a neuroprotective role in allostasis in mouse hippocampus during chronic restraint stress (CRS), and the molecular mechanisms involved. Quantitative RT-PCR/in situ hybridization and Western blots were used to assay for mRNA and protein. After mild CRS (1 h/d for 7 d), CPE protein and mRNA were significantly elevated in the hippocampal CA3 region, compared to naïve littermates. In addition, luciferase reporter assays identified a functional glucocorticoid regulatory element within the cpe promoter that mediated the up-regulation of CPE expression in primary hippocampal neurons following dexamethasone treatment, suggesting that circulating plasma glucocorticoids could evoke a similar effect on CPE in the hippocampus in vivo. Overexpression of CPE in hippocampal neurons, or CRS in mice, resulted in elevated prosurvival BCL2 protein/mRNA and p-AKT levels in the hippocampus; however, CPE(-/-) mice showed a decrease. Thus, during mild CRS, CPE expression is up-regulated, possibly contributed by glucocorticoids, to mediate neuroprotection of the hippocampus by enhancing BCL2 expression through AKT signaling, and thereby maintaining allostasis.

  6. Structural basis for the recognition of muramyltripeptide by Helicobacter pylori Csd4, a d,l-carboxypeptidase controlling the helical cell shape

    PubMed Central

    Kim, Hyoun Sook; Kim, Jieun; Im, Ha Na; An, Doo Ri; Lee, Mijoon; Hesek, Dusan; Mobashery, Shahriar; Kim, Jin Young; Cho, Kun; Yoon, Hye Jin; Han, Byung Woo; Lee, Byung Il; Suh, Se Won

    2014-01-01

    Helicobacter pylori infection causes a variety of gastrointestinal diseases, including peptic ulcers and gastric cancer. Its colonization of the gastric mucosa of the human stomach is a prerequisite for survival in the stomach. Colonization depends on its motility, which is facilitated by the helical shape of the bacterium. In H. pylori, cross-linking relaxation or trimming of peptidoglycan muropeptides affects the helical cell shape. Csd4 has been identified as one of the cell shape-determining peptidoglycan hydrolases in H. pylori. It is a Zn2+-dependent d,l-carboxypeptidase that cleaves the bond between the γ-d-Glu and the mDAP of the non-cross-linked muramyl­tripeptide (muramyl-l-Ala-γ-d-Glu-mDAP) of the peptidoglycan to produce the muramyldipeptide (muramyl-l-Ala-γ-d-Glu) and mDAP. Here, the crystal structure of H. pylori Csd4 (HP1075 in strain 26695) is reported in three different states: the ligand-unbound form, the substrate-bound form and the product-bound form. H. pylori Csd4 consists of three domains: an N-terminal d,l-carboxypeptidase domain with a typical carboxy­peptidase fold, a central β-barrel domain with a novel fold and a C-terminal immunoglobulin-like domain. The d,l-carboxypeptidase domain recognizes the substrate by interacting primarily with the terminal mDAP moiety of the muramyltripeptide. It undergoes a significant structural change upon binding either mDAP or the mDAP-containing muramyl­tripeptide. It it also shown that Csd5, another cell-shape determinant in H. pylori, is capable of interacting not only with H. pylori Csd4 but also with the dipeptide product of the reaction catalyzed by Csd4. PMID:25372672

  7. Advanced Plasma Propulsion for Human Missions to Jupiter

    NASA Technical Reports Server (NTRS)

    Donahue, Benjamin B.; Pearson, J. Boise

    1999-01-01

    This paper will briefly identify a promising fusion plasma power source, which when coupled with a promising electric thruster technology would provide for an efficient interplanetary transfer craft suitable to a 4 year round trip mission to the Jovian system. An advanced, nearly radiation free Inertial Electrostatic Confinement scheme for containing fusion plasma was judged as offering potential for delivering the performance and operational benefits needed for such high energy human expedition missions, without requiring heavy superconducting magnets for containment of the fusion plasma. Once the Jovian transfer stage has matched the heliocentric velocity of Jupiter, the energy requirements for excursions to its outer satellites (Callisto, Ganymede and Europa) by smaller excursion craft are not prohibitive. The overall propulsion, power and thruster system is briefly described and a preliminary vehicle mass statement is presented.

  8. Reaction Mechanism of Glutamate Carboxypeptidase II Revealed by Mutagenesis, X-ray Crystallography, and Computational Methods

    SciTech Connect

    Klusak, Vojtech; Barinka, Cyril; Plechanovova, Anna; Mlcochova, Petra; Konvalinka, Jan; Rulisek, Lubomir; Lubkowski, Jacek

    2009-05-29

    Glutamate carboxypeptidase II (GCPII, EC 3.4.17.21) is a zinc-dependent exopeptidase and an important therapeutic target for neurodegeneration and prostate cancer. The hydrolysis of N-acetyl-l-aspartyl-l-glutamate (N-Ac-Asp-Glu), the natural dipeptidic substrate of the GCPII, is intimately involved in cellular signaling within the mammalian nervous system, but the exact mechanism of this reaction has not yet been determined. To investigate peptide hydrolysis by GCPII in detail, we constructed a mutant of human GCPII [GCPII(E424A)], in which Glu424, a putative proton shuttle residue, is substituted with alanine. Kinetic analysis of GCPII(E424A) using N-Ac-Asp-Glu as substrate revealed a complete loss of catalytic activity, suggesting the direct involvement of Glu424 in peptide hydrolysis. Additionally, we determined the crystal structure of GCPII(E424A) in complex with N-Ac-Asp-Glu at 1.70 {angstrom} resolution. The presence of the intact substrate in the GCPII(E424A) binding cavity substantiates our kinetic data and allows a detailed analysis of GCPII/N-Ac-Asp-Glu interactions. The experimental data are complemented by the combined quantum mechanics/molecular mechanics calculations (QM/MM) which enabled us to characterize the transition states, including the associated reaction barriers, and provided detailed information concerning the GCPII reaction mechanism. The best estimate of the reaction barrier was calculated to be {Delta}G {approx} 22({+-}5) kcal{center_dot}mol{sup -1}, which is in a good agreement with the experimentally observed reaction rate constant (k{sub cat} {approx} 1 s{sup -1}). Combined together, our results provide a detailed and consistent picture of the reaction mechanism of this highly interesting enzyme at the atomic level.

  9. The pro region is not required for the expression or intracellular routeing of carboxypeptidase E.

    PubMed Central

    Song, L; Fricker, L D

    1997-01-01

    Carboxypeptidase E (CPE) is initially synthesized as a larger precursor containing an additional 14-residue propeptide that is highly conserved between human and rat. Previous studies have established that the proenzyme is enzymically active and that deletion of the pro region does not affect the expression of the active enzyme. In the present study the function of the pro region was examined both by deleting this region from CPE and by attaching this region to the N-terminus of albumin. CPE lacking the pro region is sorted into the regulated secretory pathway in AtT-20 cells, based on confocal microscopy and examination of the stimulated secretion of the protein. Stimulation of AtT-20 cells with either forskolin or phorbol 12-myristate 13-acetate induces the secretion of wild-type CPE and of CPE lacking the pro region to similar extents, indicating a similar efficiency of sorting of the mutant. When the pro region of proalbumin is replaced with the pro region of CPE followed by expression in AtT-20 cells, the protein is not sorted into the regulated pathway, based on the lack of stimulated secretion. Confocal microscopy suggests that the proCPE/albumin protein is retained in the endoplasmic reticulum to a greater extent than is proalbumin. Pulse-chase analysis indicates that the pro region of CPE is not efficiently removed from the N-terminus of albumin, and the small amount of propeptide cleavage that does occur takes place soon before secretion of the protein. In contrast, confocal microscopy indicates that the majority of the propeptide is removed from CPE, and that this cleavage occurs in the trans-Golgi network or soon after sorting into the secretory vesicles. Taken together, these results suggest that the pro region of CPE is not required for the expression or intracellular routeing of this protein. PMID:9173892

  10. An acyltransferase catalyzing the formation of diacylglucose is a serine carboxypeptidase-like protein

    PubMed Central

    Li, Alice X.; Steffens, John C.

    2000-01-01

    1-O-β-acyl acetals serve as activated donors in group transfer reactions involved in plant natural product biosynthesis and hormone metabolism. However, the acyltransferases that mediate transacylation from 1-O-β-acyl acetals have not been identified. We report the identification of a cDNA encoding a 1-O-β-acylglucose-dependent acyltransferase functioning in glucose polyester biosynthesis by Lycopersicon pennellii. The acyltransferase cDNA encodes a serine carboxypeptidase-like protein, with a conserved Ser-His-Asp catalytic triad. Expression of the acyltransferase cDNA in Saccharomyces cerevisiae conferred the ability to disproportionate 1-O-β-acylglucose to diacylglucose. The disproportionation reaction is regiospecific, catalyzing the conversion of two equivalents of 1-O-β-acylglucose to 1,2-di-O-acylglucose and glucose. Diisopropyl fluorophosphate, a transition-state analog inhibitor of serine carboxypeptidases, inhibited acyltransferase activity and covalently labeled the purified acyltransferase, suggesting the involvement of an active serine in the mechanism of the transacylation. The acyltransferase exhibits no carboxypeptidase activity; conversely, the serine carboxypeptidases we have tested show no ability to transacylate using 1-O-acyl-β-glucoses. This acyltransferase may represent one member of a broader class of enzymes recruited from proteases that have adapted a common catalytic mechanism of catabolism and modified it to accommodate a wide range of group transfer reactions used in biosynthetic reactions of secondary metabolism. The abundance of serine carboxypeptidase-like proteins in plants suggests that this motif has been used widely for metabolic functions. PMID:10829071

  11. An iron stable isotope comparison between human erythrocytes and plasma.

    PubMed

    von Blanckenburg, Friedhelm; Oelze, Marcus; Schmid, Dietmar G; van Zuilen, Kirsten; Gschwind, Hans-Peter; Slade, Alan J; Stitah, Sylvie; Kaufmann, Daniel; Swart, Piet

    2014-11-01

    We present precise iron stable isotope ratios measured by multicollector-ICP mass spectrometry (MC-ICP-MS) of human red blood cells (erythrocytes) and blood plasma from 12 healthy male adults taken during a clinical study. The accurate determination of stable isotope ratios in plasma first required substantial method development work, as minor iron amounts in plasma had to be separated from a large organic matrix prior to mass-spectrometric analysis to avoid spectroscopic interferences and shifts in the mass spectrometer's mass-bias. The (56)Fe/(54)Fe ratio in erythrocytes, expressed as permil difference from the "IRMM-014" iron reference standard (δ(56/54)Fe), ranges from -3.1‰ to -2.2‰, a range typical for male Caucasian adults. The individual subject erythrocyte iron isotope composition can be regarded as uniform over the 21 days investigated, as variations (±0.059 to ±0.15‰) are mostly within the analytical precision of reference materials. In plasma, δ(56/54)Fe values measured in two different laboratories range from -3.0‰ to -2.0‰, and are on average 0.24‰ higher than those in erythrocytes. However, this difference is barely resolvable within one standard deviation of the differences (0.22‰). Taking into account the possible contamination due to hemolysis (iron concentrations are only 0.4 to 2 ppm in plasma compared to approx. 480 ppm in erythrocytes), we model the pure plasma δ(56/54)Fe to be on average 0.4‰ higher than that in erythrocytes. Hence, the plasma iron isotope signature lies between that of the liver and that of erythrocytes. This difference can be explained by redox processes involved during cycling of iron between transferrin and ferritin.

  12. Electrochemical determination of the total antioxidant capacity of human plasma.

    PubMed

    Ziyatdinova, G K; Budnikov, H C; Pogorel'tzev, V I

    2005-04-01

    Electrochemical reduction of oxygen at a glassy carbon electrode in a 0.05 mol L(-1) solution of (C2H5)4NI in dimethylformamide leads to generation of the superoxide anion-radical. This product of reversible one-electron oxygen reduction reacts with antioxidants, a process which is based on protonation of the anion-radical by the antioxidant. Rate constants of this interaction have been calculated. Human plasma antioxidants also react with electrochemically generated superoxide anion-radical. A voltammetric method is proposed for estimation of the total antioxidant capacity (TAC) of plasma on the basis of on this reaction. The TAC of plasma was also determined using constant-current coulometry with electro-generated bromine as the active species. A correlation was observed between TAC data obtained by voltammetry (O2*-, in alpha-tocopherol units) and coulometry (Br2 as titrant). TAC of plasma from patients with purulent infections was determined. Statistically significant differences were found between TAC of patients and control group. Treatment of purulent infections increases the TAC of plasma. So, use of electrochemical methods (voltammetry and coulometry) for determination of TAC can be used for estimation of the effectiveness of treatment.

  13. 21 CFR 866.5700 - Whole human plasma or serum immunological test system.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

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

  14. 21 CFR 866.5700 - Whole human plasma or serum immunological test system.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

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

  15. 21 CFR 866.5700 - Whole human plasma or serum immunological test system.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

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

  16. 21 CFR 866.5700 - Whole human plasma or serum immunological test system.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

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

  17. 21 CFR 866.5700 - Whole human plasma or serum immunological test system.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

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

  18. Prostaglandins in human seminal plasma. Prostaglandins and related factors 46.

    PubMed

    Hamberg, M; Samuelsson, B

    1966-01-25

    This study on human seminal plasma sought after the compounds which either possess the dienone chromophore or can be converted into it by treatment with sodium hydroxide. In addition, this investigation led to the isolation of 8 more (PGs) prostaglandins which were present in higher concentrations than the previously recognized PGs. Samples of human seminal plasma were subjected to silicic acid chromatography, reversed phase partition chromatography, thin layer chromatography, and gas liquid chromatography which isolated those 8 PGs not previously recognized. 4 of these compounds, PGE1-217, PGE2-217, PGE1-278, and PGE2-278 were known from earlier studies but had not been isolated from natural sources. The other 4 were 19 hydroxy derivatives of the 4 abovementioned compounds. The concentrations of the previously recognized PGs were recently determined and it was found that the 19 hydroxy derivatives were present in concentrations 4 times higher than the PGE compounds.

  19. [Chemistry and clinical significance of human plasma proteins].

    PubMed

    Haupt, H

    1990-10-01

    Enormous progress has been made in the course of the past few years in the various fields of plasma protein research. The primary and disulfide bridge structures are now known for almost all of the 120 proteins thus far isolated from human plasma, including trace and ultratrace proteins as well as a number of genetic variants. Genetic cloning and the derivation of the amino-acid sequence from the nucleotide sequence have played a decisive role here. However, we are only in possession of the exact three-dimensional structure for a small number of plasma proteins. The major problem in this respect is, at present, the lack of suitable protein crystals for X-ray structure analysis. We still do not know the physiological function of a large number of plasma proteins, despite the fact that they, in part, have been well characterised both physically and chemically and could be assigned to their respective protein families on the basis of their amino-acid sequence. The development of techniques for protein structure determination is relatively well advanced today, yet we lack methods of illuminating the structure-function relationship. There are more than 20 different highly purified protein preparations in virus-safe form available today for substitution therapy. To this effect new purification procedures have been developed which pay particular attention to virus elimination and inactivation. Should present indications be confirmed, one may assume that further plasma proteins (e. g. proteinase inhibitors, apolipoproteins, fibronectin) could be of significance in therapy and prophylaxis. Unlimited amounts of human blood are not available. Gene technology offers a promising alternative, at least for the production of plasma protein administered to patients in small amounts. Work is being done intensively on various blood coagulation factors and proteinase inhibitors at the moment, and factor VIII: C is already being successfully used for the treatment of patients with

  20. Plasma proteomic alterations in non-human primates and humans after chronic alcohol self-administration

    PubMed Central

    Freeman, Willard M.; VanGuilder, Heather D.; Guidone, Elizabeth; Krystal, John H.; Grant, Kathleen A.; Vrana, Kent E.

    2011-01-01

    Objective diagnostics of excessive alcohol use are valuable tools in the identification and monitoring of subjects with alcohol use disorders. A number of potential biomarkers of alcohol intake have been proposed, but none have reached widespread clinical usage, often due to limited diagnostic sensitivity and specificity. In order to identify novel potential biomarkers, we performed proteomic biomarker target discovery in plasma samples from non-human primates that chronically self-administer high levels of ethanol. 2-dimensional in-gel electrophoresis (2D-DIGE) was used to quantify plasma proteins from within subject samples collected before exposure to ethanol and after three months of excessive ethanol self-administration. Highly abundant plasma proteins were depleted from plasma samples to increase proteomic coverage. Altered plasma levels of SAA4, RBP, ITIH4, clusterin, and fibronectin, identified by 2D-DIGE analysis, were confirmed in unmanipulated, whole plasma from these animals by immunoblotting. Examination of these target plasma proteins in human subjects with excessive alcohol consumption (and control subjects) revealed increased levels of SAA4 and clusterin and decreased levels of fibronectin compared to controls. These proteins not only serve as targets for further development as biomarker candidates or components of biomarker panels, but also add to the growing understanding of dysregulated immune function and lipoprotein metabolism with chronic, excessive alcohol consumption. PMID:21303580

  1. Radioimmunoassay of factor V in human plasma and platelets

    SciTech Connect

    Tracy, P.B.; Eide, L.L.; Bowie, E.J.W.; Mann, K.G.

    1982-07-01

    Homogeneous, single-chain human factor V was used to develop a double antibody competition radioimmunoassay to measure factor V concentrations in plasma and platelets. Standard curves were constructed that allow for the detection of as little as 20 ng factor V/ml of plasma. Normal factor V concentrations range from 4 to 14 ..mu..g/ml of plasma with an average value of 7.0 +/- 2.0 ..mu..g/ml (n = 64). No correlation was observed between antigen levels and age or sex. The radioimmunoassay data are consistent with factor V clotting assays, providing freshly drawn plasma is used in the bioassay. Radioimmunoassay of washed platelets indicate that 0.63-1.93 ..mu..g of factor V is present per 2.5 X 10/sup 8/ platelets (6412-14128 molecules of factor V per platelet). When normalized to individual hematocrits and platelet count, the data indicated that platelets contribute approximately 18%-25% of the factor V found in whole blood. In addition, two individuals with functionally deficient factor V were examined and found to be deficient in both antigen and activity.

  2. Inhibition of plasma vasopressin after drinking in dehydrated humans

    NASA Technical Reports Server (NTRS)

    Geelen, G.; Keil, L. C.; Kravik, S. E.; Wade, C. E.; Thrasher, T. N.; Barnes, P. R.; Pyka, G.; Nesvig, C.; Greenleaf, J. E.

    1984-01-01

    The effects of nonosmotic and nonvolumetric factors on vasopressin secretion in dehydrated humans has been investigated experimentally, before and after drinking. The subjects of the experiment were five adult men and three adult women weighing 69-77 kg. In order to determine the influence of nonosmotic and nonvolumetric factors on vasopressin secretion, measurements were obtained of the following blood hematological indices: serum Na(+) content; serum K(+) content; osmolality; and hemoglobin. Measurements of hematocrit, plasma arginine vasopressin (AVP), aldosterone, and renin activity were also obtained. It is found that dehydration increased mean serum Na(+) content, osmolality,and AVP. No significant changes were observed in renin activity, hemoglobin, hematocrit, or plasma volume, while plasma aldosterone increased from 11.1 ng/dl after dehydration to 15.6 ng/dl between 30 and 60 min after drinking. A rapid fall of AVP content following rehydration occurred in the absence of changes in the primary regulators of AVP osmolality and plasma volume, with no change in blood pressure. On the basis of the experimental results, it is suggested that oropharyngeal factors may be the mechanism, for the observed decrease in AVP following rehydration.

  3. Augmentation of human monocyte opsonin-independent phagocytosis by fragments of human plasma fibronectin.

    PubMed Central

    Czop, J K; Kadish, J L; Austen, K F

    1981-01-01

    Human plasma fibronectin isolated by gelatin-affinity chromatography increases in a dose-dependent fashion the number of human monocytes that ingest particulate activators of the human alternative complement pathway in a fully synthetic medium. The fibronectin effect is selective for these particulate activators, does not extend to particles whose ingestion is dependent upon opsonization with IgG, and is not observed with pretreatment of the monocytes. Affinity chromatography with monoclonal antibody to plasma fibronectin of 440,000 daltons reveals that only 12-53% of the protein in a phagocytically active gelatin-affinity-purified fibronectin preparations is bound to the antibody. The protein eluted after affinity chromatography with monoclonal antibody of active preparations, which represented 10-43% of the protein applied, exhibits a 2- to 10-fold increment of activity per microgram of protein above the starting gelatin-affinity-purified material. Thus, the activity that augments the percent of human monocytes ingesting particulate activators of the alternative pathway is antigenically defined as plasma fibronectin. Preparations containing only intact 440,000-dalton fibronectin are also bound to and eluted from the monoclonal antibody, but they fail to augment phagocytosis. When inactive 440,000-dalton plasma fibronectin is subjected to limited trypsin cleavage, phagocytosis-enhancing activity develops that is bound to and elutes from the affinity column prepared with monoclonal antibody, thereby indicating that the enhancing activity of plasma fibronectin resides in cleavage fragments. PMID:6943567

  4. The Complex Exogenous RNA Spectra in Human Plasma: An Interface with Human Gut Biota?

    PubMed Central

    Wang, Kai; Li, Hong; Yuan, Yue; Etheridge, Alton; Zhou, Yong; Huang, David; Wilmes, Paul; Galas, David

    2012-01-01

    Human plasma has long been a rich source for biomarker discovery. It has recently become clear that plasma RNA molecules, such as microRNA, in addition to proteins are common and can serve as biomarkers. Surveying human plasma for microRNA biomarkers using next generation sequencing technology, we observed that a significant fraction of the circulating RNA appear to originate from exogenous species. With careful analysis of sequence error statistics and other controls, we demonstrated that there is a wide range of RNA from many different organisms, including bacteria and fungi as well as from other species. These RNAs may be associated with protein, lipid or other molecules protecting them from RNase activity in plasma. Some of these RNAs are detected in intracellular complexes and may be able to influence cellular activities under in vitro conditions. These findings raise the possibility that plasma RNAs of exogenous origin may serve as signaling molecules mediating for example the human-microbiome interaction and may affect and/or indicate the state of human health. PMID:23251414

  5. Red wine activates plasma membrane redox system in human erythrocytes.

    PubMed

    Tedesco, Idolo; Moccia, Stefania; Volpe, Silvestro; Alfieri, Giovanna; Strollo, Daniela; Bilotto, Stefania; Spagnuolo, Carmela; Di Renzo, Massimo; Aquino, Rita P; Russo, Gian Luigi

    2016-01-01

    In the present study, we report that polyphenols present in red wine obtained by a controlled microvinification process are able to protect human erythrocytes from oxidative stress and to activate Plasma Membrane Redox System (PMRS). Human plasma obtained from healthy subjects was incubated in the presence of whole red wine at a concentration corresponding to 9.13-73 μg/ml gallic acid equivalents to verify the capacity to protect against hypochlorous acid (HOCl)-induced plasma oxidation and to minimize chloramine formation. Red wine reduced hemolysis and chloramine formation induced by HOCl of 40 and 35%, respectively. PMRS present on human erythrocytes transfers electrons from intracellular molecules to extracellular electron acceptors. We demonstrated that whole red wine activated PMRS activity in human erythrocytes isolated from donors in a dose-dependent manner with a maximum at about 70-100 μg/ml gallic acid equivalents. We also showed that red wine increased glutathione (GSH) levels and erythrocytic antioxidant capacity, measured by 2,2-diphenyl-1-picrylhydrazyl (DPPH) quenching assay. Furthermore, we reported that GSH played a crucial role in regulating PMRS activity in erythrocytes. In fact, the effect of iodoacetamide, an alkylating agent that induces depletion of intracellular GSH, was completely counteracted by red wine. Bioactive compounds present in red wine, such as gallic acid, resveratrol, catechin, and quercetin were unable to activate PMRS when tested at the concentrations normally present in aged red wines. On the contrary, the increase of PMRS activity was associated with the anthocyanin fraction, suggesting the capacity of this class of compounds to positively modulate PMRS enzymatic activity.

  6. Obese carboxypeptidase E knockout mice exhibit multiple defects in peptide hormone processing contributing to low bone mineral density

    PubMed Central

    Cawley, Niamh X.; Yanik, Tulin; Woronowicz, Alicja; Chang, Weizhong; Marini, Joan C.

    2010-01-01

    Carboxypeptidase E (CPE) is a prohormone/proneuropeptide processing enzyme, and mice bearing CPE mutations exhibit an obese and diabetic phenotype. Studies on CPE knockout (KO) mice revealed poor prohormone processing, resulting in deficiencies in peptide hormones/neuropeptides such as insulin, gonadotropin-releasing hormone, and cocaine- and amphetamine-regulated transcript (CART). Here, we show that CPE KO mice, an obese animal model, have low bone mineral density (BMD) accompanied by elevated plasma CTX-1 (carboxy-terminal collagen crosslinks), and osteocalcin, indicators of increased bone turnover. Receptor activator for NF-κB ligand (RANKL) expression was elevated ∼2-fold relative to osteoprotegerin in the femur of KO animals, suggesting increased osteoclastic activity in the KO mice. In the hypothalamus, mature CART, a peptide involved in eating behavior and implicated in bone metabolism, was undetectable. The melanocortin and neuropeptide Y (NPY) systems in the hypothalamus have also been implicated in bone remodeling, since MC4R KO and NPY KO mice have increased BMD. However, reduction of α-MSH, the primary ligand of MC4R by up to 94% and the lack of detectable NPY in the hypothalamus of CPE KO do not recapitulate the single-gene KO phenotypes. This study highlights the complex physiological interplay between peptides involved in energy metabolism and bone formation and furthermore suggests the possibility that patients, bearing CPE and CART mutations leading to inactive forms of these molecules, may be at a higher risk of developing osteoporosis. PMID:20460579

  7. An express immunological method for detection of human seminal plasma.

    PubMed

    Lolov, S R; Yomtova, V M; Tsankov, Y; Kehayov, I R; Kyurkchiev, S D

    1992-04-01

    Monoclonal antibodies (Mabs) against human seminal plasma (HSP) were produced and during screening procedures dissociation constants of the antigen/antibody complexes were determined. Mab 1E5 was selected for further studies because of its high reactivity in an enzyme-linked immunoassay (ELISA) and high affinity for its corresponding antigen. The specificity of Mab 1E5 was checked in absorption ELISA with human organ extracts and some biological secretions. It was established that the 1E5-corresponding epitope was a thermostable peptide moiety which could be detected in HSP, only. This monoclonal antibody was used for the development of an express method for detection of human semen. The assay was applied for screening of 57 cases of suspected rape. A complete correlation was found between the results obtained by the proposed test and by routine microscopic methods. The newly designed immunoassay is reliable, it is easily performed and it is less time-consuming. PMID:1618453

  8. Oxidation of polymines by diamine oxidase from human seminal plasma.

    PubMed Central

    Hölttä, E; Pulkkinen, P; Elfving, K; Jänne, J

    1975-01-01

    1. Diamine oxidase [amine-oxygen oxidoreductase (deaminating)(pyridoxal-containing), EC 1.4.3.6] was purified from human seminal plasma more than 1,700-fold. The enzyme appeared to be homogeneous on polyacrylamide-gel electrophoresis at two different pH values. 2. The general properties of the enzyme were comparable with those described for other diamine oxidases from different mammalian sources. The molecular weight of the enzyme was calculated to be about 182,000. 3. The enzyme had highest affinity for diamines, but polyamines spermidine and spermine were also degraded at concentrations that can be considered physiological in human semen. 3. The possible degradation of spermine by diamine oxidase in human semen in vivo may give rise to the formation of cytotoxic aldehydes that conceivably can influence the motility and survival of the spermatozoa. PMID:239684

  9. Effects of water immersion on plasma catecholamines in normal humans

    NASA Technical Reports Server (NTRS)

    Epstein, M.; Johnson, G.; Denunzio, A. G.

    1983-01-01

    An investigation was conducted in order to determine whether water immersion to the neck (NI) alters plasma catecholamines in normal humans. Eight normal subjects were studied during a seated control study (C) and during 4 hr of NI, and the levels of norepinephrine (NE) and epinephrine (E) as determined by radioenzymatic assay were measured hourly. Results show that despite the induction of a marked natriuresis and diuresis indicating significant central hypervolemia, NI failed to alter plasma NE or E levels compared with those of either C or the corresponding prestudy 1.5 hr. In addition, the diuresis and natriuresis was found to vary independently of NE. These results indicate that the response of the sympathetic nervous system to acute volume alteration may differ from the reported response to chronic volume expansion.

  10. Novel Carboxypeptidase A6 (CPA6) Mutations Identified in Patients with Juvenile Myoclonic and Generalized Epilepsy

    PubMed Central

    Sapio, Matthew R.; Vessaz, Monique; Thomas, Pierre; Genton, Pierre; Fricker, Lloyd D.; Salzmann, Annick

    2015-01-01

    Carboxypeptidase A6 (CPA6) is a peptidase that removes C-terminal hydrophobic amino acids from peptides and proteins. The CPA6 gene is expressed in the brains of humans and animals, with high levels of expression during development. It is translated with a prodomain (as proCPA6), which is removed before secretion. The active form of CPA6 binds tightly to the extracellular matrix (ECM) where it is thought to function in the processing of peptides and proteins. Mutations in the CPA6 gene have been identified in patients with temporal lobe epilepsy and febrile seizures. In the present study, we screened for CPA6 mutations in patients with juvenile myoclonic epilepsy and identified two novel missense mutations: Arg36His and Asn271Ser. Patients harboring these mutations also presented with generalized epilepsy. Neither of the novel mutations was found in a control population. Asn271 is highly conserved in CPA6 and other related metallocarboxypeptidases. Arg36 is present in the prodomain and is not highly conserved. To assess structural consequences of the amino acid substitutions, both mutants were modeled within the predicted structure of the enzyme. To examine the effects of these mutations on enzyme expression and activity, we expressed the mutated enzymes in human embryonic kidney 293T cells. These analyses revealed that Asn271Ser abolished enzymatic activity, while Arg36His led to a ~50% reduction in CPA6 levels in the ECM. Pulse-chase using radio-labeled amino acids was performed to follow secretion. Newly-synthesized CPA6 appeared in the ECM with peak levels between 2-8 hours. There was no major difference in time course between wild-type and mutant forms, although the amount of radiolabeled CPA6 in the ECM was lower for the mutants. Our experiments demonstrate that these mutations in CPA6 are deleterious and provide further evidence for the involvement of CPA6 mutations in the predisposition for several types of epilepsy. PMID:25875328

  11. Purification and characterization of a thermostable carboxypeptidase from the extreme thermophilic archaebacterium Sulfolobus solfataricus.

    PubMed

    Colombo, S; D'Auria, S; Fusi, P; Zecca, L; Raia, C A; Tortora, P

    1992-06-01

    A carboxypeptidase was purified to electrophoretic homogeneity from the thermoacidophilic archaebacterium Sulfolobus solfataricus. Molecular masses assessed by SDS/PAGE and gel filtration were 42 kDa and 170 kDa, respectively, which points to a tetrameric structure for the molecule. An isoelectric point of 5.9 was also determined. The enzyme was proven to be a metalloprotease, as shown by the inhibitory effects exerted by EDTA and o-phenanthroline; furthermore, dialysis against EDTA led to a complete loss of activity, which could be restored by addition of Zn2+ in the micromolar range, and, to a lesser extent, by Co2+. The enzyme was endowed with a broad substrate specificity, as shown by its ability to release basic, acidic and aromatic amino acids from the respective benzoylglycylated and benzyloxycarbonylated amino acids. An esterase activity of the carboxypeptidase was also demonstrated on different esterified amino acids and dipeptides blocked at the N-terminus. The enzyme displayed broad pH optima ranging over 5.5-7.0, or 5.5-9.0, when using an acidic or a basic benzyloxycarbonylated amino acid, respectively. With regard to thermostability, it was proven to be completely stable on incubation for 15 min at 85 degrees C. Furthermore, thanks to its relatively low activation energy, i.e. 31.0 kJ/mol, it was still significantly active at room temperature. At 40 degrees C, the enzyme could withstand 0.1% SDS and different organic solvents: particularly ethanol up to 99%. Amino acid and N-terminal sequence analyses did not evidence any similarity to carboxypeptidases A nor thermolysin. A weak similarity was only found with bovine carboxypeptidase B.

  12. Spectrofluorometric determination of methocarbamol in pharmaceutical preparations and human plasma.

    PubMed

    Walash, Mohamed; Belal, Fathalla; Eid, Manal; EL Abass, Samah Abo

    2011-03-01

    A simple, sensitive and rapid spectrofluorometric method for determination of methocarbamol in pharmaceutical formulations and spiked human plasma has been developed. The proposed method is based on the measurement of the native fluorescence of methocarbamol in methanol at 313 nm after excitation at 277 nm. The relative fluorescence intensity-concentration plot was rectilinear over the range of 0.05-2.0 μg/mL, with good correlation (r=0.9999), limit of detection of 0.007 μg/ mL and a lower limit of quantification of 0.022 μg/ mL. The described method was successfully applied for the determination of methocarbamol in its tablets without interference from co-formulated drugs, such as aspirin, diclofenac, paracetamol and ibuprofen, The results obtained were in good agreement with those obtained using the official method (USP 30).The high sensitivity of the method allowed the determination of the studied drug in spiked human plasma with average percentage recovery of 99.42 ± 3.84.

  13. Production and utilization of detyrosinated tubulin in developing Artemia larvae: evidence for a tubulin-reactive carboxypeptidase.

    PubMed

    Xiang, H; MacRae, T H

    1995-01-01

    The reversible, enzymatically driven removal and readdition of its carboxy-terminal tyrosine are major posttranslational modifications of alpha-tubulin. To study these processes isoform-specific antibodies were produced and subsequently used to characterize tyrosinated and detyrosinated tubulin in the brine shrimp, Artemia. Tyrosinated tubulin existed in relatively constant amounts on western blots of cell-free protein extracts from Artemia at all developmental stages examined, whereas detyrosinated tubulin was present after 20-24 h of postgastrula growth. In agreement with the blots, the detyrosinated isoform was observed in immunofluorescently stained larvae after 24 h of incubation, appearing first in structures of a transient nature, namely spindles and midbodies. The elongated muscle cells encircling the gut and the epithelium bordering the gut lumen were stained extensively with antibody to detyrosinated tubulin. Detyrosination was accompanied by the appearance of a tubulin-reactive carboxypeptidase, which used both nonpolymerized and polymerized tubulin as substrate. The enzyme bound to microtubules very poorly, if at all, under conditions used in this work. Several inhibitors of carboxypeptidase A had no effect on the carboxypeptidase from Artemia and revealed similarities between this enzyme and others thought to be tubulin specific. The use of inhibitors also indicated that the carboxypeptidase from Artemia recognized aspects of tubulin structure in addition to the carboxy-terminal tyrosine. Our results support the idea that detyrosinated tubulin appears in microtubules of varying stability, and they demonstrate that Artemia possess a carboxypeptidase with the potential to detyrosinate tubulin during growth of larvae. PMID:8714688

  14. Purification of human plasma platelet-activating factor acetylhydrolase

    SciTech Connect

    Stafforini, D.M.; Prescott, S.M.; McIntyre, T.M.

    1986-05-01

    Platelet-activating factor (PAF;1-0-alkyl-2-acetyl-sn-glycero-3-phosphocholine is synthesized by a variety of cells. It induces hypotension, and activates platelets, neutrophils, and macrophages at nanomolar concentrations. Removal of the acetate abolishes biological activity, and is catalyzed by a specific PAF acetylhydrolase present in plasma and tissues. The authors developed a rapid assay, based on separation of (/sup 3/H)acetate from (/sup 3/H-acetyl)PAF by reversed-phase chromatography. In human plasma the enzyme exhibits an apparent Km of 5.7..mu..M, with a Vmax of 0.027..mu..mol/h/mg. Ultracentrifugation in density gradients showed that 30% of the activity is associated with high density lipoproteins (HDL) and 70% with low density lipoproteins (LDL). The enzyme was purified from LDL by precipitation with Na phosphotungstate and MgCl/sub 2/, solubilization with Tween 20, column chromatography and electrophoresis. This procedure resulted in a preparation that was 21,000-fold purified from plasma (spec. act. 575..mu..mol/h/mg) with a recovery of 10%. The purified enzyme has a molecular weight of about 43,000, a broad pH optimum (peak 7.5-8.0), and a pl of 4.6. It has greater activity when PAF is in a micellar, as compared to monomeric, and exhibits surface dilution kinetics, which may be important in vivo. The purification and characterization of this enzyme will allow detailed studies of its role in PAF metabolism.

  15. Oleaster oil positively modulates plasma lipids in humans.

    PubMed

    Belarbi, Meriem; Bendimerad, Soraya; Sour, Souad; Soualem, Zoubida; Baghdad, Choukri; Hmimed, Sara; Chemat, Farid; Visioli, Francesco

    2011-08-24

    The olive tree had been domesticated during the early Neolithic in the Near East, and more than 1000 different cultivars have been identified to date. However, examples of wild olive trees (Olea europaea oleaster) can still be found in the Mediterranean basin. Evidence of oleaster use for oil production can be found in historical and sacred texts, such as the Odyssey, the Holey Koran, and the Holey Bible. While the nutritional and healthful properties of olive oil are actively being explored, there are no data on the human actions of oleaster oil. Therefore, we investigated the effect of prolonged, i.e., 1 month, consumption of oleaster oil on the lipid profile of a 40 healthy Algerian subjects (aged 27.9 ± 3.85 years), as compared to nonconsumers from the same area. Plasma urea, creatinine, and uric acid concentrations and glycemia did not significantly differ, at the end of the study, between controls and oleaster-oil-supplemented subjects. Conversely, we recorded significant decreases of plasma triglyceride concentration (-24.8%; p < 0.05), total cholesterol (-12.13%; p < 0.05), and low-density lipoprotein-cholesterol (LDL-C) (-24.39%; p < 0.05) in oleaster-oil-treated subjects. Concomitantly, high-density lipoprotein-cholesterol (HDL-C) concentrations were significantly increased (17.94%; p < 0.05) by oleaster oil administration. In conclusion, we show that oil obtained from feral olive trees, i.e., oleasters, improves the plasma lipid profile of healthy volunteers.

  16. Oleaster oil positively modulates plasma lipids in humans.

    PubMed

    Belarbi, Meriem; Bendimerad, Soraya; Sour, Souad; Soualem, Zoubida; Baghdad, Choukri; Hmimed, Sara; Chemat, Farid; Visioli, Francesco

    2011-08-24

    The olive tree had been domesticated during the early Neolithic in the Near East, and more than 1000 different cultivars have been identified to date. However, examples of wild olive trees (Olea europaea oleaster) can still be found in the Mediterranean basin. Evidence of oleaster use for oil production can be found in historical and sacred texts, such as the Odyssey, the Holey Koran, and the Holey Bible. While the nutritional and healthful properties of olive oil are actively being explored, there are no data on the human actions of oleaster oil. Therefore, we investigated the effect of prolonged, i.e., 1 month, consumption of oleaster oil on the lipid profile of a 40 healthy Algerian subjects (aged 27.9 ± 3.85 years), as compared to nonconsumers from the same area. Plasma urea, creatinine, and uric acid concentrations and glycemia did not significantly differ, at the end of the study, between controls and oleaster-oil-supplemented subjects. Conversely, we recorded significant decreases of plasma triglyceride concentration (-24.8%; p < 0.05), total cholesterol (-12.13%; p < 0.05), and low-density lipoprotein-cholesterol (LDL-C) (-24.39%; p < 0.05) in oleaster-oil-treated subjects. Concomitantly, high-density lipoprotein-cholesterol (HDL-C) concentrations were significantly increased (17.94%; p < 0.05) by oleaster oil administration. In conclusion, we show that oil obtained from feral olive trees, i.e., oleasters, improves the plasma lipid profile of healthy volunteers. PMID:21761860

  17. A sensitive radioimmunoassay for fludrocortisone in human plasma

    NASA Technical Reports Server (NTRS)

    Mitsky, V. P.; Workman, R. J.; Nicholson, W. E.; Vernikos, J.; Robertson, R. M.; Robertson, D.

    1994-01-01

    Fludrocortisone has been a mainstay of therapy for orthostatic hypotension for many years. Clinical experience suggests that there exists a substantial interindividual variation in responsiveness to the drug. To assess this, we have developed an assay that permits measurement of the low concentrations of fludrocortisone found in human plasma. Fludrocortisone was detected by radioimmunoassay. A polyclonal rabbit antibody, raised against dexamethasone which cross-reacts strongly with fludrocortisone, was reacted with either standard or unknown samples in the presence of [125I]fludrocortisone-3-TyrNH2 (synthesized by coupling tyrosine amide to fludrocortisone-3-oxime and iodinating with chloramine T oxidation). The ED10, ED50, and ED80 were 0.34, 5.0, and 30 ng/mL of plasma, respectively. The cross reactivity with other 9-fluorinated steroids was found as follows: dexamethasone, 340%; betamethasone, 230%; and triamicinolone, 8%. To preclude an erroneous result, subjects who were pregnant or receiving any steroid medication were excluded from the study. The percent cross-reactivity with the main naturally occurring steroids was as follows: 11-desoxycortisol 3.2%, cortisol 1.1%, DOC 0.3%, pregnenolone 0.1%, corticosterone 0.06%, progesterone 0.05%, and aldosterone < 0.05%. The only compound with potential for interference, because of its high level in the circulation in the early morning, was cortisol.(ABSTRACT TRUNCATED AT 250 WORDS).

  18. Human plasma fibronectin inhibits adherence of Streptococcus pyogenes to hexadecane.

    PubMed Central

    Courtney, H S; Ofek, I; Simpson, W A; Whitnack, E; Beachey, E H

    1985-01-01

    The effect of human plasma fibronectin on the adherence of Streptococcus pyogenes to hexadecane droplets was investigated. Fibronectin blocked the adherence of streptococci to hexadecane in a dose-dependent manner. The inhibitory effect resulted from the binding of fibronectin to the streptococcal cells; radiolabeled fibronectin failed to bind to the hexadecane but bound readily to untreated streptococci. Chemical treatments of streptococci that decreased streptococcal binding of fibronectin also decreased their binding to hexadecane. Pretreatment of fibronectin with lipoteichoic acid blocked the binding of fibronectin to streptococci and abolished its ability to inhibit streptococcal adherence to hexadecane in a dose-related manner. In contrast, wheat germ agglutinin, which binds to N-acetylglucosamine on the surface of S. pyogenes cells, failed to alter hexadecane adherence. The data suggest that fibronectin binds to lipoteichoic acid on the surface of the streptococci, thereby preventing lipoteichoic acid from interacting with the hexadecane phase. PMID:3880729

  19. [Purification of kininogen II (LMW) from human plasma].

    PubMed

    Díaz, A; Henriques, O B

    1976-01-01

    Plasma supernatant in which kallikrein has been activated and removed by glass powder whilst kininogen I (HMW) has been consumed by the activated kallikrein, was used for the preparation of kininogen II. It was purified by chromatography on DEAE-cellulose followed by gel filtration on G-200 Sephadex. The purification of kininogen II was assessed from determinations of the amount of kinin released (expressed as bradykinin) as measured on the isolated guinea pig ileum, using samples incubated with human salivary kallikrein or trypsin. A preparation of kininogen II containing an activity equivalent to 8 microgram Br/mg protein, was obtained. Salivary kallikrein released approximately three times more kinin from the substrate as compared to trypsin. PMID:1032267

  20. Cloning and expression of the carboxypeptidase gene from Aspergillus saitoi and determination of the catalytic residues by site-directed mutagenesis.

    PubMed Central

    Chiba, Y; Midorikawa, T; Ichishima, E

    1995-01-01

    Carboxypeptidase from Aspergillus saitoi removes acidic, neutral and basic amino acids as well as proline from the C-terminal position at pH 2-5. cpdS, a cDNA encoding A. saitoi carboxypeptidase, was cloned and expressed. Analysis of the 1816-nucleotide sequence revealed a single open reading frame coding for 523 amino acids. When A. saitoi carboxypeptidase cDNA was expressed in yeast cells, carboxypeptidase activity was detected in the cell extract and was immunostained with a 72 kDa protein with polyclonal anti-(A. saitoi carboxypeptidase) serum. The recombinant enzyme treated with glycopeptidase F migrated with an apparent molecular mass of 60 kDa on SDS/PAGE, which was the same as that of the de-N-glycosylated carboxypeptidase from A. saitoi. Site-directed mutagenesis of the cpdS indicated that Ser-153, Asp-357 and His-436 residues were essential for the enzymic catalysis. It can be concluded that A. saitoi carboxypeptidase has a catalytic triad comprising Asp-His-Ser and is a member of serine carboxypeptidase family (EC 3.4.16.1). Images Figure 3 Figure 4 Figure 5 PMID:7772020

  1. Relationship of plasma leptin to plasma cytokines and human survivalin sepsis and septic shock.

    PubMed

    Arnalich, F; López, J; Codoceo, R; Jim nez, M; Madero, R; Montiel, C

    1999-09-01

    Leptin production is increased in rodents by administration of endotoxin or cytokines. To investigate whether circulating leptin is related to cytokine release and survival in human sepsis, plasma concentrations of leptin, interleukin (IL)-6, IL-1beta, tumor necrosis factor (TNF)-alpha, soluble TNF receptor type I, IL-1 receptor antagonist (IL-1ra), and the inflammatory modulator IL-10 were measured as soon as severe sepsis (n=28) or septic shock (n=14) developed and every 6 h for 24 h. Patients with sepsis or septic shock had leptin concentrations 2.3- and 4.2-fold greater, respectively, than the control group. There was an independent association for leptin with IL-1ra and IL-10 in both patient groups. By discriminant analysis, leptin and IL-6 were independent predictors of death. These findings suggest that increases in leptin levels may be a host defense mechanism during sepsis.

  2. Crystallization and preliminary X-ray diffraction study of porcine carboxypeptidase B

    SciTech Connect

    Akparov, V. Kh.; Timofeev, V. I. Kuranova, I. P.

    2015-05-15

    Crystals of porcine pancreatic carboxypeptidase B have been grown in microgravity by the capillary counter-diffusion method through a gel layer. The X-ray diffraction study showed that the crystals belong to sp. gr. P4{sub 1}2{sub 1}2 and have the following unit-cell parameters: a = b = 79.58 Å, c = 100.51 Å; α = β = γ = 90.00°. The X-ray diffraction data set suitable for the determination of the three-dimensional structure at atomic resolution was collected from one of the grown crystals at the SPring 8 synchrotron facility to 0.98 Å resolution.

  3. Phytochelatins are synthesized by two vacuolar serine carboxypeptidases in Saccharomyces cerevisiae.

    PubMed

    Wünschmann, Jana; Beck, Andreas; Meyer, Laurent; Letzel, Thomas; Grill, Erwin; Lendzian, Klaus J

    2007-04-17

    Phytochelatins (PCs) are cysteine-rich peptides that chelate heavy metal ions, thereby mediating heavy metal tolerance in plants, fission yeast, and Caenorhabditis elegans. They are synthesized from glutathione by PC synthase, a specific dipeptidyltransferase. While Saccharomyces cerevisiae synthesizes PCs upon exposure to heavy metal ions, the S. cerevisiae genome does not encode a PC synthase homologue. How PCs are synthesized in yeast is unclear. This study shows that the vacuolar serine carboxypeptidases CPY and CPC are responsible for PC synthesis in yeast. The finding of a PCS-like activity of these enzymes in vivo discloses another route for PC biosynthesis in eukaryotes.

  4. A mutant of Escherichia coli defective in penicillin-binding protein 5 and lacking D-alanine carboxypeptidase IA.

    PubMed Central

    Nishimura, Y; Suzuki, H; Hirota, Y; Park, J T

    1980-01-01

    A mutant of Escherichia coli defective in penicillin-binding protein 5 activity was isolated. The mutation (pfv) was shown to be located at 14.0 min on the E. coli chromosome map. Loss of penicillin-binding protein 5 in the pfv mutant was associated with the loss of D-alanine carboxypeptidase IA activity and increased sensitivity to beta-lactam antibiotics. We conclude that penicillin-binding protein 5 catalyzes the major D-alanine carboxypeptidase IA activity and that the enzyme activity, in vivo, protects E. coli cells from killing by low inhibitory concentrations of beta-lactam antibiotics. PMID:6995448

  5. Crystal Structure of Human Plasma Platelet-Activating Factor Acetylhydrolase

    SciTech Connect

    Samanta, U.; Bahnson, B

    2008-01-01

    Human plasma platelet-activating factor (PAF) acetylhydrolase functions by reducing PAF levels as a general anti-inflammatory scavenger and is linked to anaphylactic shock, asthma, and allergic reactions. The enzyme has also been implicated in hydrolytic activities of other pro-inflammatory agents, such as sn-2 oxidatively fragmented phospholipids. This plasma enzyme is tightly bound to low and high density lipoprotein particles and is also referred to as lipoprotein-associated phospholipase A{sub 2}. The crystal structure of this enzyme has been solved from x-ray diffraction data collected to a resolution of 1.5{angstrom}. It has a classic lipase {alpha}/{beta}-hydrolase fold, and it contains a catalytic triad of Ser{sup 273}, His{sup 351}, and Asp{sup 296}. Two clusters of hydrophobic residues define the probable interface-binding region, and a prediction is given of how the enzyme is bound to lipoproteins. Additionally, an acidic patch of 10 carboxylate residues and a neighboring basic patch of three residues are suggested to play a role in high density lipoprotein/low density lipoprotein partitioning. A crystal structure is also presented of PAF acetylhydrolase reacted with the organophosphate compound paraoxon via its active site Ser{sup 273}. The resulting diethyl phosphoryl complex was used to model the tetrahedral intermediate of the substrate PAF to the active site. The model of interface binding begins to explain the known specificity of lipoprotein-bound substrates and how the active site can be both close to the hydrophobic-hydrophilic interface and at the same time be accessible to the aqueous phase.

  6. Fitzgerald factor (high molecular weight kininogen) clotting activity in human plasma in health and disease in various animal plasmas.

    PubMed

    Saito, H; Goldsmith, G; Waldmann, R

    1976-12-01

    Fitzgerald factor (high molecular weight kininogen) is an agent in normal human plasma that corrects the impaired in vitro surface-mediated plasma reactions of blood coagulation, fibrinolysis, and kinin generation observed in Fitzgerald trait plasma. To assess the possible pathophysiologic role of Fitzgerald factor, its titer was measured by a functional clot-promoting assay. Mean +/- SD in 42 normal adults was 0.99+/-0.25 units/ml, one unit being the activity in 1 ml of normal pooled plasma. No difference in titer was noted between normal men and women, during pregnancy, or after physical exercise. Fitzgerald factor activity was significantly reduced in the plasmas of eight patients with advanced hepatic cirrhosis (0.40+/-0.09 units/ml) and of ten patients with disseminated intravascular coagulation (0.60+/-0.30 units/ml), but was normal in plasmas of patients with other congenital clotting factor deficiencies, nephrotic syndrome, rheumatoid arthritis, systemic lupus erythematosus, or sarcoidosis, or under treatment with warfarin. The plasmas of 21 mammalian species tested appeared to contain Fitzgerald factor activity, but those of two avian, two repitilian, and one amphibian species did not correct the coagulant defect in Fitzgerald trait plasmas. PMID:1000085

  7. The cytosolic carboxypeptidases CCP2 and CCP3 catalyze posttranslational removal of acidic amino acids

    PubMed Central

    Tort, Olivia; Tanco, Sebastián; Rocha, Cecilia; Bièche, Ivan; Seixas, Cecilia; Bosc, Christophe; Andrieux, Annie; Moutin, Marie-Jo; Avilés, Francesc Xavier; Lorenzo, Julia; Janke, Carsten

    2014-01-01

    The posttranslational modification of carboxy-terminal tails of tubulin plays an important role in the regulation of the microtubule cytoskeleton. Enzymes responsible for deglutamylating tubulin have been discovered within a novel family of mammalian cytosolic carboxypeptidases. The discovery of these enzymes also revealed the existence of a range of other substrates that are enzymatically deglutamylated. Only four of six mammalian cytosolic carboxypeptidases had been enzymatically characterized. Here we complete the functional characterization of this protein family by demonstrating that CCP2 and CCP3 are deglutamylases, with CCP3 being able to hydrolyze aspartic acids with similar efficiency. Deaspartylation is a novel posttranslational modification that could, in conjunction with deglutamylation, broaden the range of potential substrates that undergo carboxy-terminal processing. In addition, we show that CCP2 and CCP3 are highly regulated proteins confined to ciliated tissues. The characterization of two novel enzymes for carboxy-terminal protein modification provides novel insights into the broadness of this barely studied process. PMID:25103237

  8. Fibulin-1 purification from human plasma using affinity chromatography on Factor H-Sepharose.

    PubMed

    DiScipio, Richard G; Liddington, Robert C; Schraufstatter, Ingrid U

    2016-05-01

    A method is reported to purify Fibulin-1 from human plasma resulting in a 36% recovery. The steps involve removal of the cryoglobulin and the vitamin K dependent proteins followed by polyethylene glycol and ammonium sulfate precipitations, DEAE-Sephadex column chromatography and finally Factor H-Sepharose affinity purification. The procedure is designed to be integrated into an overall scheme for the isolation of over 30 plasma proteins from a single batch of human plasma. Results from mass spectroscopy, SDS-PAGE, and Western blotting indicate that human plasma Fibulin-1 is a single chain of the largest isotype. Functional binding assays demonstrated calcium ion dependent interaction of Fibulin-1 for fibrinogen, fibronectin, and Factor H. The procedure described is the first to our knowledge that enables a large scale purification of Fibulin-1 from human plasma. PMID:26826315

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

    PubMed

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

    2016-06-01

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

  10. [Activity of peptidyl-dipeptidase A and carboxypeptidase N in the serum of patients with Alzheimer disease].

    PubMed

    Solov'ev, V B; Gengin, M T

    2007-01-01

    The activity of peptidyl-dipeptidase A and carboxypeptidase N taking part in peptide metabolism in the serum of patients with Alzheimer disease were studied. The role of these enzymes in the metabolism of neuropeptides and beta-amyloid at the Alzheimer disease was discussed.

  11. Catalytic properties of recombinant dipeptidyl carboxypeptidase from Escherichia coli: a comparative study with angiotensin I-converting enzyme.

    PubMed

    Cunha, Carlos Eduardo L; Magliarelli, Helena de Fátima; Paschoalin, Thaysa; Nchinda, Aloysius T; Lima, Jackson C; Juliano, Maria A; Paiva, Paulo B; Sturrock, Edward D; Travassos, Luiz R; Carmona, Adriana K

    2009-09-01

    Dipeptidyl carboxypeptidase from Escherichia coli (EcDcp) is a zinc metallopeptidase with catalytic properties closely resembling those of angiotensin I-converting enzyme (ACE). However, EcDcp and ACE are classified in different enzyme families (M3 and M2, respectively) due to differences in their primary sequences. We cloned and expressed EcDcp and studied in detail the enzyme's S(3) to S(1)' substrate specificity using positional-scanning synthetic combinatorial (PS-SC) libraries of fluorescence resonance energy transfer (FRET) peptides. These peptides contain ortho-aminobenzoic acid (Abz) and 2,4-dinitrophenyl (Dnp) as donor/acceptor pair. In addition, using FRET substrates developed for ACE [Abz-FRK(Dnp)P-OH, Abz-SDK(Dnp)P-OH and Abz-LFK(Dnp)-OH] as well as natural ACE substrates (angiotensin I, bradykinin, and Ac-SDKP-OH), we show that EcDcp has catalytic properties very similar to human testis ACE. EcDcp inhibition studies were performed with the ACE inhibitors captopril (K(i)=3 nM) and lisinopril (K(i)=4.4 microM) and with two C-domain-selective ACE inhibitors, 5-S-5-benzamido-4-oxo-6-phenylhexanoyl-L-tryptophan (kAW; K(i)=22.0 microM) and lisinopril-Trp (K(i)=0.8 nM). Molecular modeling was used to provide the basis for the differences found in the inhibitors potency. The phylogenetic relationship of EcDcp and related enzymes belonging to the M3 and M2 families was also investigated and the results corroborate the distinct origins of EcDcp and ACE.

  12. Hormones and endocrine disruptors in human seminal plasma.

    PubMed

    Hampl, R; Kubatova, J; Heracek, J; Sobotka, V; Starka, L

    2013-07-01

    Seminal plasma represents a unique environment for maturation, nutrition, and protection of male germ cells from damaging agents. It contains an array of organic as well as inorganic chemicals, encompassing a number of biologically and immunologically active compounds, including hormones. Seminal plasma contains also various pollutants transferred from outer environment known as endocrine disruptors. They interfere with hormones at the receptor level, act as inhibitors of their biosynthesis, and affect hormone regulation.In this minireview, the main groups of hormones detected in seminal plasma are summarized. Seminal gonadal steroids were investigated mostly with aim to use them as biomarkers of impaired spermatogenesis (sperm count, motility, morphology). Concentrations of hormones in the seminal plasma often differ considerably from the blood plasma levels in dependence on their origin. In some instances (dihydrotestosterone, estradiol), their informative value is higher than determination in blood.Out of peptide hormones detected in seminal plasma, peptides of transforming growth factor beta family, especially antimullerian hormone, and oligopeptides related to thyrotropin releasing hormone have the high informative value, while assessment of seminal gonadotropins and prolactin does not bring advantage over determination in blood.Though there is a large body of information about the endocrine disruptors' impact on male reproduction, especially with their potential role in decline of male reproductive functions within the last decades, there are only scarce reports on their presence in seminal plasma. Herein, the main groups of endocrine disruptors found in seminal plasma are reviewed, and the use of their determination for investigation of fertility disorders is discussed.

  13. High-resolution structure of the M14-type cytosolic carboxypeptidase from Burkholderia cenocepacia refined exploiting PDB-REDO strategies

    SciTech Connect

    Rimsa, Vadim; Eadsforth, Thomas C.; Joosten, Robbie P.; Hunter, William N.

    2014-02-01

    The structure of a bacterial M14-family carboxypeptidase determined exploiting microfocus synchrotron radiation and highly automated refinement protocols reveals its potential to act as a polyglutamylase. A potential cytosolic metallocarboxypeptidase from Burkholderia cenocepacia has been crystallized and a synchrotron-radiation microfocus beamline allowed the acquisition of diffraction data to 1.9 Å resolution. The asymmetric unit comprises a tetramer containing over 1500 amino acids, and the high-throughput automated protocols embedded in PDB-REDO were coupled with model–map inspections in refinement. This approach has highlighted the value of such protocols for efficient analyses. The subunit is constructed from two domains. The N-terminal domain has previously only been observed in cytosolic carboxypeptidase (CCP) proteins. The C-terminal domain, which carries the Zn{sup 2+}-containing active site, serves to classify this protein as a member of the M14D subfamily of carboxypeptidases. Although eukaryotic CCPs possess deglutamylase activity and are implicated in processing modified tubulin, the function and substrates of the bacterial family members remain unknown. The B. cenocepacia protein did not display deglutamylase activity towards a furylacryloyl glutamate derivative, a potential substrate. Residues previously shown to coordinate the divalent cation and that contribute to peptide-bond cleavage in related enzymes such as bovine carboxypeptidase are conserved. The location of a conserved basic patch in the active site adjacent to the catalytic Zn{sup 2+}, where an acetate ion is identified, suggests recognition of the carboxy-terminus in a similar fashion to other carboxypeptidases. However, there are significant differences that indicate the recognition of substrates with different properties. Of note is the presence of a lysine in the S1′ recognition subsite that suggests specificity towards an acidic substrate.

  14. Utilization of a depsipeptide substrate for trapping acyl—enzyme intermediates of penicillin-sensitive D-alanine carboxypeptidases

    PubMed Central

    Rasmussen, James R.; Strominger, Jack L.

    1978-01-01

    The penicillin-sensitive D-alanine carboxypeptidases of Bacillus subtilis, Escherichia coli, and Staphylococcus aureus catalyzed the hydrolysis of the D-lactic acid residue from the depsipeptide diacetyl-L-lysyl-D-alanyl-D-lactic acid. The ester substrate was hydrolyzed faster than the peptide analogue, diacetyl-L-lysyl-D-alanyl-D-alanine, by the B. subtilis (15-fold) and E. coli (4-fold) carboxypeptidases, presumably because acylation (k2), which is the rate-limiting step of the peptidase reaction, occurred more rapidly during cleavage of the ester bond than during cleavage of the amide bond. No rate acceleration was observed with the S. aureus carboxypeptidase for which deacylation (k3) is already the rate-determining step with the peptide substrate. The efficiency of utilization of the depsipeptide (Vmax/Km) was greatly enhanced (19- to 147-fold) for all three enzymes. After incubation of the B. subtilis carboxypeptidase and [14C]diacetyl-L-lysyl-D-alanyl-D-lactic acid at pH 5.0 and lowering of the pH to 3.0, a radioactive acyl-enzyme intermediate containing 0.43 mol of substrate per mol of enzyme was isolated by Sephadex G-50 chromatography. After acetone precipitation, the acyl group of the denatured acyl-enzyme complex appeared to be bound to the protein by an ester bond. Acyl enzymes were also detected for the S. aureus and E. coli carboxypeptidases after sodium dodecyl sulfate/polyacrylamide gel electrophoresis and fluorography of enzyme incubated with [14C]depsipeptide and precipitated with acetone. Images PMID:415311

  15. Differential Responses of Plasma Adropin Concentrations To Dietary Glucose or Fructose Consumption In Humans.

    PubMed

    Butler, Andrew A; St-Onge, Marie-Pierre; Siebert, Emily A; Medici, Valentina; Stanhope, Kimber L; Havel, Peter J

    2015-10-05

    Adropin is a peptide hormone encoded by the Energy Homeostasis Associated (ENHO) gene whose physiological role in humans remains incompletely defined. Here we investigated the impact of dietary interventions that affect systemic glucose and lipid metabolism on plasma adropin concentrations in humans. Consumption of glucose or fructose as 25% of daily energy requirements (E) differentially affected plasma adropin concentrations (P < 0.005) irrespective of duration, sex or age. Glucose consumption reduced plasma adropin from 3.55 ± 0.26 to 3.28 ± 0.23 ng/ml (N = 42). Fructose consumption increased plasma adropin from 3.63 ± 0.29 to 3.93 ± 0.34 ng/ml (N = 45). Consumption of high fructose corn syrup (HFCS) as 25% E had no effect (3.43 ± 0.32 versus 3.39 ± 0.24 ng/ml, N = 26). Overall, the effect of glucose, HFCS and fructose on circulating adropin concentrations were similar to those observed on postprandial plasma triglyceride concentrations. Furthermore, increases in plasma adropin levels with fructose intake were most robust in individuals exhibiting hypertriglyceridemia. Individuals with low plasma adropin concentrations also exhibited rapid increases in plasma levels following consumption of breakfasts supplemented with lipids. These are the first results linking plasma adropin levels with dietary sugar intake in humans, with the impact of fructose consumption linked to systemic triglyceride metabolism. In addition, dietary fat intake may also increase circulating adropin concentrations.

  16. Differential Responses of Plasma Adropin Concentrations To Dietary Glucose or Fructose Consumption In Humans.

    PubMed

    Butler, Andrew A; St-Onge, Marie-Pierre; Siebert, Emily A; Medici, Valentina; Stanhope, Kimber L; Havel, Peter J

    2015-01-01

    Adropin is a peptide hormone encoded by the Energy Homeostasis Associated (ENHO) gene whose physiological role in humans remains incompletely defined. Here we investigated the impact of dietary interventions that affect systemic glucose and lipid metabolism on plasma adropin concentrations in humans. Consumption of glucose or fructose as 25% of daily energy requirements (E) differentially affected plasma adropin concentrations (P < 0.005) irrespective of duration, sex or age. Glucose consumption reduced plasma adropin from 3.55 ± 0.26 to 3.28 ± 0.23 ng/ml (N = 42). Fructose consumption increased plasma adropin from 3.63 ± 0.29 to 3.93 ± 0.34 ng/ml (N = 45). Consumption of high fructose corn syrup (HFCS) as 25% E had no effect (3.43 ± 0.32 versus 3.39 ± 0.24 ng/ml, N = 26). Overall, the effect of glucose, HFCS and fructose on circulating adropin concentrations were similar to those observed on postprandial plasma triglyceride concentrations. Furthermore, increases in plasma adropin levels with fructose intake were most robust in individuals exhibiting hypertriglyceridemia. Individuals with low plasma adropin concentrations also exhibited rapid increases in plasma levels following consumption of breakfasts supplemented with lipids. These are the first results linking plasma adropin levels with dietary sugar intake in humans, with the impact of fructose consumption linked to systemic triglyceride metabolism. In addition, dietary fat intake may also increase circulating adropin concentrations. PMID:26435060

  17. Inhibition of platelet (/sup 3/H)- imipramine binding by human plasma protein fractions

    SciTech Connect

    Strijewski, A.; Chudzik, J.; Tang, S.W.

    1988-01-01

    Inhibition of high-affinity (/sup 3/H)-imipramine binding to platelet membranes by human plasma fractions and isolated plasma proteins was investigated. Several plasma proteins were found to contribute to the observed apparent inhibition and this contribution was assessed in terms of inhibitor units. Alpha/sub 1/ acid glycoprotein, high density and low density lipoprotein, IgG and ..cap alpha../sub 1/-antitrypsin were identified as effective non-specific inhibitors. Alpha-1-acid glycoprotein was confirmed to be the most potent plasma protein inhibitor. Cohn fractions were evaluated for the presence of the postulated endocoid of (/sup 3/H)-imipramine binding site.

  18. Reaction pathway and free energy profile determined for specific recognition of oligosaccharide moiety of carboxypeptidase Y.

    PubMed

    Senkara-Barwijuk, Elżbieta; Kobiela, Tomasz; Lebed, Kateryna; Lekka, Małgorzata

    2012-01-01

    The interaction of mannose specific lectin (from Lens culinaris, LcL) with the carbohydrate moiety of carboxypeptidase Y (CaY) was studied using both atomic force microscope (AFM) and quartz crystal microbalance with dissipation monitoring (QCM-D). The AFM enables to determine the positions of energy barriers present in the energy landscape of the single complex undergoing dissociation. The QCM-D measurements allow the estimation of the quantitative parameters characterizing the kinetics of the studied molecular interaction (namely the association and dissociation rate constants and the association constant). The use of both methods not only delivers the complementary characterization of kinetic and thermodynamic parameters but also permits to investigate the mechanism of the binding and unbinding of the molecules. The results for LcL were compared with those obtained for concanavalin A i.e. lectin, which interacts with the carbohydrate moiety on a similar way.

  19. Carborane-containing urea-based inhibitors of glutamate carboxypeptidase II: Synthesis and structural characterization.

    PubMed

    Youn, Sihyun; Kim, Kyung Im; Ptacek, Jakub; Ok, Kiwon; Novakova, Zora; Kim, YunHye; Koo, JaeHyung; Barinka, Cyril; Byun, Youngjoo

    2015-11-15

    Glutamate carboxypeptidase II (GCPII) is a zinc metalloprotease on the surface of astrocytes which cleaves N-acetylaspartylglutamate to release N-acetylaspartate and glutamate. GCPII inhibitors can decrease glutamate concentration and play a protective role against apoptosis or degradation of brain neurons. Herein, we report the synthesis and structural analysis of novel carborane-based GCPII inhibitors. We determined the X-ray crystal structure of GCPII in complex with a carborane-containing inhibitor at 1.79Å resolution. The X-ray analysis revealed that the bulky closo-carborane cluster is located in the spacious entrance funnel region of GCPII, indicating that the carborane cluster can be further structurally modified to identify promising lead structures of novel GCPII inhibitors.

  20. Analysis of non-thermal plasma-induced cell injury in human lung cancer cell lines

    NASA Astrophysics Data System (ADS)

    Kurita, Hirofumi; Sano, Kaori; Wada, Motoi; Mizuno, Kazue; Ono, Ryo; Yasuda, Hachiro; Takashima, Kazunori; Mizuno, Akira

    2015-09-01

    Recent progress of biomedical application of atmospheric pressure plasma shows that the biological effects are mainly due to reactive oxygen and nitrogen species (RONS) in liquid produced by the plasma exposure. To elucidate the cellular responses induced by exposure to the plasma, we focused on identification and quantification of reactive chemical species in plasma-exposed cell culture medium, and cell injury in mammalian cells after treatment of the plasma-exposed medium. In this study, we examined human lung cancer cell lines. The contribution of H2O2 to the cellular responses was considered. Here, an atmospheric pressure plasma jet (APPJ) sustained by a pulsed power supply in argon was used. After APPJ exposure to cell culture medium, RONS detection in liquid was conducted. It showed that OH radical, ONOO-, NO2-, NO3-, and H2O2 were produced in the plasma-exposed medium. Cellular responses of human lung cancer cell lines to the plasma-exposed medium in a concentration-dependence manner were also studied. It showed that the plasma-exposed medium and the H2O2 treatment gave similar reduction in viability and induction of apoptosis. This work was partly supported by MEXT KAKENHI Grant Number 24108005 and JSPS KAKENHI Grant Number 26390096.

  1. Highly sensitive radioimmunoassay of atrial natriuretic peptide (ANP) in human plasma and urine

    SciTech Connect

    Marumo, F.; Sakamoto, H.; Ando, K.; Ishigami, T.; Kawakami, M.

    1986-05-29

    A highly sensitive radioimmunoassay has been established for measurement of human plasma and urine concentrations of atrial natriuretic peptide (ANP) and requires no extraction or concentration process. An antiserum was prepared from rabbits immunized with ..cap alpha..-human ANP (..cap alpha..-hANP) coupled with bovine-thyroglobulin. The sensitivity of this method was 0.2 pg/tube of synthetic ..cap alpha..-hANP utilized as authentic standard. Recovery of ..cap alpha..-hANP spiked to plasma and urine was 97.7 +/- 15.4% and 97.1 +/- 9.5% (mean +/- SD), respectively. Plasma and urinary ANP concentrations versus assay data showed satisfactory linearity. In 124 health subjects, the plasma ANP concentration was 31.7 +/- 12.0 pg/ml. Two different molecular forms of ANP in plasma and a single form in urine were found by gel permeation chromatography.

  2. Determination of human plasma xanthine oxidase activity by high-performance liquid chromatography.

    PubMed

    Yamamoto, T; Moriwaki, Y; Takahashi, S; Tsutsumi, Z; Yamakita, J; Nasako, Y; Hiroishi, K; Higashino, K

    1996-06-01

    An assay for human plasma xanthine oxidase activity was developed with pterin as the substrate and the separation of product (isoxanthopterin) by high-performance liquid chromatography with a fluorescence detector. The reaction mixture consists of 60 microliters of plasma and 240 microliters of 0.2 M Tris-HCl buffer (pH 9.0) containing 113 microM pterin. With this assay, the activity of plasma xanthine oxidase could be easily determined despite its low activity. As a result, it could be demonstrated that the intravenous administration of heparin or the oral administration of ethanol did not increase plasma xanthine oxidase activity in normal subjects, and also that plasma xanthine oxidase activity was higher in patients with hepatitis C virus infection than in healthy subjects or patients with gout. In addition, a single patient with von Gierke's disease showed a marked increase in the plasma activity of this enzyme, relative to that apparent in normal subjects. PMID:8811453

  3. Bacterial Inactivation of Wound Infection in a Human Skin Model by Liquid-Phase Discharge Plasma

    PubMed Central

    Kim, Paul Y.; Kim, Yoon-Sun; Koo, Il Gyo; Jung, Jae Chul; Kim, Gon Jun; Choi, Myeong Yeol; Yu, Zengqi; Collins, George J.

    2011-01-01

    Background We investigate disinfection of a reconstructed human skin model contaminated with biofilm-formative Staphylococcus aureus employing plasma discharge in liquid. Principal Findings We observed statistically significant 3.83-log10 (p<0.001) and 1.59-log10 (p<0.05) decreases in colony forming units of adherent S. aureus bacteria and 24 h S. aureus biofilm culture with plasma treatment. Plasma treatment was associated with minimal changes in histological morphology and tissue viability determined by means of MTT assay. Spectral analysis of the plasma discharge indicated the presence of highly reactive atomic oxygen radicals (777 nm and 844 nm) and OH bands in the UV region. The contribution of these and other plasma-generated agents and physical conditions to the reduction in bacterial load are discussed. Conclusions These findings demonstrate the potential of liquid plasma treatment as a potential adjunct therapy for chronic wounds. PMID:21897870

  4. Potentiation of the anaphylatoxins in vivo using an inhibitor of serum carboxypeptidase N (SCPN). I. Lethality and pathologic effects on pulmonary tissue.

    PubMed Central

    Huey, R.; Bloor, C. M.; Kawahara, M. S.; Hugli, T. E.

    1983-01-01

    Carboxypeptidase N (EC 3.4.12.7) (SCPN) is a plasma enzyme that efficiently inactivates the anaphylatoxins C3a and C4a and significantly reduces C5a spasmogenic activity by removing the C-terminal arginyl residue from each of these factors. The arginine analog DL-2-mercaptomethyl-3-guanidinoethylthiopropanoic acid (SCPN-INH) is a potent competitive inhibitor of SCPN with a Ki for this carboxypeptidase in serum of 2 x 10(-9) M. Therefore, we have used the SCPN inhibitor to potentiate biologic activity of the anaphylatoxins in vivo. Infusion via the carotid artery of about 40 mg of SCPN-INH into each of 8 adult guinea pigs inactivated the SCPN for at least 3 hours and caused no measurable toxic effects. When cobra venom factor (CVF) is infused into guinea pigs, it activates the alternative pathway of complement, thereby generating the anaphylatoxins C3a and C5a. Ordinarily, infusion of CVF is nonlethal, because the generated anaphylatoxins are rapidly converted to C3a des Arg and C5a des Arg by SCPN. However, CVF (200 micrograms) plus SCPN-INH delivered intravenously in 5 animals induced a lethal reaction in less than 5 minutes. The authors conclude that the lethal effect is due largely to the anaphylatoxins. Histologic sections of the lungs from treated animals show dramatic structural changes consistent with peripheral small airway constriction, bronchial constriction, and vasoconstriction of small muscular arteries. Also, cell aggregates are present in blood vessels. Other histologic changes include severe congestion, pulmonary edema, and an interstitial infiltrate of mononuclear cells. Large doses of chlorpheniramine prevent this lethal reaction. Lethality is apparently attributable to asphyxia and is dependent on the level of CVF administered: eg, 100 micrograms CVF was not lethal in 4 animals given SCPN inhibitor, although signs of respiratory distress were observed. On histologic examination of lungs from guinea pigs given CVF and SCPN-INH, the features are

  5. Hydrogen peroxide: A central player in physical plasma-induced oxidative stress in human blood cells.

    PubMed

    Bekeschus, S; Kolata, J; Winterbourn, C; Kramer, A; Turner, R; Weltmann, K D; Bröker, B; Masur, K

    2014-05-01

    Plasma medicine is an interdisciplinary field and recent clinical studies showed benefits of topical plasma application to chronic wounds. Whereas most investigations have focused on plasma-skin cell interaction, immune cells are omnipresent in most tissues as well. They not only elicit specific immune responses but also regulate inflammation, which is central in healing and regeneration. Plasma generates short-lived radicals and species in the gas phase. Mechanisms of plasma-cell interactions are not fully understood but it is hypothesized that reactive oxygen and nitrogen species (RONS) mediate effects of plasma on cells. In this study human blood cells were investigated after cold atmospheric plasma treatment with regard to oxidation and viability. Plasma generates hydrogen peroxide (H2O2) and the responses were similar in cells treated with concentration-matched H2O2. Both treatments gave an equivalent reduction in viability and this was completely abrogated if catalase was added prior to plasma exposure. Further, five oxidation probes were utilized and fluorescence increase was observed in plasma-treated cells. Dye-dependent addition of catalase diminished most but not all of the probe fluorescence, assigning H2O2 a dominant but not exclusive role in cellular oxidation by plasma. Investigations for other species revealed generation of nitrite and formation of 3-nitrotyrosine but not 3-chlorotyrosine after plasma treatment indicating presence of RNS which may contribute to cellular redox changes observed. Together, these results will help to clarify how oxidative stress associates with physical plasma treatment in wound relevant cells. PMID:24528134

  6. Alternative pathways of thromboplastin-dependent activation of human factor X in plasma

    SciTech Connect

    Marlar, R.A.; Griffin, J.H.

    1981-01-01

    To determine the interrelationships of the major coagulation pathways, the activation of 3H-labeled factor X in normal and various deficient human plasmas was evaluated when clotting was triggered by dilute rabbit or human thromboplastin. Various dilutions of thromboplastin and calcium were added to plasma samples containing 3H-factor X, and the time course of factor X activation was determined. At a 1/250 dilution of rabbit brain thromboplastin, the rate of factor X activation in plasmas deficient in factor VIII or factor IX was 10% of the activation rate of normal plasma or of factor XI deficient plasma. Reconstitution of the deficient plasmas with factors VIII or IX, respectively, reconstituted normal factor X activation. Similar results were obtained when various dilutions of human thromboplastin replaced the rabbit thromboplastin. From these plasma experiments, it is inferred that the dilute thromboplastin-dependent activation of factor X requires factors VII, IX, and VIII. An alternative extrinsic pathway that involves factors IX and VIII may be the physiologic extrinsic pathway and hence help to explain the consistent clinical observations of bleeding diatheses in patients deficient in factors IX or VIII.

  7. Separation of Albumin, Ceruloplasmin, and Transferrin from Human Plasma.

    ERIC Educational Resources Information Center

    Barnes, Grady; Frieden, Earl

    1982-01-01

    Procedures are provided for separating the principal metalloproteins (albumin, ceruloplasmin, and transferrin) from plasma using column chromatographic techniques. The experiment can be completed in two separate three-hour laboratory periods during which column chromatography is illustrated and the effect of pH on charge and affinity of a protein…

  8. Plasma oxytocin concentrations following MDMA or intranasal oxytocin in humans.

    PubMed

    Kirkpatrick, Matthew G; Francis, Sunday M; Lee, Royce; de Wit, Harriet; Jacob, Suma

    2014-08-01

    MDMA (±3,4-methylenedioxymethamphetamine, 'ecstasy') is reportedly used recreationally because it increases feelings of sociability and interpersonal closeness. Prior work suggests that the pro-social effects of MDMA may be mediated by release of oxytocin. A direct examination of plasma levels of oxytocin after acute doses of oxytocin and MDMA, in the same individuals, would provide further evidence for the idea that MDMA produces its pro-social effects by increasing oxytocin. Fourteen healthy MDMA users participated in a 4-session, double-blind study in which they received oral MDMA (0.75 and 1.5mg/kg), intranasal oxytocin (20IU or 40IU), and placebo. Plasma oxytocin concentrations, as well as cardiovascular and subjective effects were assessed before and at several time points after drug administration. MDMA (1.5mg/kg only) increased plasma oxytocin levels to a mean peak of 83.7pg/ml at approximately 90-120min, compared to 18.6pg/ml after placebo. Intranasal oxytocin (40IU, but not 20IU) increased plasma oxytocin levels to 48.0pg/ml, 30-60min after nasal spray administration. MDMA dose-dependently increased heart rate, blood pressure, feelings of euphoria (e.g., 'High' and 'Like Drug'), and feelings of sociability, whereas oxytocin had no cardiovascular or subjective effects. The subjective and cardiovascular responses to MDMA were not related to plasma oxytocin levels, although the N was small for this analysis. Future studies examining the effects of oxytocin antagonists on responses to MDMA will help to determine the mechanism by which MDMA produces pro-social effects.

  9. Plasma atriopeptin response to prolonged cycling in humans.

    PubMed

    Perrault, H; Cantin, M; Thibault, G; Brisson, G R; Brisson, G; Beland, M

    1991-03-01

    The exercise-induced increase in plasma atriopeptin (ANP) has been related to exercise intensity. The independent effect of duration on the ANP response to dynamic exercise remains incompletely documented. The purpose of this study was to describe the time course of plasma ANP concentration during a 90-min cycling exercise protocol and to examine this in light of concurrent variations in plasma arginine vasopressin (AVP), aldosterone (ALD), and catecholamine (norepinephrine and epinephrine) concentrations as well as plasma renin activity (PRA). Seven male and four female healthy college students (23 +/- 2 yr) completed a prolonged exercise protocol on a cycle ergometer at an intensity of 67% of maximal O2 uptake. Venous blood was sampled through an indwelling catheter at rest, after 15, 30, 45, 60, and 90 min of exercise, and after 30 min of passive upright recovery. Results (means +/- SE) indicate an increase in ANP from rest (22 +/- 2.6 pg/ml) at 15 min of exercise (45.3 +/- 7.4 pg/ml) with a further increase at 30 min (59.4 +/- 9.8 pg/ml) and a leveling-off thereafter until completion of the exercise protocol (51.7 +/- 10.7 pg/ml). In plasma ALD and PRA, a significant increase was found from rest (ALD, 21.4 +/- 6.4 ng/dl), PRA, 2.5 +/- 0.5 ng.ml-1.h-1 after 30 min of cycling, which continued to increase until completion of the exercise (ALD 46.6 +/- 8.7 ng/dl, PRA 9.5 +/- 0.9 ng.ml-1.h-1.(ABSTRACT TRUNCATED AT 250 WORDS)

  10. Plasma atriopeptin response to prolonged cycling in humans.

    PubMed

    Perrault, H; Cantin, M; Thibault, G; Brisson, G R; Brisson, G; Beland, M

    1991-03-01

    The exercise-induced increase in plasma atriopeptin (ANP) has been related to exercise intensity. The independent effect of duration on the ANP response to dynamic exercise remains incompletely documented. The purpose of this study was to describe the time course of plasma ANP concentration during a 90-min cycling exercise protocol and to examine this in light of concurrent variations in plasma arginine vasopressin (AVP), aldosterone (ALD), and catecholamine (norepinephrine and epinephrine) concentrations as well as plasma renin activity (PRA). Seven male and four female healthy college students (23 +/- 2 yr) completed a prolonged exercise protocol on a cycle ergometer at an intensity of 67% of maximal O2 uptake. Venous blood was sampled through an indwelling catheter at rest, after 15, 30, 45, 60, and 90 min of exercise, and after 30 min of passive upright recovery. Results (means +/- SE) indicate an increase in ANP from rest (22 +/- 2.6 pg/ml) at 15 min of exercise (45.3 +/- 7.4 pg/ml) with a further increase at 30 min (59.4 +/- 9.8 pg/ml) and a leveling-off thereafter until completion of the exercise protocol (51.7 +/- 10.7 pg/ml). In plasma ALD and PRA, a significant increase was found from rest (ALD, 21.4 +/- 6.4 ng/dl), PRA, 2.5 +/- 0.5 ng.ml-1.h-1 after 30 min of cycling, which continued to increase until completion of the exercise (ALD 46.6 +/- 8.7 ng/dl, PRA 9.5 +/- 0.9 ng.ml-1.h-1.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:1827790

  11. Helium generated cold plasma finely regulates activation of human fibroblast-like primary cells.

    PubMed

    Brun, Paola; Pathak, Surajit; Castagliuolo, Ignazio; Palù, Giorgio; Brun, Paola; Zuin, Matteo; Cavazzana, Roberto; Martines, Emilio

    2014-01-01

    Non-thermal atmospheric pressure plasmas are being developed for a wide range of health care applications, including wound healing. However in order to exploit the potential of plasma for clinical applications, the understanding of the mechanisms involved in plasma-induced activation of fibroblasts, the cells active in the healing process, is mandatory. In this study, the role of helium generated plasma in the tissue repairing process was investigated in cultured human fibroblast-like primary cells, and specifically in hepatic stellate cells and intestinal subepithelial myofibroblasts. Five minutes after treatment, plasma induced formation of reactive oxygen species (ROS) in cultured cells, as assessed by flow cytometric analysis of fluorescence-activated 2',7'-dichlorofluorescein diacetate probe. Plasma-induced intracellular ROS were characterized by lower concentrations and shorter half-lives with respect to hydrogen peroxide-induced ROS. Moreover ROS generated by plasma treatment increased the expression of peroxisome proliferator activated receptor (PPAR)-γ, nuclear receptor that modulates the inflammatory responses. Plasma exposure promoted wound healing in an in vitro model and induced fibroblast migration and proliferation, as demonstrated, respectively, by trans-well assay and partitioning between daughter cells of carboxyfluorescein diacetate succinimidyl ester fluorescent dye. Plasma-induced fibroblast migration and proliferation were found to be ROS-dependent as cellular incubation with antioxidant agents (e.g. N-acetyl L-cysteine) cancelled the biological effects. This study provides evidence that helium generated plasma promotes proliferation and migration in liver and intestinal fibroblast-like primary cells mainly by increasing intracellular ROS levels. Since plasma-evoked ROS are time-restricted and elicit the PPAR-γ anti-inflammatory molecular pathway, this strategy ensures precise regulation of human fibroblast activation and can be considered a

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

    SciTech Connect

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

    1983-04-01

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

  13. Helium generated cold plasma finely regulates activation of human fibroblast-like primary cells.

    PubMed

    Brun, Paola; Pathak, Surajit; Castagliuolo, Ignazio; Palù, Giorgio; Brun, Paola; Zuin, Matteo; Cavazzana, Roberto; Martines, Emilio

    2014-01-01

    Non-thermal atmospheric pressure plasmas are being developed for a wide range of health care applications, including wound healing. However in order to exploit the potential of plasma for clinical applications, the understanding of the mechanisms involved in plasma-induced activation of fibroblasts, the cells active in the healing process, is mandatory. In this study, the role of helium generated plasma in the tissue repairing process was investigated in cultured human fibroblast-like primary cells, and specifically in hepatic stellate cells and intestinal subepithelial myofibroblasts. Five minutes after treatment, plasma induced formation of reactive oxygen species (ROS) in cultured cells, as assessed by flow cytometric analysis of fluorescence-activated 2',7'-dichlorofluorescein diacetate probe. Plasma-induced intracellular ROS were characterized by lower concentrations and shorter half-lives with respect to hydrogen peroxide-induced ROS. Moreover ROS generated by plasma treatment increased the expression of peroxisome proliferator activated receptor (PPAR)-γ, nuclear receptor that modulates the inflammatory responses. Plasma exposure promoted wound healing in an in vitro model and induced fibroblast migration and proliferation, as demonstrated, respectively, by trans-well assay and partitioning between daughter cells of carboxyfluorescein diacetate succinimidyl ester fluorescent dye. Plasma-induced fibroblast migration and proliferation were found to be ROS-dependent as cellular incubation with antioxidant agents (e.g. N-acetyl L-cysteine) cancelled the biological effects. This study provides evidence that helium generated plasma promotes proliferation and migration in liver and intestinal fibroblast-like primary cells mainly by increasing intracellular ROS levels. Since plasma-evoked ROS are time-restricted and elicit the PPAR-γ anti-inflammatory molecular pathway, this strategy ensures precise regulation of human fibroblast activation and can be considered a

  14. Association between Human Plasma Chondroitin Sulfate Isomers and Carotid Atherosclerotic Plaques.

    PubMed

    Zinellu, Elisabetta; Lepedda, Antonio Junior; Cigliano, Antonio; Pisanu, Salvatore; Zinellu, Angelo; Carru, Ciriaco; Bacciu, Pietro Paolo; Piredda, Franco; Guarino, Anna; Spirito, Rita; Formato, Marilena

    2012-01-01

    Several studies have evidenced variations in plasma glycosaminoglycans content in physiological and pathological conditions. In normal human plasma GAGs are present mainly as undersulfated chondroitin sulfate (CS). The aim of the present study was to evaluate possible correlations between plasma CS level/structure and the presence/typology of carotid atherosclerotic lesion. Plasma CS was purified from 46 control subjects and 47 patients undergoing carotid endarterectomy showing either a soft or a hard plaque. The concentration and structural characteristics of plasma CS were assessed by capillary electrophoresis of constituent unsaturated fluorophore-labeled disaccharides. Results showed that the concentration of total CS isomers was increased by 21.4% (P < 0.01) in plasma of patients, due to a significant increase of undersulfated CS. Consequently, in patients the plasma CS charge density was significantly reduced with respect to that of controls. After sorting for plaque typology, we found that patients with soft plaques and those with hard ones differently contribute to the observed changes. In plasma from patients with soft plaques, the increase in CS content was not associated with modifications of its sulfation pattern. On the contrary, the presence of hard plaques was associated with CS sulfation pattern modifications in presence of quite normal total CS isomers levels. These results suggest that the plasma CS content and structure could be related to the presence and the typology of atherosclerotic plaque and could provide a useful diagnostic tool, as well as information on the molecular mechanisms responsible for plaque instability.

  15. High-resolution structure of the M14-type cytosolic carboxypeptidase from Burkholderia cenocepacia refined exploiting PDB_REDO strategies

    PubMed Central

    Rimsa, Vadim; Eadsforth, Thomas C.; Joosten, Robbie P.; Hunter, William N.

    2014-01-01

    A potential cytosolic metallocarboxypeptidase from Burk­holderia cenocepacia has been crystallized and a synchrotron-radiation microfocus beamline allowed the acquisition of diffraction data to 1.9 Å resolution. The asymmetric unit comprises a tetramer containing over 1500 amino acids, and the high-throughput automated protocols embedded in PDB_REDO were coupled with model–map inspections in refinement. This approach has highlighted the value of such protocols for efficient analyses. The subunit is constructed from two domains. The N-terminal domain has previously only been observed in cytosolic carboxypeptidase (CCP) proteins. The C-terminal domain, which carries the Zn2+-containing active site, serves to classify this protein as a member of the M14D subfamily of carboxypeptidases. Although eukaryotic CCPs possess deglutamylase activity and are implicated in processing modified tubulin, the function and substrates of the bacterial family members remain unknown. The B. cenocepacia protein did not display deglutamylase activity towards a furylacryloyl glutamate derivative, a potential substrate. Residues previously shown to coordinate the divalent cation and that contribute to peptide-bond cleavage in related enzymes such as bovine carboxypeptidase are conserved. The location of a conserved basic patch in the active site adjacent to the catalytic Zn2+, where an acetate ion is identified, suggests recognition of the carboxy-terminus in a similar fashion to other carboxypeptidases. However, there are significant differences that indicate the recognition of substrates with different properties. Of note is the presence of a lysine in the S1′ recognition subsite that suggests specificity towards an acidic substrate. PMID:24531462

  16. Transgenic mice expressing high plasma concentrations of human apolipoprotein B100 and lipoprotein(a).

    PubMed Central

    Linton, M F; Farese, R V; Chiesa, G; Grass, D S; Chin, P; Hammer, R E; Hobbs, H H; Young, S G

    1993-01-01

    The B apolipoproteins, apo-B48 and apo-B100, are key structural proteins in those classes of lipoproteins considered to be atherogenic [e.g., chylomicron remnants, beta-VLDL, LDL, oxidized LDL, and Lp(a)]. Here we describe the development of transgenic mice expressing high levels of human apo-B48 and apo-B100. A 79.5-kb human genomic DNA fragment containing the entire human apo-B gene was isolated from a P1 bacteriophage library and microinjected into fertilized mouse eggs. 16 transgenic founders expressing human apo-B were generated, and the animals with the highest expression had plasma apo-B100 levels nearly as high as those of normolipidemic humans (approximately 50 mg/dl). The human apo-B100 in transgenic mouse plasma was present largely in lipoproteins of the LDL class as shown by agarose gel electrophoresis, chromatography on a Superose 6 column, and density gradient ultracentrifugation. When the human apo-B transgenic founders were crossed with transgenic mice expressing human apo(a), the offspring that expressed both transgenes had high plasma levels of human Lp(a). Both the human apo-B and Lp(a) transgenic mice will be valuable resources for studying apo-B metabolism and the role of apo-B and Lp(a) in atherosclerosis. Images PMID:8254057

  17. Impaired processing of FLP and NLP peptides in carboxypeptidase E (EGL-21)-deficient Caenorhabditis elegans as analyzed by mass spectrometry.

    PubMed

    Husson, Steven J; Janssen, Tom; Baggerman, Geert; Bogert, Brigitte; Kahn-Kirby, Amanda H; Ashrafi, Kaveh; Schoofs, Liliane

    2007-07-01

    Biologically active peptides are synthesized from inactive pre-proproteins or peptide precursors by the sequential actions of processing enzymes. Proprotein convertases cleave the precursor at pairs of basic amino acids, which are then removed from the carboxyl terminus of the generated fragments by a specific carboxypeptidase. Caenorhabditis elegans strains lacking proprotein convertase EGL-3 display a severely impaired neuropeptide profile (Husson et al. 2006, J. Neurochem.98, 1999-2012). In the present study, we examined the role of the C. elegans carboxypeptidase E orthologue EGL-21 in the processing of peptide precursors. More than 100 carboxy-terminally extended neuropeptides were detected in egl-21 mutant strains. These findings suggest that EGL-21 is a major carboxypeptidase involved in the processing of FMRFamide-like peptide (FLP) precursors and neuropeptide-like protein (NLP) precursors. The impaired peptide profile of egl-3 and egl-21 mutants is reflected in some similar phenotypes. They both share a severe widening of the intestinal lumen, locomotion defects, and retention of embryos. In addition, egl-3 animals have decreased intestinal fat content. Taken together, these results suggest that EGL-3 and EGL-21 are key enzymes for the proper processing of neuropeptides that control egg-laying, locomotion, fat storage and the nutritional status.

  18. NtSCP1 from tobacco is an extracellular serine carboxypeptidase III that has an impact on cell elongation.

    PubMed

    Bienert, Manuela Désirée; Delannoy, Mélanie; Navarre, Catherine; Boutry, Marc

    2012-03-01

    The leaf extracellular space contains several peptidases, most of which are of unknown function. We isolated cDNAs for two extracellular serine carboxypeptidase III genes from tobacco (Nicotiana tabacum), NtSCP1 and NtSCP2, belonging to a phylogenetic clade not yet functionally characterized in plants. NtSCP1 and NtSCP2 are orthologs derived from the two ancestors of tobacco. Reverse transcription-polymerase chain reaction analysis showed that NtSCP1 and NtSCP2 are expressed in root, stem, leaf, and flower tissues. Expression analysis of the β-glucuronidase reporter gene fused to the NtSCP1 transcription promoter region confirmed this expression profile. Western blotting of NtSCP1 and expression of an NtSCP1-green fluorescent protein fusion protein showed that the protein is located in the extracellular space of tobacco leaves and culture cells. Purified His-tagged NtSCP1 had carboxypeptidase activity in vitro. Transgenic tobacco plants overexpressing NtSCP1 showed a reduced flower length due to a decrease in cell size. Etiolated seedlings of these transgenic plants had shorter hypocotyls. These data provide support for a role of an extracellular type III carboxypeptidase in the control of cell elongation.

  19. Scavenging of hydroxyl radicals generated in human plasma following X-ray irradiation.

    PubMed

    Hosokawa, Yoichiro; Sano, Tomoaki

    2015-11-01

    There are various antioxidant materials that scavenge free radicals in human plasma. It is possible that the radical-scavenging function causes a radiation protective effect in humans. This study estimated the hydroxyl (OH) radical-scavenging activity induced by X-ray irradiation in human plasma. The test subjects included 111 volunteers (75 males and 36 females) ranging from 22 to 35 years old (average, 24.0). OH radicals generated in irradiated human plasma were measured by electron spin resonance (ESR). The relationships between the amount of the OH radical and chemical and biological parameters [total protein, total cholesterol, triglycerides and hepatitis B surface (HBs) antibodies] were estimated in the plasma of the 111 volunteers by a multivariate analysis. The presence of HBs antibodies had the greatest influence on OH radical-scavenging activity. One volunteer who did not have the HBs antibody was given an inoculation of the hepatitis B vaccine. There was a remarkable decrease in the amount of OH radical generated from plasma after the HBs antibody was produced. The results indicate that the HBs antibody is an important factor for the scavenging of OH radicals initiated by X-ray irradiation in the human body.

  20. Characterization of the human plasma phosphoproteome using linear ion trap mass spectrometry and multiple search engines.

    PubMed

    Carrascal, Montserrat; Gay, Marina; Ovelleiro, David; Casas, Vanessa; Gelpí, Emilio; Abian, Joaquin

    2010-02-01

    Major plasma protein families play different roles in blood physiology and hemostasis and in immunodefense. Other proteins in plasma can be involved in signaling as chemical messengers or constitute biological markers of the status of distant tissues. In this respect, the plasma phosphoproteome holds potentially relevant information on the mechanisms modulating these processes through the regulation of protein activity. In this work we describe for the first time a collection of phosphopeptides identified in human plasma using immunoaffinity separation of the seven major serum protein families from other plasma proteins, SCX fractionation, and TiO(2) purification prior to LC-MS/MS analysis. One-hundred and twenty-seven phosphosites in 138 phosphopeptides mapping 70 phosphoproteins were identified with FDR < 1%. A high-confidence collection of phosphosites was obtained using a combined search with the OMSSA, SEQUEST, and Phenyx search engines.

  1. Preparation of factor IX deficient human plasma by immunoaffinity chromatography using a monoclonal antibody.

    PubMed

    Goodall, A H; Kemble, G; O'Brien, D P; Rawlings, E; Rotblat, F; Russell, G C; Janossy, G; Tuddenham, E G

    1982-03-01

    A murine hybridoma clone is described that grows continuously in culture and produces a monoclonal antibody we have called Royal Free Monoclonal Antibody to factor IX No. 1 (RFF-IX/1). This has high affinity for a coagulation site on factor IX. RFF-IX/1 immobilised on sepharose can be used to deplete factor IX from normal human plasma. This immunoaffinity depleted plasma is indistinguishable from severe Christmas disease plasma and can be used as the substrate in a one stage coagulation assay for factor IX. The affinity column has high capacity and can be regenerated so that large scale production from normal plasma of factor IX deficient plasma as a diagnostic reagent is now feasible.

  2. Determination of flurbiprofen in human plasma by gas chromatography with mass spectrometry and its pharmacokinetics.

    PubMed

    Yilmaz, Bilal; Sahin, Huseyin; Akba, Vedat; Erdem, Ali Fuat

    2014-01-01

    This paper describes a GCIMS method for the determination of flurbiprofen in human plasma. Flurbiprofen and internal standard ibuprofen were extracted from plasma by using a liquid-liquid extraction method. Derivatization was carried out using N-Methyl-N-(trimethylsilyl)trifluoroacetamide. The calibration curve was linear between the concentration range of 0.10 and 5.0 microg/mL. Intraday and interday precision values for flurbiprofen in plasma were less than 5.49%, and accuracy (relative error) was better than 5.33%. The extraction recoveries of flurbiprofen from human plasma were between 93.6 and 98.6%. The LOD and LOQ of flurbiprofen were 0.03 and 0.10 microg/mL, respectively. This assay was applied to determine the pharmacokinetic parameters of flurbiprofen in healthy Turkish volunteers who had been given 100 mg of flurbiprofen.

  3. Microwave digestion preparation and ICP determination of boron in human plasma

    NASA Technical Reports Server (NTRS)

    Ferrando, A. A.; Green, N. R.; Barnes, K. W.; Woodward, B.

    1993-01-01

    A microwave digestion procedure, followed by Inductively Coupled Argon Plasma Spectroscopy, is described for the determination of boron (B) in human plasma. The National Institute of Standards and Technology (NIST) currently does not certify the concentration of B in any substance. The NIST citrus leaves 1572 (CL) Standard Reference Material (SRM) and wheat flour 1567a (WF) were chosen to determine the efficacy of digestion. CL and WF values compare favorably to those obtained from an open-vessel, wet digestion followed by ICP, and by neutron activation and mass spectrometric measurements. Plasma samples were oxidized by doubled-distilled ultrapure HNO3 in 120 mL PFA Teflon vessels. An MDS-81D microwave digestion procedure allows for rapid and relatively precise determination of B in human plasma, while limiting handling hazards and sources of contamination.

  4. The impact of red cabbage fermentation on bioavailability of anthocyanins and antioxidant capacity of human plasma.

    PubMed

    Wiczkowski, Wieslaw; Szawara-Nowak, Dorota; Romaszko, Jerzy

    2016-01-01

    The effect of red cabbage fermentation on anthocyanin bioavailability and plasma antioxidant capacity in humans was studied. In a randomized crossover study, 13 volunteers consumed fresh and fermented red cabbage. Blood and urine samples were collected before and after consumption. Analyses of anthocyanins by HPLC-MS/MS and plasma antioxidant capacity by photochemiluminescence assay were conducted. Red cabbage products contained 20 different nonacylated and acylated anthocyanins with the main structure of cyanidin triglucosides. The anthocyanins ingested were present in physiological fluids in form of 18 native anthocyanins and 12 metabolites (methylated, glucuronided, sulfated). Among cyanidin metabolites identified, methylated forms were predominant. Bioavailability of anthocyanin from fresh red cabbage was over 10% higher than from fermented red cabbage. Upon fresh cabbage consumption, volunteers plasma showed higher antioxidant capacity than after fermented cabbage intake. The study has shown that fermentation process affects red cabbage anthocyanins bioavailability and human plasma antioxidant capacity.

  5. Spectrofluorimetric Determination of α-Tocopherol in Capsules and Human Plasma

    PubMed Central

    Demirkaya-Miloglu, Fatma; Kadioglu, Y.; Senol, O.; Yaman, M. E.

    2013-01-01

    A simple, sensitive and rapid spectrofluorimetric method for determination of α-tocopherol in pharmaceutical capsule and human plasma was developed and validated. The native fluorescence of α-tocopherol was measured at 334 nm with excitation at 291 nm, after extraction of α-tocopherol from human plasma hexane:dichloromethane mixture. The calibration curves were linear (R≥0.9993) in the concentration range of 0.25-2.5 μg/ml of α-tocopherol in both standard solutions and plasma samples. The developed method was directly and easily applied for determination of α-tocopherol in the plasma of healthy volunteers and different type of bladder cancer and stomach cancer patients and also pharmaceutical capsule. PMID:24403657

  6. Free amino acids in human blood plasma during space flights.

    PubMed

    Ushakov, A S; Vlasova, T F

    1976-10-01

    The present investigation presents results of studying free amino acids of peripheral plasma in cosmonauts who made space flights of different duration onboard the spacecraft Soyuz-12, Soyuz-16 and the orbital station Salyut-4. The study showed changes in the content of free amino acids which varied for different amino acids. Most pronounced changes were found in the content of glutamic and aspartic acids, sulfur-containing amino acids and arginine.

  7. Effects of Sleep Restriction on the Human Plasma Metabolome

    PubMed Central

    Bell, Lauren N.; Kilkus, Jennifer M.; Booth, John N.; Bromley, Lindsay E.; Imperial, Jacqueline G.; Penev, Plamen D.

    2013-01-01

    This study examined the effects of recurrent sleep restriction on the plasma metabolome of adults with familial risk of type 2 diabetes. Eleven healthy adults (6M/5F; mean [SD] age: 26 [3] years; BMI 23.5 [2.3] kg/m2) with parental history of type 2 diabetes participated in a two-condition, two-period randomized crossover study at the Clinical Resource Center at an academic hospital. Each participant completed two 8-night inpatient sessions with restricted (5.5-h time-in-bed) vs. adequate (8.5-h time-in-bed) sleep opportunity while daily food intake and physical activity were carefully controlled. A combination of two UHPLC/MS/MS platforms and one GC/MS platform was used to measure 362 biochemicals in fasting plasma samples collected from study participants the morning after each 8-night sleep treatment. Relative concentrations of 12 amino acids and related metabolites were increased when sleep was curtailed. Sleep restriction also induced elevations in several fatty acid, bile acid, steroid hormone, and tricarboxylic acid cycle intermediates. In contrast, circulating levels of glucose, some monosaccharides, gluconate, and five-carbon sugar alcohols tended to decline when sleep was reduced. Recurrent sleep curtailment affected multiple pathways of intermediary metabolism in adults at risk for type 2 diabetes. An elevation in plasma amino acids and related biochemicals was the most pronounced metabolic signature seen in response to 8 nights of sleep restriction. PMID:23954406

  8. Aortic carboxypeptidase-like protein (ACLP) enhances lung myofibroblast differentiation through transforming growth factor β receptor-dependent and -independent pathways.

    PubMed

    Tumelty, Kathleen E; Smith, Barbara D; Nugent, Matthew A; Layne, Matthew D

    2014-01-31

    Idiopathic pulmonary fibrosis (IPF) is a chronic and fatal lung disease characterized by the overgrowth, hardening, and scarring of lung tissue. The exact mechanisms of how IPF develops and progresses are unknown. IPF is characterized by extracellular matrix remodeling and accumulation of active TGFβ, which promotes collagen expression and the differentiation of smooth muscle α-actin (SMA)-positive myofibroblasts. Aortic carboxypeptidase-like protein (ACLP) is an extracellular matrix protein secreted by fibroblasts and myofibroblasts and is expressed in fibrotic human lung tissue and in mice with bleomycin-induced fibrosis. Importantly, ACLP knockout mice are significantly protected from bleomycin-induced fibrosis. The goal of this study was to identify the mechanisms of ACLP action on fibroblast differentiation. As primary lung fibroblasts differentiated into myofibroblasts, ACLP expression preceded SMA and collagen expression. Recombinant ACLP induced SMA and collagen expression in mouse and human lung fibroblasts. Knockdown of ACLP slowed the fibroblast-to-myofibroblast transition and partially reverted differentiated myofibroblasts by reducing SMA expression. We hypothesized that ACLP stimulates myofibroblast formation partly through activating TGFβ signaling. Treatment of fibroblasts with recombinant ACLP induced phosphorylation and nuclear translocation of Smad3. This phosphorylation and induction of SMA was dependent on TGFβ receptor binding and kinase activity. ACLP-induced collagen expression was independent of interaction with the TGFβ receptor. These findings indicate that ACLP stimulates the fibroblast-to-myofibroblast transition by promoting SMA expression via TGFβ signaling and promoting collagen expression through a TGFβ receptor-independent pathway. PMID:24344132

  9. Differential Responses of Plasma Adropin Concentrations To Dietary Glucose or Fructose Consumption In Humans

    PubMed Central

    Butler, Andrew A.; St-Onge, Marie-Pierre; Siebert, Emily A.; Medici, Valentina; Stanhope, Kimber L.; Havel, Peter J.

    2015-01-01

    Adropin is a peptide hormone encoded by the Energy Homeostasis Associated (ENHO) gene whose physiological role in humans remains incompletely defined. Here we investigated the impact of dietary interventions that affect systemic glucose and lipid metabolism on plasma adropin concentrations in humans. Consumption of glucose or fructose as 25% of daily energy requirements (E) differentially affected plasma adropin concentrations (P < 0.005) irrespective of duration, sex or age. Glucose consumption reduced plasma adropin from 3.55 ± 0.26 to 3.28 ± 0.23 ng/ml (N = 42). Fructose consumption increased plasma adropin from 3.63 ± 0.29 to 3.93 ± 0.34 ng/ml (N = 45). Consumption of high fructose corn syrup (HFCS) as 25% E had no effect (3.43 ± 0.32 versus 3.39 ± 0.24 ng/ml, N = 26). Overall, the effect of glucose, HFCS and fructose on circulating adropin concentrations were similar to those observed on postprandial plasma triglyceride concentrations. Furthermore, increases in plasma adropin levels with fructose intake were most robust in individuals exhibiting hypertriglyceridemia. Individuals with low plasma adropin concentrations also exhibited rapid increases in plasma levels following consumption of breakfasts supplemented with lipids. These are the first results linking plasma adropin levels with dietary sugar intake in humans, with the impact of fructose consumption linked to systemic triglyceride metabolism. In addition, dietary fat intake may also increase circulating adropin concentrations. PMID:26435060

  10. Vampire bat salivary plasminogen activator is quiescent in human plasma in the absence of fibrin unlike human tissue plasminogen activator.

    PubMed

    Gardell, S J; Hare, T R; Bergum, P W; Cuca, G C; O'Neill-Palladino, L; Zavodny, S M

    1990-12-15

    The vampire bat salivary plasminogen activator (Bat-PA) is a potent PA that exhibits remarkable selectivity toward fibrin-bound plasminogen (Gardell et al, J Biol Chem 256: 3568, 1989). Herein, we describe the activity of recombinant DNA-derived Bat-PA (rBat-PA) in a human plasma milieu. rBat-PA and recombinant human single-chain tissue plasminogen activator (rt-PA) are similarly efficacious at lysing plasma clots. In stark contrast to rt-PA, the addition of 250 nmol/L rBat-PA to plasma in the absence of a clot failed to deplete plasminogen, alpha 2-antiplasmin and fibrinogen. The lytic activities exhibited by finger-domain minus Bat-PA (F- rBat-PA) and finger and epidermal growth factor-like domains minus Bat-PA (FG- rBat-PA) were less than rBat-PA, especially at low concentrations of PA; nevertheless, these truncated forms also possessed a strict requirement for a fibrin cofactor. The loss of PA activity following the addition of rBat-PA to plasma was slower than that observed when either rt-PA or two-chain rt-PA was added. The efficacy, fibrin selectivity, and decreased susceptibility to inactivation exhibited by rBat-PA in vitro in a human plasma milieu suggests that rBat-PA may be superior to rt-PA for the treatment of thrombotic complications. PMID:2124935

  11. The effects of plasma-processing conditions on the morphology of adherent human blood platelets

    SciTech Connect

    Murugesan, R.; Hanley, E.; Lauer, J. L.; Shohet, J. L.; Albrecht, R. M.; Heintz, J. A.; Oliver, J. A.

    2008-05-01

    Hematocompatibility and nonfouling properties of materials are crucial for the development of small-scale biomedical devices. This study examines the adhesion and morphology of purified human platelets on plasma-polymerized tetraglyme-coated glass substrates. The effect of varying the plasma-processing parameters on platelet responses was determined using scanning electron microscopy. Images of platelets on the coated surfaces show that a significant reduction in platelet adhesion and spreading can be achieved as the processing parameters are varied.

  12. Simultaneous extraction of. beta. -endorphin and leu- and met-enkephalins from human and rat plasma

    SciTech Connect

    Bhathena, S.J.; Smith, P.M.; Kennedy, B.W. ); Voyles, N.R.; Recant, L. )

    1989-01-01

    A simple, rapid and reliable procedure is described to simultaneously concentrated and purify {beta}-endorphin, leu-and met-enkephalins from small volumes of human and rat plasma before radioimmunoassay is performed. It uses C{sub 18} Sep-Pak reverse phase cartridges. The effectiveness of different protease inhibitors in preventing degradation of opiates by plasma and different solvent systems for eluting opiates is also evaluated.

  13. Regulation of human amnion cell growth and morphology by sera, plasma, and growth factors.

    PubMed

    Gaffney, E V; Grimaldi, M A

    1981-01-01

    The requirements of human epithelial cells derived from the amnion membrane for serum factors were investigated. The growth promoting effects of human whole blood serum (WBS), platelet-poor defibrinogenated plasma, and plasma-derived serum (PDS) were examined in primary cultures of these ectodermal cells. The numbers of population doublings recorded after 10 days in the presence of 10% WBS, defibrinogenated plasma, or PDS were 2.3, 2.0 or 1.5, respectively. Although dialysis of sera or plasma had little effect on growth promotion, it markedly decreased the capacity of plasma to maintain cells in culture beyond 10 days. The differences in growth activities could not be attributed to the presence of anticoagulant in plasma and PDS or to the presence of excess calcium in PDS. Platelet lysates and purified platelet-derived growth factor had no effect on growth. Amnion cell growth was enhanced by epidermal growth factor (EGF) or hydrocortisone, but the glucocorticoid did not condition cells to respond to growth factors. Insulin and fibroblast growth factor singly or in combination had no effect on cell replication. Giant cell formation accompanied maintenance in hydrocortisone with defibrinogenated plasma and PDS. Discrete regions of dense population appeared in the presence of hydrocortisone, EGF, and undialyzed supplements.

  14. Formation of tissue factor activity following incubation of recombinant human tissue factor apoprotein with plasma lipoproteins

    SciTech Connect

    Sakai, T.; Kisiel, W. )

    1990-11-01

    Incubation of recombinant human tissue factor apoprotein (Apo-TF) with human plasma decreased the recalcified clotting time of this plasma in a time-and dose-dependent manner suggesting relipidation of the Apo-TF by plasma lipoproteins. Incubation of Apo-TF with purified preparations of human very low density, low density and high density lipoproteins resulted in tissue factor activity in a clotting assay. The order of effectiveness was VLDL greater than LDL much greater than HDL. Tissue factor activity generated by incubation of a fixed amount of Apo-TF with plasma lipoproteins was lipoprotein concentration-dependent and saturable. The association of Apo-TF with lipoprotein particles was supported by gel filtration studies in which {sup 125}I-Apo-TF coeluted with the plasma lipoprotein in the void volume of a Superose 6 column in the presence and absence of calcium ions. In addition, void-volume Apo-TF-lipoprotein fractions exhibited tissue factor activity. These results suggest that the factor VIII-bypassing activity of bovine Apo-TF observed in a canine hemophilic model may be due, in part, to its association with plasma lipoproteins and expression of functional tissue factor activity.

  15. Inhibition of carboxypeptidase A by D-penicillamine: mechanism and implications for drug design.

    PubMed

    Chong, C R; Auld, D S

    2000-06-27

    Zinc metalloprotease inhibitors are usually designed to inactivate the enzyme by forming a stable ternary complex with the enzyme and active-site zinc. D-Cysteine inhibits carboxypeptidase, ZnCPD, by forming such a complex, with a K(i) of 2.3 microM. In contrast, the antiarthritis drug D-penicillamine, D-PEN, which differs from D-Cys only by the presence of two methyl groups on the beta-carbon, inhibits ZnCPD by promoting the release of the active-site zinc. We have given the name catalytic chelator to such inhibitors. Inhibition is a two-step process characterized by formation of a complex with the enzyme (K(i(initial)) = 1.2 mM) followed by release of the active-site zinc at rates up to 420-fold faster than the spontaneous release. The initial rate of substrate hydrolysis at completion of the second step also depends on D-PEN concentration, reflecting formation of a thermodynamic equilibrium governed by the stability constants of chelator and apocarboxypeptidase for zinc (K(i(final)) = 0.25 mM). The interaction of D-PEN and D-Cys with the active-site metal has been examined by replacing the active-site zinc by a chromophoric cobalt atom. Both inhibitors perturb the d-d transitions of CoCPD in the 500-600 nm region within milliseconds of mixing but only the CoCPD.D-Cys complex displays a strong S --> Co(II) charge-transfer band at 340 nm indicative of a metal-sulfur bond. While the D-Cys complex is stable, the CoCPD.D-PEN complex breaks down to apoenzyme and Co(D-PEN)(2) with a half-life of 0.5 s. D-PEN is the first drug found to inhibit a metalloprotease by increasing the dissociation rate constant of the active-site metal. The ability of D-PEN to catalyze metal removal from carboxypeptidase A and other zinc proteases suggests a possible mechanism of action in arthritis and Wilson's disease and may also underlie complications associated with its clinical use.

  16. Biochemical quantification of sympathetic nervous activity in humans using radiotracer methodology: fallibility of plasma noradrenaline measurements

    SciTech Connect

    Esler, M.; Leonard, P.; O'Dea, K.; Jackman, G.; Jennings, G.; Korner, P.

    1982-01-01

    We have developed radiotracer techniques for studying noradrenaline kinetics, to assess better sympathetic nervous system function in humans. Tritiated l-noradrenaline was infused intravenously (0.35 microCi/m2/min) to plateau plasma concentration. Noradrenaline plasma clearance was calculated from plasma tritiated noradrenaline concentration at steady state, and the rate of spillover of noradrenaline to plasma derived from plasma noradrenaline specific radioactivity. Mean noradrenaline spillover at rest in 34 normal subjects was 0.33 micrograms/m2/min (range 0.17-0.61 micrograms/m2/min). Predictably, noradrenaline spillover was reduced in patients with subnormal sympathetic nervous system activity, 0.16 +/- 0.09 micrograms/m2/min in eight patients with idiopathic peripheral autonomic insufficiency, and 0.11 +/- 0.07 micrograms/m2/min (mean +/- SD) in six patients with essential hypertension treated with clonidine (0.45 mg daily). Noradrenaline line plasma clearance in normal subjects was 1.32 +/- 0.28 L/m2/min. Clearance fell with age, causing the previously described rise in plasma noradrenaline concentration with aging. Unexpected effects of drugs were encountered, for example chronic beta-adrenergic blockade in patients with essential hypertension reduced noradrenaline clearance. Plasma noradrenaline concentration measurements were not in agreement with noradrenaline release rate values, and do not reliably indicate sympathetic nervous system activity, in instances such as these where noradrenaline clearance is abnormal.

  17. Determination of bevantolol in human plasma by high performance liquid chromatography using solid phase extraction technique.

    PubMed

    Trung, Tran Quoc; Long, Pham Hai; Al-Abd, Ahmed M; Ku, Hyo Jeong; Lee, Ho Yoon; Hwang, Sung Joo; Kim, Kyeong Ho

    2007-07-01

    A method was developed and fully validated for the determination of bevantolol, an adrenergic-receptor blocker, in human plasma. Bevantolol and betaxolol as internal standard (I.S) were extracted from 1 mL of human plasma by solid phase extraction technique using Sep-pak silica cartridge. Chromatographic separation was accomplished under isocratic conditions using a reverse-phase C8 analytical column and mixture of dibasic ammonium phosphate (pH 5.7; 50 mM)-acetonitrile (75:25, v/v) as mobile phase, with a detection wavelength at 220 nm. The method was proved to be specific by testing six different human plasma sources. Linearity was established for the concentration ranges of 40-1600 ng/mL with correlation coefficent of 0.9995. The lower limit of quantification 40 ng/mL with precision of 10.9% as C.V%.

  18. Validation of a HPLC/FLD Method for Quantification of Tocotrienols in Human Plasma.

    PubMed

    Che, Hui-Ling; Tan, Doryn Meam-Yee; Meganathan, Puvaneswari; Gan, Yee-Lin; Abdul Razak, Ghazali; Fu, Ju-Yen

    2015-01-01

    Quantification of tocotrienols in human plasma is critical when the attention towards tocotrienols on its distinctive properties is arising. We aim to develop a simple and practical normal-phase high performance liquid chromatography method to quantify the amount of four tocotrienol homologues in human plasma. Using both the external and internal standards, tocotrienol homologues were quantified via a normal-phase high performance liquid chromatography with fluorescence detector maintained at the excitation wavelength of 295 nm and the emission wavelength of 325 nm. The four tocotrienol homologues were well separated within 30 minutes. A large interindividual variation between subjects was observed as the absorption of tocotrienols is dependent on food matrix and gut lipolysis. The accuracies of lower and upper limit of quantification ranged between 92% and 109% for intraday assays and 90% and 112% for interday assays. This method was successfully applied to quantify the total amount of four tocotrienol homologues in human plasma. PMID:26604927

  19. Aptamer RA36 inhibits of human, rabbit, and rat plasma coagulation activated with thrombin or snake venom coagulases.

    PubMed

    Savchik, E Yu; Kalinina, T B; Drozd, N N; Makarov, V A; Zav'yalova, E G; Lapsheva, E N; Mudrik, N N; Babij, A V; Pavlova, G V; Golovin, A V; Kopylov, A M

    2013-11-01

    RA36 DNA aptamer is a direct anticoagulant prolonging clotting time of human, rabbit, and rat plasma in the thrombin time test. Anticoagulant activity of RA36 is lower than that of recombinant hirudin. During inhibition of human plasma clotting activated with echitox (coagulase from Echis multisquamatus venom), the aptamer presumably binds to meisothrombin exosite I. The sensitivity of human plasma to the aptamer 5-fold surpasses that of rat plasma. Analysis of RA36 binding to coagulase of Agkistrodon halys venom (ancistron) is required for proving the effect of aptamer on polymerization of human fibrinogen. PMID:24319726

  20. Two independent apolipoprotein A5 haplotypes influence human plasma triglyceride levels.

    PubMed

    Pennacchio, Len A; Olivier, Michael; Hubacek, Jaroslav A; Krauss, Ronald M; Rubin, Edward M; Cohen, Jonathan C

    2002-11-15

    The recently identified apolipoprotein A5 gene (APOA5) has been shown to play an important role in determining plasma triglyceride concentrations in humans and mice. We previously identified an APOA5 haplotype (designated APOA5*2) that is present in approximately 16% of Caucasians and is associated with increased plasma triglyceride concentrations. In this report we describe another APOA5 haplotype (APOA5*3) containing the rare allele of the single nucleotide polymorphism c.56C>G that changes serine to tryptophan at codon 19 and is independently associated with high plasma triglyceride levels in three different populations. In a sample of 264 Caucasian men and women with plasma triglyceride concentrations above the 90th percentile or below the 10th percentile, the APOA5*3 haplotype was more than three-fold more common in the group with high plasma triglyceride levels. In a second independently ascertained sample of Caucasian men and women (n=419) who were studied while consuming their self-selected diets as well as after high-carbohydrate diets and high-fat diets, the APOA5*3 haplotype was associated with increased plasma triglyceride levels on all three dietary regimens. In a third population comprising 2660 randomly selected individuals, the APOA5*3 haplotype was found in 12% of Caucasians, 14% of African-Americans and 28% of Hispanics and was associated with increased plasma triglyceride levels in both men and women in each ethnic group. These findings establish that the APOA5 locus contributes significantly to inter-individual variation in plasma triglyceride levels in humans. Together, the APOA5*2 and APOA5*3 haplotypes are found in 25-50% of African-Americans, Hispanics and Caucasians and support the contribution of common human variation to quantitative phenotypes in the general population.

  1. Two independent apolipoprotein a5 Haplotypes influence human plasma triglyceride levels

    SciTech Connect

    Pennacchio, Len A.; Olivier, Michael; Hubacek, Jaroslav A.; Krauss, Ronald M.; Rubin, Edward M.; Cohen, Jonathan C.

    2002-09-16

    The recently identified apolipoprotein A5 gene (APOA5) has been shown to play an important role in determining plasma triglyceride concentrations in humans and mice. We previously identified an APOA5 haplotype (designated APOA5*2) that is present in {approx}16 percent of Caucasians and is associated with increased plasma triglyceride concentrations. In this report we describe another APOA5 haplotype (APOA5*3) containing the rare allele of the single nucleotide polymorphism c.56C>G that changes serine to tryptophan at codon 19 and is independently associated with high plasma triglyceride levels in three different populations. In a sample of 264 Caucasian men and women with plasma triglyceride concentrations above the 90th percentile or below the 10th percentile, the APOA5*3 haplotype was more than three-fold more common in the group with high plasma triglyceride levels. In a second independently ascertained sample of Caucasian men and women (n 1/4 419) who were studied while consuming their self-selected diets as well as after high-carbohydrate diets and high-fat diets, the APOA5*3 haplotype was associated with increased plasma triglyceride levels on all three dietary regimens. In a third population comprising 2660 randomly selected individuals, the APOA5*3 haplotype was found in 12 percent of Caucasians, 14 percent of African-Americans and 28 percent of Hispanics and was associated with increased plasma triglyceride levels in both men and women in each ethnic group. These findings establish that the APOA5 locus contributes significantly to inter-individual variation in plasma triglyceride levels in humans. Together, the APOA5*2 and APOA5*3 haplotypes are found in 25 50 percent of African-Americans, Hispanics and Caucasians and support the contribution of common human variation to quantitative phenotypes in the general population.

  2. Plasma adrenal and gonadal sex steroids in human pubertal development.

    PubMed

    Ducharme, J R; Forest, M G; De Peretti, E; Sempé, M; Collu, R; Bertrand, J

    1976-03-01

    Plasma free dehydroepiandrosterone (DHA), androstenedione (delta), testosterone (T), dihydrotestosterone (DHT), estrone (E1), and estradiol (E2) were measured by radioimmunoassay in 55 boys and 54 girls 3.5 to 16.3 years of age. Plasma DHA increased significantly between 6 and 8 years of age in girls and between 8 and 10 years of age in boys. A further significant increase was noted between 10 and 12 years of age in both sexes. Delta rose significantly between 8 and 10 years of age in girls and between 10 and 12 years in boys. In contrast, no significant increase in T, DHT, or E1, was noted prior to 12 years of age in both sexes. However, E2 showed a significant increase between 10 and 12 years of age in girls. This early rise in the course of pubertal development of the two sex steroids predominantly of adrenal origin, DHA and delta, and its occurence 1 to 2 years earlier in girls than in boys, as does puberty itself, suggest a possible role for these steroids in the mechanisms involved in triggering the hypothalamic-pituitary-gonadal axis at puberty.

  3. Peanut consumption increases levels of plasma very long chain fatty acids in humans.

    PubMed

    Lam, Christina; Wong, Derek; Cederbaum, Stephen; Lim, Bennie; Qu, Yong

    2012-11-01

    Peanut consumption has been suspected of raising plasma very long chain fatty acid (VLCFA) levels in humans. The effect of peanut consumption on VLCFAs was studied in six human subjects. After 3 to 4h of peanut butter ingestion, plasma C26:0 and C26:0/C22:0 were found to be significantly elevated to levels seen in patients with peroxisomal disorders. These levels returned to normal within 12h. Peanut consumption needs to be accounted for when interpreting VLCFAs. PMID:22864056

  4. Properties of salt-resistant lipase and lipoprotein lipase purified from human post-heparin plasma.

    PubMed Central

    Ostlund-Lindqvist, A M

    1979-01-01

    Lipoprotein lipase and salt-resistant lipase were isolated from human post-heparin plasma. The proteins of human post-plasma lipoprotein lipase and salt-resistant lipase were identified and demonstrated to be immunologically different. Significant differences between the two enzymes in their relative amino acid composition were demonstrated, which indicates that the two enzymes are different proteins. When analysed by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis, the enzymes seemed to have monomer molecular weights similar to that of lipoprotein lipase purified from bovine milk. Images Fig. 1. Fig. 3. PMID:113002

  5. Effects of oral aspartame on plasma phenylalanine in humans and experimental rodents. Short note.

    PubMed

    Wurtman, R J; Maher, T J

    1987-01-01

    All aspartame does given to humans cause greater elevations in plasma (and, presumably, brain) phenylalanine than in plasma tyrosine. In contrast, doses of aspartame usually used in experiments on rodents preferentially elevate tyrosine. Since phenylalanine can inhibit brain catecholamine synthesis while tyrosine is the antidote for this effect, we determined the aspartame dose that would be needed to elevate phenylalanine more than tyrosine in rodents, using published data. In general rodents need 60 times as much aspartame, on a mg/kg basis, as humans to obtain comparable elevations in phenylalanine with respect to tyrosine.

  6. Radioimmunoassay for Luteinizing Hormone in Human plasma Or Serum: Physiological Studies*

    PubMed Central

    Odell, W. D.; Ross, G. T.; Rayford, P. L.

    1967-01-01

    It is not practical to quantitate gonadotropin in the blood of normal men and women by utilizing bioassays. We have developed a method for sensitive, precise, and specific radioimmunoassay of luteinizing hormone (LH) in human serum or plasma. Antisera were developed against human chorionic gonadotropin, and one of these was selected for extensive cross-reaction with human LH. Highly purified LH was radioiodinated by the method of Greenwood, Hunter, and Glover. Separation of antibody-bound from free LH-131I was accomplished by a double antibody technique. Dose-response curves for the purifed LH, an impure urinary LH preparation, pituitary powder, and LH in plasma were all identical. Immunoassay and bioassay of impure urinary and pituitary gonadotropin preparations in terms of a common standard resulted in an index of discrimination of close to unity. LH levels in plasma from 32 adult men and 30 women outside the midcycle ranged from 0.6 to 3.2 mμg per ml (1 mμg of our laboratory LH standard is equivalent to 8 mU of the Second International Reference Preparation of Human Menopausal Gonadotropin). Levels were remarkably constant in men from day to day and in women except at midcycle, when a sharp peak occurred lasting less than 24 hours. In all women studied who had a midcycle LH peak, mean plasma LH levels during the follicular phase of the menstrual cycle were higher than mean values obtained during the luteal phase. Prepubertal children had detectable plasma LH, and mean values were only slightly less than in adults. Plasma from castrate men or women or postmenopausal women contained 4.5 to 10.5 mμg per ml. Clomiphene treatment of four men resulted in a doubling of plasma LH in 5 days. PMID:6018762

  7. In silico approaches to identify the potential inhibitors of glutamate carboxypeptidase II (GCPII) for neuroprotection.

    PubMed

    Naushad, Shaik Mohammad; Janaki Ramaiah, M; Stanley, Balraj Alex; Prasanna Lakshmi, S; Vishnu Priya, J; Hussain, Tajamul; Alrokayan, Salman A; Kutala, Vijay Kumar

    2016-10-01

    To develop a potential inhibitor for glutamate carboxypeptidase II (GCPII) effective against all the eight common genetic variants reported, PyMOL molecular visualization system was used to generate models of variants using the crystal structure of GCPII i.e. 2OOT as a template. High-throughput virtual screening of 29 compounds revealed differential efficacy across the eight genetic variants (pIC50: 4.70 to 10.22). Pharmacophore analysis and quantitative structure-activity relationship (QSAR) studies revealed a urea-based N-acetyl aspartyl glutamate (NAAG) analogue as more potent inhibitor, which was effective across all the genetic variants of GCPII as evidenced by glide scores (-4.32 to -7.08) and protein-ligand interaction plots (13 interactions in wild GCPII). This molecule satisfied Lipinski rule of five and rule of three for drug-likeliness. Being a NAAG-analogue, this molecule might confer neuroprotection by inhibiting glutamatergic neurotransmission mediated by N-acetylated alpha-linked acidic dipeptidase (NAALADase), a splice variant of GCPII. PMID:27430729

  8. Phenotypic Characterization of Mice Heterozygous for a Null Mutation of Glutamate Carboxypeptidase II

    PubMed Central

    Han, Liqun; Picker, Jonathan D.; Schaevitz, Laura R.; Tsai, Guochuan; Feng, Jiamin; Jiang, Zhichun; Chu, Hillary C.; Basu, Alo C.; Berger-Sweeney, Joanne; Coyle, Joseph T.

    2009-01-01

    Glutamate is the major excitatory neurotransmitter in the mammalian central nervous system. Disturbed glutamate signaling resulting in hypofunction of NMDA receptors has been implicated in the pathophysiology of schizophrenia. Glutamate Carboxypeptidase II (GCP II) hydrolyzes N-acetyl-alpha L-aspartyl-L-glutamate (NAAG) into glutamate and N-acetyl-aspartate (NAA). NAAG is a neuropeptide that is an NMDA receptor antagonist as well as an agonist for the metabotropic glutamate receptor-3 (mGluR3), which inhibits glutamate release. The aggregate effect of NAAG is thus to attenuate NMDA receptor activation. To manipulate the expression of GCP II, loxP sites were inserted flanking exon 1 and 2, which were excised by crossing with a Cre-expressing mouse. The mice heterozygous for this deletion showed a 50% reduction in the expression level of protein and functional activity of GCP II in brain samples. Heterozygous mutant crosses did not yield any homozygous null animals at birth or as embryos (N >200 live births and fetuses). These data are consistent with the previous report that GCP II homozygous mutant mice generated by removing exons 9 and 10 of GCP II gene were embryonically lethal and confirm our hypothesis that GCP II plays an essential role early in embryonic development. Heterozygous mice, however, developed normally to adulthood and exhibited increased locomotor activity, reduced social interaction, and a subtle cognitive deficit in working memory. PMID:19347959

  9. Serine carboxypeptidase 46 Regulates Grain Filling and Seed Germination in Rice (Oryza sativa L.)

    PubMed Central

    Li, Zhiyong; Tang, Liqun; Qiu, Jiehua; Zhang, Wen; Wang, Yifeng; Tong, Xiaohong; Wei, Xiangjin; Hou, Yuxuan

    2016-01-01

    Serine carboxypeptidase (SCP) is one of the largest groups of enzymes catalyzing proteolysis for functional protein maturation. To date, little is known about the function of SCPs in rice. In this study, we present a comprehensive analysis of the gene structure and expression profile of 59 rice SCPs. SCP46 is dominantly expressed in developing seeds, particularly in embryo, endosperm and aleurone layers, and could be induced by ABA. Functional characterization revealed that knock-down of SCP46 resulted in smaller grain size and enhanced seed germination. Furthermore, scp46 seed germination became less sensitive to the ABA inhibition than the Wild-type did; suggesting SCP46 is involved in ABA signaling. As indicated by RNA-seq and qRT-PCR analysis, numerous grain filling and seed dormancy related genes, such as SP, VP1 and AGPs were down-regulated in scp46. Yeast-two-hybrid assay also showed that SCP46 interacts with another ABA-inducible protein DI19-1. Taken together, we suggested that SCP46 is a master regulator of grain filling and seed germination, possibly via participating in the ABA signaling. The results of this study shed novel light into the roles of SCPs in rice. PMID:27448032

  10. Structure and function of a novel LD-carboxypeptidase a involved in peptidoglycan recycling.

    PubMed

    Das, Debanu; Hervé, Mireille; Elsliger, Marc-André; Kadam, Rameshwar U; Grant, Joanna C; Chiu, Hsiu-Ju; Knuth, Mark W; Klock, Heath E; Miller, Mitchell D; Godzik, Adam; Lesley, Scott A; Deacon, Ashley M; Mengin-Lecreulx, Dominique; Wilson, Ian A

    2013-12-01

    Approximately 50% of cell wall peptidoglycan in Gram-negative bacteria is recycled with each generation. The primary substrates used for peptidoglycan biosynthesis and recycling in the cytoplasm are GlcNAc-MurNAc(anhydro)-tetrapeptide and its degradation product, the free tetrapeptide. This complex process involves ∼15 proteins, among which the cytoplasmic enzyme ld-carboxypeptidase A (LdcA) catabolizes the bond between the last two l- and d-amino acid residues in the tetrapeptide to form the tripeptide, which is then utilized as a substrate by murein peptide ligase (Mpl). LdcA has been proposed as an antibacterial target. The crystal structure of Novosphingobium aromaticivorans DSM 12444 LdcA (NaLdcA) was determined at 1.89-Å resolution. The enzyme was biochemically characterized and its interactions with the substrate modeled, identifying residues potentially involved in substrate binding. Unaccounted electron density at the dimer interface in the crystal suggested a potential site for disrupting protein-protein interactions should a dimer be required to perform its function in bacteria. Our analysis extends the identification of functional residues to several other homologs, which include enzymes from bacteria that are involved in hydrocarbon degradation and destruction of coral reefs. The NaLdcA crystal structure provides an alternate system for investigating the structure-function relationships of LdcA and increases the structural coverage of the protagonists in bacterial cell wall recycling.

  11. Active Compounds Against Anopheles minimus Carboxypeptidase B for Malaria Transmission-Blocking Strategy.

    PubMed

    Mongkol, Watcharakorn; Arunyawat, Uraiwan; Surat, Wunrada; Kubera, Anchanee

    2015-11-01

    Malaria transmission-blocking compounds have been studied to block the transmission of malaria parasites, especially the drug-resistant Plasmodium. Carboxypeptidase B (CPB) in the midgut of Anopheline mosquitoes has been demonstrated to be essential for the sexual development of Plasmodium in the mosquito. Thus, the CPB is a potential target for blocking compounds. The aim of this research was to screen compounds from the National Cancer Institute (NCI) diversity dataset and U.S. Food and Drug Administration (FDA)-approved drugs that could reduce the Anopheles CPB activity. The cDNA fragment of cpb gene from An. minimus (cpbAmi) was amplified and sequenced. The three-dimensional structure of CPB was predicted from the deduced amino acid sequence. The virtual screening of the compounds from NCI diversity set IV and FDA-approved drugs was performed against CPBAmi. The inhibition activity against CPBAmi of the top-scoring molecules was characterized in vitro. Three compounds-NSC-1014, NSC-332670, and aminopterin with IC50 at 0.99 mM, 1.55 mM, and 0.062 mM, respectively-were found to significantly reduce the CPBAmi activity.

  12. Glutamate Carboxypeptidase II in Diagnosis and Treatment of Neurologic Disorders and Prostate Cancer

    PubMed Central

    Bařinka, C.; Rojas, C.; Slusher, B.; Pomper, M.

    2012-01-01

    Glutamate carboxypeptidase II (GCPII) is a membrane-bound binuclear zinc metallopeptidase with the highest expression levels found in the nervous and prostatic tissue. Throughout the nervous system, glia-bound GCPII is intimately involved in the neuron-neuron and neuron-glia signaling via the hydrolysis of N-acetylaspartylglutamate (NAAG), the most abundant mammalian peptidic neurotransmitter. The inhibition of the GCPII-controlled NAAG catabolism has been shown to attenuate neurotoxicity associated with enhanced glutamate transmission and GCPII-specific inhibitors demonstrate efficacy in multiple preclinical models including traumatic brain injury, stroke, neuropathic and inflammatory pain, amyotrophic lateral sclerosis, and schizophrenia. The second major area of pharmacological interventions targeting GCPII focuses on prostate carcinoma; GCPII expression levels are highly increased in androgen-independent and metastatic disease. Consequently, the enzyme serves as a potential target for imaging and therapy. This review offers a summary of GCPII structure, physiological functions in healthy tissues, and its association with various pathologies. The review also outlines the development of GCPII-specific small-molecule compounds and their use in preclinical and clinical settings. PMID:22214450

  13. Structure and Function of a Novel ld-Carboxypeptidase A Involved in Peptidoglycan Recycling

    PubMed Central

    Das, Debanu; Hervé, Mireille; Elsliger, Marc-André; Kadam, Rameshwar U.; Grant, Joanna C.; Chiu, Hsiu-Ju; Knuth, Mark W.; Klock, Heath E.; Miller, Mitchell D.; Godzik, Adam; Lesley, Scott A.; Deacon, Ashley M.

    2013-01-01

    Approximately 50% of cell wall peptidoglycan in Gram-negative bacteria is recycled with each generation. The primary substrates used for peptidoglycan biosynthesis and recycling in the cytoplasm are GlcNAc-MurNAc(anhydro)-tetrapeptide and its degradation product, the free tetrapeptide. This complex process involves ∼15 proteins, among which the cytoplasmic enzyme ld-carboxypeptidase A (LdcA) catabolizes the bond between the last two l- and d-amino acid residues in the tetrapeptide to form the tripeptide, which is then utilized as a substrate by murein peptide ligase (Mpl). LdcA has been proposed as an antibacterial target. The crystal structure of Novosphingobium aromaticivorans DSM 12444 LdcA (NaLdcA) was determined at 1.89-Å resolution. The enzyme was biochemically characterized and its interactions with the substrate modeled, identifying residues potentially involved in substrate binding. Unaccounted electron density at the dimer interface in the crystal suggested a potential site for disrupting protein-protein interactions should a dimer be required to perform its function in bacteria. Our analysis extends the identification of functional residues to several other homologs, which include enzymes from bacteria that are involved in hydrocarbon degradation and destruction of coral reefs. The NaLdcA crystal structure provides an alternate system for investigating the structure-function relationships of LdcA and increases the structural coverage of the protagonists in bacterial cell wall recycling. PMID:24123814

  14. N-Substituted Glutamyl Sulfonamides As Inhibitors Of Glutamate Carboxypeptidase II (GCP2)

    PubMed Central

    Blank, Brian R.; Alayoglu, Pinar; Engen, William; Choi, Joseph K.; Berkman, Clifford E.; Anderson, Marc O.

    2011-01-01

    Glutamate carboxypeptidase II (GCP2) is a membrane-bound cell-surface peptidase which is implicated in several neurological disorders, and is also over-expressed in prostate tumor cells. There is significant interest in the inhibition of GCP2 as a means of neuroprotection, while GCP2 inhibition as a method to treat prostate cancer remains a topic of further investigation. The key zinc-binding functional group of the well characterized classes of GCP2 inhibitors (phophonates and phosphoramidates) is tetrahedral and negatively charged at neutral pH, while glutamyl urea class of inhibitors possess a planar and neutral zinc-binding group. This study introduces a new class of GCP2 inhibitors, N-substituted glutamyl sulfonamides, which possess a neutral tetrahedral zinc-binding motif. A library containing 15 secondary sulfonamides and 4 tertiary (N-methyl) sulfonamides was prepared and evaluated for inhibitory potency against purified GCP2 enzyme activity. While most inhibitors lacked potency at 100 μM, short alkyl sulfonamides exhibited promising low micromolar potency, with the optimal inhibitor in this series being glutamyl N-propylsulfonamide (2g). Lastly, molecular docking was used to develop a model to formulate an explanation for the relative inhibitory potencies employed for this class of inhibitors. PMID:21219587

  15. Electrochemical detection of protein kinase activity based on carboxypeptidase Y digestion triggered signal amplification.

    PubMed

    Yin, Huanshun; Wang, Xinxu; Guo, Yunlong; Zhou, Yunlei; Ai, Shiyun

    2015-04-15

    An effective assay method for monitoring protein kinase activity and screening inhibitors is greatly beneficial to kinase-related drug discovery, early diagnosis of diseases, and therapeutic effect evaluation. Herein, we develop a simple electrochemical method for detecting the activity of casein kinase II (CK2) based on phosphorylation against carboxypeptidase Y (CPY) digestion triggered signal amplification, where CK2 catalyzed phosphorylation event protects the substrate peptide from the digestion of CPY, maintains the repulsive force of the substrate peptide towards the redox probe, and results in a weak electrochemical signal. Whereas, without phosphorylation, the substrate peptide is digested by CPY and a strong electrochemical signal is obtained. The detection feasibility is demonstrated for the assay of CK2 activity with low detection limit of 0.047unit/mL. Moreover, the biosensor was used for the analysis of kinase inhibition. Based on the electrochemical signal dependent inhibitor concentration, the IC50 value of ellagic acid was estimated to be 39.77nM. The proposed method is also successfully applied to analyze CK2 activity in cell lysates, proving the applicability in complex biological samples.

  16. Neuroprotection mediated by glutamate carboxypeptidase II (NAALADase) inhibition requires TGF-beta.

    PubMed

    Thomas, A G; Liu, W; Olkowski, J L; Tang, Z; Lin, Q; Lu, X C; Slusher, B S

    2001-10-26

    Inhibition of glutamate carboxypeptidase (GCP) II (EC 3.4.17.21), also termed N-acetylated alpha-linked acidic dipeptidase (NAALADase), has been shown to protect against ischemic injury presumably via decreasing glutamate and increasing N-acetyl-aspartyl-glutamate (NAAG). NAAG is a potent and selective mGlu3 receptor agonist. Activation of glial mGlu3 receptors has been shown to protect against NMDA toxicity by releasing transforming growth factors, TGF-betas. We hypothesized that GCP II inhibition could be neuroprotective also via TGF-betas, due to increased NAAG. To verify this, Enzyme-Linked Immunosorbent Assays (ELISAs) were performed on media from both control and ischemic cultures treated with the GCP II inhibitor, 2-(phosphonomethyl)-pentanedioic acid (2-PMPA). We found that 2-PMPA attenuated ischemia-induced declines in TGF-beta. To further assess the role of TGF-betas in 2-PMPA-mediated neuroprotection, a neutralizing antibody to TGF-beta (TGF-beta Ab) was used. In both in vitro and in vivo models of cerebral ischemia, TGF-beta Ab reversed the neuroprotection by 2-PMPA. Antibodies to other growth factors had no effect. Data suggests that neuroprotection by GCP II inhibition may be partially mediated by promoting TGF-beta release. PMID:11698060

  17. Mice lacking glutamate carboxypeptidase II develop normally, but are less susceptible to traumatic brain injury.

    PubMed

    Gao, Yang; Xu, Siyi; Cui, Zhenwen; Zhang, Mingkun; Lin, Yingying; Cai, Lei; Wang, Zhugang; Luo, Xingguang; Zheng, Yan; Wang, Yong; Luo, Qizhong; Jiang, Jiyao; Neale, Joseph H; Zhong, Chunlong

    2015-07-01

    Glutamate carboxypeptidase II (GCPII) is a transmembrane zinc metallopeptidase found mainly in the nervous system, prostate and small intestine. In the nervous system, glia-bound GCPII mediates the hydrolysis of the neurotransmitter N-acetylaspartylglutamate (NAAG) into glutamate and N-acetylaspartate. Inhibition of GCPII has been shown to attenuate excitotoxicity associated with enhanced glutamate transmission under pathological conditions. However, different strains of mice lacking the GCPII gene are reported to exhibit striking phenotypic differences. In this study, a GCPII gene knockout (KO) strategy involved removing exons 3-5 of GCPII. This generated a new GCPII KO mice line with no overt differences in standard neurological behavior compared to their wild-type (WT) littermates. However, GCPII KO mice were significantly less susceptible to moderate traumatic brain injury (TBI). GCPII gene KO significantly lessened neuronal degeneration and astrocyte damage in the CA2 and CA3 regions of the hippocampus 24 h after moderate TBI. In addition, GCPII gene KO reduced TBI-induced deficits in long-term spatial learning/memory tested in the Morris water maze and motor balance tested via beam walking. Knockout of the GCPII gene is not embryonic lethal and affords histopathological protection with improved long-term behavioral outcomes after TBI, a result that further validates GCPII as a target for drug development consistent with results from studies using GCPII peptidase inhibitors. PMID:25872793

  18. Serine carboxypeptidase 46 Regulates Grain Filling and Seed Germination in Rice (Oryza sativa L.).

    PubMed

    Li, Zhiyong; Tang, Liqun; Qiu, Jiehua; Zhang, Wen; Wang, Yifeng; Tong, Xiaohong; Wei, Xiangjin; Hou, Yuxuan; Zhang, Jian

    2016-01-01

    Serine carboxypeptidase (SCP) is one of the largest groups of enzymes catalyzing proteolysis for functional protein maturation. To date, little is known about the function of SCPs in rice. In this study, we present a comprehensive analysis of the gene structure and expression profile of 59 rice SCPs. SCP46 is dominantly expressed in developing seeds, particularly in embryo, endosperm and aleurone layers, and could be induced by ABA. Functional characterization revealed that knock-down of SCP46 resulted in smaller grain size and enhanced seed germination. Furthermore, scp46 seed germination became less sensitive to the ABA inhibition than the Wild-type did; suggesting SCP46 is involved in ABA signaling. As indicated by RNA-seq and qRT-PCR analysis, numerous grain filling and seed dormancy related genes, such as SP, VP1 and AGPs were down-regulated in scp46. Yeast-two-hybrid assay also showed that SCP46 interacts with another ABA-inducible protein DI19-1. Taken together, we suggested that SCP46 is a master regulator of grain filling and seed germination, possibly via participating in the ABA signaling. The results of this study shed novel light into the roles of SCPs in rice.

  19. PLASTOCHRON3/GOLIATH encodes a glutamate carboxypeptidase required for proper development in rice.

    PubMed

    Kawakatsu, Taiji; Taramino, Graziana; Itoh, Jun-Ichi; Allen, Justin; Sato, Yutaka; Hong, Soon-Kwan; Yule, Ryan; Nagasawa, Nobuhiro; Kojima, Mikiko; Kusaba, Makoto; Sakakibara, Hitoshi; Sakai, Hajime; Nagato, Yasuo

    2009-06-01

    Most aerial parts of the plant body are products of the continuous activity of the shoot apical meristem (SAM). Leaves are the major component of the aerial plant body, and their temporal and spatial distribution mainly determines shoot architecture. Here we report the identification of the rice gene PLASTOCHRON3 (PLA3)/GOLIATH (GO) that regulates various developmental processes including the rate of leaf initiation (the plastochron). PLA3/GO encodes a glutamate carboxypeptidase, which is thought to catabolize small acidic peptides and produce small signaling molecules. pla3 exhibits similar phenotypes to pla1 and pla2- a shortened plastochron, precocious leaf maturation and rachis branch-to-shoot conversion in the reproductive phase. However, in contrast to pla1 and pla2, pla3 showed pleiotropic phenotypes including enlarged embryo, seed vivipary, defects in SAM maintenance and aberrant leaf morphology. Consistent with these pleiotropic phenotypes, PLA3 is expressed in the whole plant body, and is involved in plant hormone homeostasis. Double mutant analysis revealed that PLA1, PLA2 and PLA3 are regulated independently but function redundantly. Our results suggest that PLA3 modulates various signaling pathways associated with a number of developmental processes. PMID:19228340

  20. T cell–dependent survival of CD20+ and CD20− plasma cells in human secondary lymphoid tissue

    PubMed Central

    Withers, David R.; Fiorini, Claudia; Fischer, Randy T.; Ettinger, Rachel; Grammer, Amrie C.

    2007-01-01

    The signals mediating human plasma cell survival in vivo, particularly within secondary lymphoid tissue, are unclear. Human tonsils grafted into immunodeficient mice were therefore used to delineate the mechanisms promoting the survival of plasma cells. Tonsillar plasma cells were maintained within the grafts and the majority were nonproliferating, indicating a long-lived phenotype. A significant depletion of graft plasma cells was observed after anti-CD20 treatment, consistent with the expression of CD20 by most of the cells. Moreover, anti-CD52 treatment caused the complete loss of all graft lymphocytes, including plasma cells. Unexpectedly, anti-CD3, but not anti-CD154, treatment caused the complete loss of plasma cells, indicating an essential role for T cells, but not CD40-CD154 interactions in plasma cell survival. The in vitro coculture of purified tonsillar plasma cells and T cells revealed a T-cell survival signal requiring cell contact. Furthermore, immunofluorescence studies detected a close association between human plasma cells and T cells in vivo. These data reveal that human tonsil contains long-lived plasma cells, the majority of which express CD20 and can be deleted with anti-CD20 therapy. In addition, an important role for contact-dependent interactions with T cells in human plasma cell survival within secondary lymphoid tissue was identified. PMID:17299094

  1. T cell-dependent survival of CD20+ and CD20- plasma cells in human secondary lymphoid tissue.

    PubMed

    Withers, David R; Fiorini, Claudia; Fischer, Randy T; Ettinger, Rachel; Lipsky, Peter E; Grammer, Amrie C

    2007-06-01

    The signals mediating human plasma cell survival in vivo, particularly within secondary lymphoid tissue, are unclear. Human tonsils grafted into immunodeficient mice were therefore used to delineate the mechanisms promoting the survival of plasma cells. Tonsillar plasma cells were maintained within the grafts and the majority were nonproliferating, indicating a long-lived phenotype. A significant depletion of graft plasma cells was observed after anti-CD20 treatment, consistent with the expression of CD20 by most of the cells. Moreover, anti-CD52 treatment caused the complete loss of all graft lymphocytes, including plasma cells. Unexpectedly, anti-CD3, but not anti-CD154, treatment caused the complete loss of plasma cells, indicating an essential role for T cells, but not CD40-CD154 interactions in plasma cell survival. The in vitro coculture of purified tonsillar plasma cells and T cells revealed a T-cell survival signal requiring cell contact. Furthermore, immunofluorescence studies detected a close association between human plasma cells and T cells in vivo. These data reveal that human tonsil contains long-lived plasma cells, the majority of which express CD20 and can be deleted with anti-CD20 therapy. In addition, an important role for contact-dependent interactions with T cells in human plasma cell survival within secondary lymphoid tissue was identified.

  2. Virucidal Effect of Cold Atmospheric Gaseous Plasma on Feline Calicivirus, a Surrogate for Human Norovirus

    PubMed Central

    Aboubakr, Hamada A.; Williams, Paul; Gangal, Urvashi; Youssef, Mohammed M.; El-Sohaimy, Sobhy A. A.; Bruggeman, Peter J.

    2015-01-01

    Minimal food-processing methods are not effective against foodborne viruses, such as human norovirus (NV). It is important, therefore, to explore novel nonthermal technologies for decontamination of foods eaten fresh, minimally processed and ready-to-eat foods, and food contact surfaces. We studied the in vitro virucidal activity of cold atmospheric gaseous plasma (CGP) against feline calicivirus (FCV), a surrogate of NV. Factors affecting the virucidal activity of CGP (a so-called radio frequency atmospheric pressure plasma jet) were the plasma generation power, the exposure time and distance, the plasma feed gas mixture, and the virus suspension medium. Exposure to 2.5-W argon (Ar) plasma caused a 5.55 log10 unit reduction in the FCV titer within 120 s. The reduction in the virus titer increased with increasing exposure time and decreasing exposure distance. Of the four plasma gas mixtures studied (Ar, Ar plus 1% O2, Ar plus 1% dry air, and Ar plus 0.27% water), Ar plus 1% O2 plasma treatment had the highest virucidal effect: more than 6.0 log10 units of the virus after 15 s of exposure. The lowest virus reduction was observed with Ar plus 0.27% water plasma treatment (5 log10 unit reduction after 120 s). The highest reduction in titer was observed when the virus was suspended in distilled water. Changes in temperature and pH and formation of H2O2 were not responsible for the virucidal effect of plasma. The oxidation of viral capsid proteins by plasma-produced reactive oxygen and nitrogen species in the solution was thought to be responsible for the virucidal effect. In conclusion, CGP exhibits virucidal activity in vitro and has the potential to combat viral contamination in foods and on food preparation surfaces. PMID:25795667

  3. Virucidal effect of cold atmospheric gaseous plasma on feline calicivirus, a surrogate for human norovirus.

    PubMed

    Aboubakr, Hamada A; Williams, Paul; Gangal, Urvashi; Youssef, Mohammed M; El-Sohaimy, Sobhy A A; Bruggeman, Peter J; Goyal, Sagar M

    2015-06-01

    Minimal food-processing methods are not effective against foodborne viruses, such as human norovirus (NV). It is important, therefore, to explore novel nonthermal technologies for decontamination of foods eaten fresh, minimally processed and ready-to-eat foods, and food contact surfaces. We studied the in vitro virucidal activity of cold atmospheric gaseous plasma (CGP) against feline calicivirus (FCV), a surrogate of NV. Factors affecting the virucidal activity of CGP (a so-called radio frequency atmospheric pressure plasma jet) were the plasma generation power, the exposure time and distance, the plasma feed gas mixture, and the virus suspension medium. Exposure to 2.5-W argon (Ar) plasma caused a 5.55 log10 unit reduction in the FCV titer within 120 s. The reduction in the virus titer increased with increasing exposure time and decreasing exposure distance. Of the four plasma gas mixtures studied (Ar, Ar plus 1% O2, Ar plus 1% dry air, and Ar plus 0.27% water), Ar plus 1% O2 plasma treatment had the highest virucidal effect: more than 6.0 log10 units of the virus after 15 s of exposure. The lowest virus reduction was observed with Ar plus 0.27% water plasma treatment (5 log10 unit reduction after 120 s). The highest reduction in titer was observed when the virus was suspended in distilled water. Changes in temperature and pH and formation of H2O2 were not responsible for the virucidal effect of plasma. The oxidation of viral capsid proteins by plasma-produced reactive oxygen and nitrogen species in the solution was thought to be responsible for the virucidal effect. In conclusion, CGP exhibits virucidal activity in vitro and has the potential to combat viral contamination in foods and on food preparation surfaces.

  4. Effective ex vivo neutralization of human immunodeficiency virus type 1 in plasma by recombinant immunoglobulin molecules.

    PubMed Central

    Gauduin, M C; Allaway, G P; Maddon, P J; Barbas, C F; Burton, D R; Koup, R A

    1996-01-01

    We tested the ability of human monoclonal antibodies (immunoglobulin G1b12 [IgG1b12] and 19b) and CD4-based molecules (CD4-IgG2 and soluble CD4 [sCD4]) to neutralize human immunodeficiency virus type 1 directly from the plasma of seropositive donors in an ex vivo neutralization assay. IgG1b12 and CD4-IgG2, at concentrations from 1 to 25 micrograms/ml, were found to be effective at reducing the HIV-1 titer in most plasma samples. When viruses recovered from plasma samples were expanded to produce virus stocks, no correlation between the neutralization sensitivities to IgG1b12 and CD4-IgG2 of the in vitro passaged stocks and those of the ex vivo neutralizations performed directly on the plasma was observed. These differences could be due to changes in neutralization sensitivity that occur after one passage of the virus in vitro, or they could be related to the presence of complement or antibodies in the plasma. Furthermore, differences in expression of adhesion molecules on plasma-derived and phytohemagglutinin-activated peripheral blood mononuclear cell-derived viruses could be involved. These studies suggest that IgG1b12 and CD4-IgG2 have broad and potent neutralizing activity in both in vitro and ex vivo neutralization assays and should be considered for use as potential immunoprophylactic or therapeutic agents. PMID:8642690

  5. Sustained secretion of immunoglobulin by long-lived human tonsil plasma cells.

    PubMed

    van Laar, Jacob M; Melchers, Marc; Teng, Y K Onno; van der Zouwen, Boris; Mohammadi, Rozbeh; Fischer, Randy; Margolis, Leonid; Fitzgerald, Wendy; Grivel, Jean-Charles; Breedveld, Ferdinand C; Lipsky, Peter E; Grammer, Amrie C

    2007-09-01

    Immunoglobulin-secreting cells comprise both short-lived proliferating plasmablasts and long-lived nonproliferating plasma cells. To determine the phenotype and functional activity of Ig-secreting cells in human lymphoid tissue, we used a tonsillar organ culture model. A significant proportion of IgA and IgG secretion was shown to be mediated by long-lived, nonproliferating plasma cells that coexpressed high levels of CD27 and CD38. The presence of such cells was further corroborated by the finding of enhanced expression in the CD19(+) B-cell population of XBP-1, IRF-4, and particularly Blimp-1 genes involved in the differentiation of plasma cells. Intact tissue seemed to be necessary for optimal functional activity of plasma cells. A strong correlation was found between concentrations of interleukin-6 and IgA or IgG, but not IgM, in culture supernatants suggesting a role for interleukin-6 in the survival of long-lived plasma cells. Taken together, the present study demonstrates that human lymphoid tissue harbors a population of nonproliferating plasma cells that are dependent on an intact microenvironment for ongoing Ig secretion.

  6. Sustained Secretion of Immunoglobulin by Long-Lived Human Tonsil Plasma Cells

    PubMed Central

    van Laar, Jacob M.; Melchers, Marc; Teng, Y. K. Onno; van der Zouwen, Boris; Mohammadi, Rozbeh; Fischer, Randy; Margolis, Leonid; Fitzgerald, Wendy; Grivel, Jean-Charles; Breedveld, Ferdinand C.; Lipsky, Peter E.; Grammer, Amrie C.

    2007-01-01

    Immunoglobulin-secreting cells comprise both short-lived proliferating plasmablasts and long-lived nonproliferating plasma cells. To determine the phenotype and functional activity of Ig-secreting cells in human lymphoid tissue, we used a tonsillar organ culture model. A significant proportion of IgA and IgG secretion was shown to be mediated by long-lived, nonproliferating plasma cells that coexpressed high levels of CD27 and CD38. The presence of such cells was further corroborated by the finding of enhanced expression in the CD19+ B-cell population of XBP-1, IRF-4, and particularly Blimp-1 genes involved in the differentiation of plasma cells. Intact tissue seemed to be necessary for optimal functional activity of plasma cells. A strong correlation was found between concentrations of interleukin-6 and IgA or IgG, but not IgM, in culture supernatants suggesting a role for interleukin-6 in the survival of long-lived plasma cells. Taken together, the present study demonstrates that human lymphoid tissue harbors a population of nonproliferating plasma cells that are dependent on an intact microenvironment for ongoing Ig secretion. PMID:17690187

  7. Plasma metabolomics in human pulmonary tuberculosis disease: a pilot study.

    PubMed

    Frediani, Jennifer K; Jones, Dean P; Tukvadze, Nestan; Uppal, Karan; Sanikidze, Eka; Kipiani, Maia; Tran, ViLinh T; Hebbar, Gautam; Walker, Douglas I; Kempker, Russell R; Kurani, Shaheen S; Colas, Romain A; Dalli, Jesmond; Tangpricha, Vin; Serhan, Charles N; Blumberg, Henry M; Ziegler, Thomas R

    2014-01-01

    We aimed to characterize metabolites during tuberculosis (TB) disease and identify new pathophysiologic pathways involved in infection as well as biomarkers of TB onset, progression and resolution. Such data may inform development of new anti-tuberculosis drugs. Plasma samples from adults with newly diagnosed pulmonary TB disease and their matched, asymptomatic, sputum culture-negative household contacts were analyzed using liquid chromatography high-resolution mass spectrometry (LC-MS) to identify metabolites. Statistical and bioinformatics methods were used to select accurate mass/charge (m/z) ions that were significantly different between the two groups at a false discovery rate (FDR) of q<0.05. Two-way hierarchical cluster analysis (HCA) was used to identify clusters of ions contributing to separation of cases and controls, and metabolomics databases were used to match these ions to known metabolites. Identity of specific D-series resolvins, glutamate and Mycobacterium tuberculosis (Mtb)-derived trehalose-6-mycolate was confirmed using LC-MS/MS analysis. Over 23,000 metabolites were detected in untargeted metabolomic analysis and 61 metabolites were significantly different between the two groups. HCA revealed 8 metabolite clusters containing metabolites largely upregulated in patients with TB disease, including anti-TB drugs, glutamate, choline derivatives, Mycobacterium tuberculosis-derived cell wall glycolipids (trehalose-6-mycolate and phosphatidylinositol) and pro-resolving lipid mediators of inflammation, known to stimulate resolution, efferocytosis and microbial killing. The resolvins were confirmed to be RvD1, aspirin-triggered RvD1, and RvD2. This study shows that high-resolution metabolomic analysis can differentiate patients with active TB disease from their asymptomatic household contacts. Specific metabolites upregulated in the plasma of patients with active TB disease, including Mtb-derived glycolipids and resolvins, have potential as biomarkers

  8. Quantitative variability of 342 plasma proteins in a human twin population

    PubMed Central

    Liu, Yansheng; Buil, Alfonso; Collins, Ben C; Gillet, Ludovic CJ; Blum, Lorenz C; Cheng, Lin-Yang; Vitek, Olga; Mouritsen, Jeppe; Lachance, Genevieve; Spector, Tim D; Dermitzakis, Emmanouil T; Aebersold, Ruedi

    2015-01-01

    The degree and the origins of quantitative variability of most human plasma proteins are largely unknown. Because the twin study design provides a natural opportunity to estimate the relative contribution of heritability and environment to different traits in human population, we applied here the highly accurate and reproducible SWATH mass spectrometry technique to quantify 1,904 peptides defining 342 unique plasma proteins in 232 plasma samples collected longitudinally from pairs of monozygotic and dizygotic twins at intervals of 2–7 years, and proportioned the observed total quantitative variability to its root causes, genes, and environmental and longitudinal factors. The data indicate that different proteins show vastly different patterns of abundance variability among humans and that genetic control and longitudinal variation affect protein levels and biological processes to different degrees. The data further strongly suggest that the plasma concentrations of clinical biomarkers need to be calibrated against genetic and temporal factors. Moreover, we identified 13 cis-SNPs significantly influencing the level of specific plasma proteins. These results therefore have immediate implications for the effective design of blood-based biomarker studies. PMID:25652787

  9. Effect of zinc concentration on the activity of angiotensin converting enzyme in human plasma and serum

    SciTech Connect

    Reeves, P.G.; Carl, G.F.; Smith, D.K.; O'Dell, B.L.

    1986-03-05

    The activity of angiotensin converting enzyme is measured clinically to assist in the diagnosis of sarcoidosis and to monitor therapy with steroids, and with antihypertensive drugs that inhibit the enzyme. Even though it has been known for some time that ACE is a zinc dependent enzyme, it was discovered only recently that zinc, in addition to endogenous levels in the assay mixture, is required for maximal activity of rat serum ACE. The present experiment was designed to determine if additional zinc is required for maximal activation of ACE in plasma and serum of human subjects. Plasma or serum samples were incubated at 37/sup 0/ in a zinc-free medium, pH 7.4, containing hippurylglyclglycine as the substrate. The addition of 20 ..mu..M zinc significantly increased ACE activity in plasma (95.4 +/- 11.9 vs 192.8 +/- 24.3 U/L) and in serum (89.9 +/- 5.6 vs 195.7 +/- 9.3 U/L) compared to samples without added zinc. Enzyme activity was increased 2.4-fold when zinc was added to plasma from a patient with low plasma zinc. These data suggest that the endogenous level of zinc in the assay mixture resulting from the addition of an aliquot of plasma or serum is insufficient to obtain maximal activity of ACE. The addition of zinc to zinc deficient plasma increased ACE activity even more.

  10. Effects of non-thermal atmospheric plasma on human periodontal ligament mesenchymal stem cells

    NASA Astrophysics Data System (ADS)

    Miletić, M.; Mojsilović, S.; Okić Đorđević, I.; Maletić, D.; Puač, N.; Lazović, S.; Malović, G.; Milenković, P.; Petrović, Z. Lj; Bugarski, D.

    2013-08-01

    Here we investigate the influences of non-thermal atmospheric plasma on human mesenchymal stem cells isolated from periodontal ligament (hPDL-MSCs). A specially redesigned plasma needle was used as the source of low-temperature plasma and its effects on different hPDL-MSC functions were investigated. Cell cultures were obtained from extracted normal impacted third molars and characterized for their phenotype and multi-potential differentiation. The hPDL-MSCs possessed all the typical MSC properties, including clonogenic ability, high proliferation rate, specific phenotype and multilineage differentiation. The data regarding the interaction of plasma with hPDL-MSCs demonstrated that plasma treatment inhibited the migration of hPDL-MSCs and induced some detachment, while not affecting their viability. Additionally, plasma significantly attenuated hPDL-MSCs' proliferation, but promoted their osteogenic differentiation. The results of this study indicated that a non-thermal plasma offers specific activity with non-destructive properties that can be advantageous for future dental applications.

  11. Non-thermal dielectric-barrier discharge plasma damages human keratinocytes by inducing oxidative stress

    PubMed Central

    KIM, KI CHEON; PIAO, MEI JING; HEWAGE, SUSARA RUWAN KUMARA MADDUMA; HAN, XIA; KANG, KYOUNG AH; JO, JIN OH; MOK, YOUNG SUN; SHIN, JENNIFER H.; PARK, YEUNSOO; YOO, SUK JAE; HYUN, JIN WON

    2016-01-01

    The aim of this study was to identify the mechanisms through which dielectric-barrier discharge plasma damages human keratinocytes (HaCaT cells) through the induction of oxidative stress. For this purpose, the cells were exposed to surface dielectric-barrier discharge plasma in 70% oxygen and 30% argon. We noted that cell viability was decreased following exposure of the cells to plasma in a time-dependent manner, as shown by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. The levels of intracellular reactive oxygen species (ROS) were determined using 2′,7′-dichlorodihydro-fluorescein diacetate and dihydroethidium was used to monitor superoxide anion production. Plasma induced the generation of ROS, including superoxide anions, hydrogen peroxide and hydroxyl radicals. N-acetyl cysteine, which is an antioxidant, prevented the decrease in cell viability caused by exposure to plasma. ROS generated by exposure to plasma resulted in damage to various cellular components, including lipid membrane peroxidation, DNA breaks and protein carbonylation, which was detected by measuring the levels of 8-isoprostane and diphenyl-1-pyrenylphosphine assay, comet assay and protein carbonyl formation. These results suggest that plasma exerts cytotoxic effects by causing oxidative stress-induced damage to cellular components. PMID:26573561

  12. Non-thermal dielectric-barrier discharge plasma damages human keratinocytes by inducing oxidative stress.

    PubMed

    Kim, Ki Cheon; Piao, Mei Jing; Madduma Hewage, Susara Ruwan Kumara; Han, Xia; Kang, Kyoung Ah; Jo, Jin Oh; Mok, Young Sun; Shin, Jennifer H; Park, Yeunsoo; Yoo, Suk Jae; Hyun, Jin Won

    2016-01-01

    The aim of this study was to identify the mechanisms through which dielectric-barrier discharge plasma damages human keratinocytes (HaCaT cells) through the induction of oxidative stress. For this purpose, the cells were exposed to surface dielectric-barrier discharge plasma in 70% oxygen and 30% argon. We noted that cell viability was decreased following exposure of the cells to plasma in a time-dependent manner, as shown by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. The levels of intracellular reactive oxygen species (ROS) were determined using 2',7'-dichlorodihydrofluorescein diacetate and dihydroethidium was used to monitor superoxide anion production. Plasma induced the generation of ROS, including superoxide anions, hydrogen peroxide and hydroxyl radicals. N-acetyl cysteine, which is an antioxidant, prevented the decrease in cell viability caused by exposure to plasma. ROS generated by exposure to plasma resulted in damage to various cellular components, including lipid membrane peroxidation, DNA breaks and protein carbonylation, which was detected by measuring the levels of 8-isoprostane and diphenyl-1-pyrenylphosphine assay, comet assay and protein carbonyl formation. These results suggest that plasma exerts cytotoxic effects by causing oxidative stress-induced damage to cellular components. PMID:26573561

  13. Characterization of plasma cell populations at autopsy after human allogeneic bone marrow transplantation.

    PubMed Central

    Cousineau, S.; Belanger, R.; Perreault, C.

    1986-01-01

    Postmortem fixed tissue sections of the lymphoid and digestive systems of eight consecutive leukemic patients dying of various diseases after bone marrow transplantation (BMT) were analyzed for the presence of the heavy chains gamma, alpha, mu, delta, and epsilon and light chains kappa and lambda, with the use of a standard immunoperoxidase method. Two distinct types of plasma cell populations were found. The first type was a widely distributed polyclonal plasma cell population, lacking IgD-positive plasma cells and germinal centers. The second type of plasma cell population, found in 6 of 8 patients, was a group of monoclonal plasma cell populations positive for the heavy chains gamma, alpha, mu, or delta. Recent immunohistologic observations of the human lymph node suggest that the first type of polyclonal plasma cell population could arise from a nonspecific expansion of sIgM+, sIgD- B lymphocytes. The lack of germinal centers, a structure closely involved in specific-antibody production, may correlate with the poor specific-antibody response documented in patients after BMT. The monoclonal plasma cell populations, found with an unexpectedly high frequency, are probably related to a functional T-cell defect. Images Figure 1 Figure 2 Figure 3 Figure 4 PMID:3089020

  14. ANGPTL3 blockade with a human monoclonal antibody reduces plasma lipids in dyslipidemic mice and monkeys.

    PubMed

    Gusarova, Viktoria; Alexa, Corey A; Wang, Yan; Rafique, Ashique; Kim, Jee Hae; Buckler, David; Mintah, Ivory J; Shihanian, Lisa M; Cohen, Jonathan C; Hobbs, Helen H; Xin, Yurong; Valenzuela, David M; Murphy, Andrew J; Yancopoulos, George D; Gromada, Jesper

    2015-07-01

    Angiopoietin-like protein 3 (ANGPTL3) is a circulating protein synthesized exclusively in the liver that inhibits LPL and endothelial lipase (EL), enzymes that hydrolyze TGs and phospholipids in plasma lipoproteins. Here we describe the development and testing of a fully human monoclonal antibody (REGN1500) that binds ANGPTL3 with high affinity. REGN1500 reversed ANGPTL3-induced inhibition of LPL activity in vitro. Intravenous administration of REGN1500 to normolipidemic C57Bl/6 mice increased LPL activity and decreased plasma TG levels by ≥50%. Chronic administration of REGN1500 to dyslipidemic C57Bl/6 mice for 8 weeks reduced circulating plasma levels of TG, LDL-cholesterol (LDL-C), and HDL-cholesterol (HDL-C) without any changes in liver, adipose, or heart TG contents. Studies in EL knockout mice revealed that REGN1500 reduced serum HDL-C through an EL-dependent mechanism. Finally, administration of a single dose of REGN1500 to dyslipidemic cynomolgus monkeys caused a rapid and pronounced decrease in plasma TG, nonHDL-C, and HDL-C. REGN1500 normalized plasma TG levels even in monkeys with a baseline plasma TG greater than 400 mg/dl. Collectively, these data demonstrate that neutralization of ANGPTL3 using REGN1500 reduces plasma lipids in dyslipidemic mice and monkeys, and thus provides a potential therapeutic agent for treatment of patients with hyperlipidemia. PMID:25964512

  15. [Binding of cortisol, fluocortolone and difluocortolone to human plasma proteins (author's transl)].

    PubMed

    Fellier, H; Gleispach, H; Esterbauer, H

    1977-10-01

    The binding properties of [3H]cortisol, [3H]fluocortolone and [3H]difluocortolone by human plasma, human albumin, human- beta- and gamma-globulins have been studied by equilibrium dialysis. Cortisol, in physiological concentrations (0,4 micromol/l), is 98% bound in human plasma at 25 degrees C, fluocortolone 96% and diflucortolone 85%. Uncer physiological conditions cortisol is mainly bound to the corticosteroid binding globulin (transcortin). 2/3 of fluocortolone is bound to transcortin and 1/3 to albumin and globulins, whereas difluocortolone is mainly bound to albumin and to globulins but not to transcortin. The binding affinities of beta- and gamma-globulins are -ery low for the corticoids investigated, but they are higher for fluocortolone and difluocortolone than for cortisol. PMID:925615

  16. Analysis of free hydroxytyrosol in human plasma following the administration of olive oil.

    PubMed

    Pastor, Antoni; Rodríguez-Morató, Jose; Olesti, Eulàlia; Pujadas, Mitona; Pérez-Mañá, Clara; Khymenets, Olha; Fitó, Montserrat; Covas, María-Isabel; Solá, Rosa; Motilva, María-José; Farré, Magí; de la Torre, Rafael

    2016-03-11

    Hydroxytyrosol (HT) from olive oil, a potent bioactive molecule with health benefits, has a poor bioavailability, its free form (free HT) being undetectable so far. This fact leads to the controversy whether attained HT concentrations after olive oil polyphenol ingestion are too low to explain the observed biological activities. Due to this, an analytical methodology to determine free HT in plasma is crucial for understanding HT biological activity. Plasma HT instability and low concentrations have been major limitations for its quantification in clinical studies. Here, we describe a method to detect and quantify free HT in human plasma by using liquid chromatography coupled to tandem mass spectrometry. The method encompasses different steps of sample preparation including plasma stabilization, protein precipitation, selective derivatization with benzylamine, and purification by solid-phase extraction. A high sensitivity (LOD, 0.3ng/mL), specificity and stability of HT is achieved following these procedures. The method was validated and its applicability was demonstrated by analyzing human plasma samples after olive oil intake. A pharmacokinetic comparison was performed measuring free HT plasma concentrations following the intake of 25mL of ordinary olive oil (nearly undetectable concentrations) versus an extra-virgin olive oil (Cmax=4.40ng/mL). To our knowledge, this is the first time that an analytical procedure for quantifying free HT in plasma after olive oil dietary doses has been reported. The present methodology opens the door to a better understanding of the relationship between HT plasma concentrations and its beneficial health effects.

  17. Determination of loperamide in human plasma and saliva by liquid chromatography-tandem mass spectrometry.

    PubMed

    Arafat, Tawfiq; Arafat, Basil; awad, Riad; awwad, Ahmad Abu

    2014-12-01

    A simple and sensitive liquid chromatography-tandem mass spectrometric method for quantification of loperamide in human plasma and saliva was developed and validated, and then successfully applied in pharmacokinetic clinical study to investigate and correlate bioavailability of Imodium(®) 2mg quartet tablet dose in both human plasma and saliva. Loperamide with labeled internal standard was extracted from its biological matrix by methanol as protein direct precipitant in single extraction step. Adequate chromatographic separation for analytes from plasma and saliva matrices was achieved using ACE C18 (50mm×2.1mm, 5μm) column, eluted by water/methanol/formic acid (30:70:0.1%, v/v), delivered isocratically at constant flow rate of 0.75ml/min. The method validation intends to investigate specificity, sensitivity, linearity, precision, accuracy, recovery, matrix effect and stability according to European guideline, and partial validation was applied on saliva, specificity, matrix effect, recovery, sensitivity, within and between day precision and accuracy. The calibration curve was linear through the range of 20-3000pg/ml in both plasma and saliva using a 50μl sample volume. The partial validation sections outcome in saliva was so close to those in plasma. The within- and between-day precisions were all below 8.7% for plasma and below 11.4% for saliva. Accuracies ranged from 94 to 105% for both matrices. In this study, 26 healthy volunteers participated in the clinical study, and 6 of gave their saliva samples in addition to plasma at the same time schedule. The pharmacokinetic parameters of Cmax, AUC0-t and AUC0-∞, Tmax and T1/2 in both plasma and saliva were calculated and correlated.

  18. Effect of organo and inorganic lithium salt on human blood plasma glutathione- A comparative study.

    PubMed

    Ullah, Hashmat; Khan, Muhammad Farid; Jan, Syed Umer; Hashmat, Farwa

    2016-03-01

    Investigation of toxicological effect of various metals is the field of interest for toxicological scientists since four to five decades and especially the toxicological effect of those drugs containing metals and there use is common because there is no other choice except to use these metal containing drugs. Inorganic as well as organic salts of lithium are commonly used in prophylaxis and treatments of many psychiatric disorders. The aim of the present study was to see the difference between the effect of organic and inorganic salt of lithium commonly used in psychiatric disorders on the GSH of human blood plasma. It is the scientific fact that ionic dissociation of organic and inorganic salts of any metal is always quite different hence to prove this fact, the effect of lithium citrate (organic salt of lithium) and lithium carbonate (inorganic salt of lithium) was investigated on human blood plasma GSH to find the difference between the effect of two. Ellman's method was used for the quantification of glutathione contents in plasma. It was found that lithium citrate decrease plasma GSH contents less than lithium carbonate indicating that organic salts of lithium are safe than inorganic salts of lithium when are used in psychiatric disorders. Further to analyze the effect of organic and inorganic salt of lithium on blood plasma GSH with the increase in incubation time was also evaluated and was found that both concentration and time dependent effect of organic salt of lithium shows that this salt has decreased plasma GSH contents of human blood less than inorganic salt of lithium either by promoting oxidation of GSH into GSSG or by lithium glutathione complex formation. These results suggest the physicians that the use of organic lithium salts is much safer than inorganic salts of lithium in terms of depletion of blood plasma GSH contents. PMID:27087067

  19. Identification of peptidase substrates in human plasma by FTMS based differential mass spectrometry

    NASA Astrophysics Data System (ADS)

    Yates, Nathan A.; Deyanova, Ekaterina G.; Geissler, Wayne; Wiener, Matthew C.; Sachs, Jeffrey R.; Wong, Kenny K.; Thornberry, Nancy A.; Sinha Roy, Ranabir; Settlage, Robert E.; Hendrickson, Ronald C.

    2007-01-01

    Approximately 2% of the human genome encodes for proteases. Unfortunately, however, the biological roles of most of these enzymes remain poorly defined, since the physiological substrates are typically unknown and are difficult to identify using traditional methods. We have developed a proteomics experiment based on FTMS profiling and differential mass spectrometry (dMS) to identify candidate endogenous substrates of proteases using fractionated human plasma as the candidate substrate pool. Here we report proof-of-concept experiments for identifying in vitro substrates of aminopeptidase P2, (APP2) and dipeptidyl peptidase 4 (DPP-4), a peptidase of therapeutic interest for the treatment of type 2 diabetes. For both proteases, previously validated peptide substrates spiked into the human plasma pool were identified. Of note, the differential mass spectrometry experiments also identified novel substrates for each peptidase in the subfraction of human plasma. Targeted MS/MS analysis of these peptides in the complex human plasma pool and manual confirmation of the amino acid sequences led to the identification of these substrates. The novel DPP-4 substrate EPLGRQLTSGP was chemically synthesized and cleavage kinetics were determined in an in vitro DPP-4 enzyme assay. The apparent second order rate constant (kcat/KM) for DPP-4-mediated cleavage was determined to be 2.3 x 105 M-1 s-1 confirming that this peptide is efficiently processed by the peptidase in vitro. Collectively, these results demonstrate that differential mass spectrometry has the potential to identify candidate endogenous substrates of target proteases from a human plasma pool. Importantly, knowledge of the endogenous substrates can provide useful insight into the biology of these enzymes and provides useful biomarkers for monitoring their activity in vivo.

  20. Determination of vancomycin in human plasma using high-performance liquid chromatography with fluorescence detection.

    PubMed

    Abu-Shandi, Khalid Hamad

    2009-09-01

    A high-performance liquid chromatographic (HPLC) method with fluorescence detection for the quantification of vancomycin in human plasma was developed and validated. The method includes an extraction of vancomycin by deproteinization with acetonitrile. The analyses were carried out at 258 nm as the emission wavelength while exciting at 225 nm on a reversed-phase column (30 cm x 4 mm i.d. x 10 microm Waters Associates microBondapak C18) using a mobile phase composed of methanol and phosphate buffer at pH 6.3. Vancomycin was quantitatively recovered from human plasma samples (>96%) with high values of precision. The separation was completed within 27 min. The calibration curve was linear over the range from 5 to 1,000 ng/mL with the detection and quantification limits of 2 ng/mL and 5 ng/mL, respectively. This method is suitable for the routine assay of plasma samples.

  1. A high-performance liquid chromatographic assay for the enantiomers of bevantolol in human plasma.

    PubMed

    Rose, S E; Randinitis, E J

    1991-06-01

    A method was developed and validated for the simultaneous analysis of (+)- and (-)-bevantolol in human plasma. The assay involves plasma protein precipitation, derivatization of racemic bevantolol to its diastereomeric thioureas with 2,3,4,5-tetra-o-acetyl-alpha-D-glucopyranosyl isothiocyanate, and solid-phase extraction of the diastereomers from 0.5 ml human plasma. Chromatographic separation was accomplished under isocratic conditions using a reversed-phase C-18 analytical column and mobile phase consisting of equal parts of 75 mM dibasic ammonium phosphate buffer (adjusted to pH 3.5 with phosphoric acid) and acetonitrile, with a detection wavelength of 220 nm. The absolute peak-height method was employed for quantitation. Retention times for the diastereomers of (+)- and (-)-bevantolol were 7.4 and 6.4 min, respectively. The method is suitable for the quantification of the enantiomers over a concentration range of 40 to 800 ng/ml per enantiomer.

  2. Relationship between plasma cholesterol levels and cholesterol esterification in isolated human mononuclear cells

    SciTech Connect

    Dallongeville, J.; Davignon, J.; Lussier-Cacan, S. )

    1990-01-01

    The authors studied the relationship between plasma lipoprotein concentrations and cholesterol esterification in freshly isolated human mononuclear cells from 27 normolipidemic and 32 hyperlipidemic individuals. Cells were either incubated for 5 hours with radiolabeled oleate immediately after isolation or were preincubated for 18 hours in the presence of exogenous cholesterol, and then incubated with ({sup 14}C)sodium-oleate-albumin complex. In the absence of exogenous cholesterol, control and hypercholesterolemic subjects had similarly low values of intracellular cholesterol esterification. In the presence of exogenous cholesterol, both hypertriglyceridemic and hypercholesterolemic subjects had higher cholesterol esterification than controls. There was a significant correlation between the rate of cholesterol esterification and plasma total cholesterol. These results suggest that plasma cholesterol levels may regulate mononuclear cell intra-cellular cholesterol esterification in humans.

  3. Determination of phenazopyridine in human plasma by GC-MS and its pharmacokinetics.

    PubMed

    Li, Kai-jun; Chen, Qin-hua; Zhang, Zhuo; Zhou, Peng; Li, Peng; Liu, Jia; Zhu, Jun

    2008-09-01

    A sensitive, selective, and simple gas chromatography-mass spectrometry method is developed for quantitation of phenazopyridine (PAP) in human plasma using internal standard (diazepam). PAP and IS are extracted from plasma by liquid-liquid extraction and analyzed on a DB-5MS column with mass selective detector. Excellent linearity is found between 5-500 ng/mL (r = 0.9992, n = 7) for PAP in human plasma. The limit of detection is 0.3 ng/mL. Intra- and Inter-day precisions expressed as the relative standard deviation for the method are 1.37-6.69% and 1.24-6.01%, respectively. Extraction efficiency is more than 90%, and recoveries are in the range of 92.65-96.21%. This method is successfully applied for the pharmacokinetics and bioequivalence of 2 formulations of PAP in 18 healthy male volunteers who received a single 200 mg dose of each formulation.

  4. Lysozyme: primary bactericidin in human plasma serum active against Bacillus subtilis.

    PubMed Central

    Selsted, M E; Martinez, R J

    1978-01-01

    The in vitro bactericidal reaction of human plasma serum against Bacillus subtilis was investigated. Human lysozyme was purified to homogeneity, and antiserum was prepared against the enzyme. The anti-lysozyme immunoglobulin G was used as a specific inhibitor in bactericidal and bacteriolytic reactions. It was found that at low serum concentrations lysozyme was the primary bactericide active against B. subtilis. At appreciably higher serum concentrations, a lysozyme-independent bactericidal activity was also demonstrated. Images PMID:97236

  5. A comprehensive analysis of the Streptococcus pyogenes and human plasma protein interaction network.

    PubMed

    Sjöholm, Kristoffer; Karlsson, Christofer; Linder, Adam; Malmström, Johan

    2014-07-01

    Streptococcus pyogenes is a major human bacterial pathogen responsible for severe and invasive disease associated with high mortality rates. The bacterium interacts with several human blood plasma proteins and clarifying these interactions and their biological consequences will help to explain the progression from mild to severe infections. In this study, we used a combination of mass spectrometry (MS) based techniques to comprehensively quantify the components of the S. pyogenes-plasma protein interaction network. From an initial list of 181 interacting human plasma proteins defined using liquid chromatography (LC)-MS/MS analysis we further subdivided the interacting protein list using selected reaction monitoring (SRM) depending on the level of enrichment and protein concentration on the bacterial surface. The combination of MS methods revealed several previously characterized interactions between the S. pyogenes surface and human plasma along with many more, so far uncharacterised, possible plasma protein interactions with S. pyogenes. In follow-up experiments, the combination of MS techniques was applied to study differences in protein binding to a S. pyogenes wild type strain and an isogenic mutant lacking several important virulence factors, and a unique pair of invasive and non-invasive S. pyogenes isolates from the same patient. Comparing the plasma protein-binding properties of the wild type and the mutant and the invasive and non-invasive S. pyogenes bacteria revealed considerable differences, underlining the significance of these protein interactions. The results also demonstrate the power of the developed mass spectrometry method to investigate host-microbial relationships with a large proteomics depth and high quantitative accuracy.

  6. Plasma protein binding of an antisense oligonucleotide targeting human ICAM-1 (ISIS 2302).

    PubMed

    Watanabe, Tanya A; Geary, Richard S; Levin, Arthur A

    2006-01-01

    In vitro ultrafiltration was used to determine the plasma protein-binding characteristics of phosphorothioate oligonucleotides (PS ODNs). Although there are binding data on multiple PS ODNs presented here, the focus of this research is on the protein-binding characteristics of ISIS 2302, a PS ODN targeting human intercellular adhesion molecule-1 (ICAM-1) mRNA, which is currently in clinical trials for the treatment of ulcerative colitis. ISIS 2302 was shown to be highly bound (> 97%) across species (mouse, rat, monkey, human), with the mouse having the least degree of binding. ISIS 2302 was highly bound to albumin and, to a lesser, extent alpha2-macroglobulin and had negligible binding to alpha1-acid glycoprotein. Ten shortened ODN metabolites (8, 10, and 12-19 nucleotides [nt] in length, truncated from the 3' end) were evaluated in human plasma. The degree of binding was reduced as the ODN metabolite length decreased. Three additional 20-nt (20-mer) PS ODNs (ISIS 3521, ISIS 2503, and ISIS 5132) of varying sequence but similar chemistry were evaluated. Although the tested PS ODNs were highly bound to plasma proteins, suggesting a commonality within the chemical class, these results suggested that the protein-binding characteristics in human plasma may be sequence dependent. Lastly, drug displacement studies with ISIS 2302 and other concomitant drugs with known protein-binding properties were conducted to provide information on potential drug interactions. Coadministered ISIS 2302 and other high-binding drugs evaluated in this study did not displace one another at supraclinical plasma concentrations and, thus, are not anticipated to cause any pharmacokinetic interaction in the clinic as a result of the displacement of binding to plasma proteins.

  7. Detection of a lysosomal carboxypeptidase and a lysosomal dipeptidase in highly-purified dipeptidyl aminopeptidase I /cathepsin C/ and the elimination of their activities from preparations used to sequence peptides.

    NASA Technical Reports Server (NTRS)

    Mcdonald, J. K.; Zeitman, B. B.; Ellis, S.

    1972-01-01

    Description of the properties of a carboxypeptidase, termed 'catheptic carboxypeptidase C,' and a dipeptidase, termed 'Ser-Met dipeptidase.' Both were found in highly purified DAP I from either beef spleen or rat liver. Methods are described for the removal or selective inactivation of these contaminating enzymes.

  8. Assessment of renal function by the stable oxygen and hydrogen isotopes in human blood plasma.

    PubMed

    Kuo, Tai-Chih; Wang, Chung-Ho; Lin, Hsiu-Chen; Lin, Yuan-Hau; Lin, Matthew; Lin, Chun-Mao; Kuo, Hsien-Shou

    2012-01-01

    Water (H(2)O) is the most abundant and important molecule of life. Natural water contains small amount of heavy isotopes. Previously, few animal model studies have shown that the isotopic composition of body water could play important roles in physiology and pathophysiology. Here we study the stable isotopic ratios of hydrogen (δ(2)H) and oxygen (δ(18)O) in human blood plasma. The stable isotopic ratio is defined and determined by δ(sample) = [(R(sample)/R(STD))-1] * 1000, where R is the molar ratio of rare to abundant, for example, (18)O/(16)O. We observe that the δ(2)H and the δ(18)O in human blood plasma are associated with the human renal functions. The water isotope ratios of the δ(2)H and δ(18)O in human blood plasma of the control subjects are comparable to those of the diabetes subjects (with healthy kidney), but are statistically higher than those of the end stage renal disease subjects (p<0.001 for both ANOVA and Student's t-test). In addition, our data indicate the existence of the biological homeostasis of water isotopes in all subjects, except the end stage renal disease subjects under the haemodialysis treatment. Furthermore, the unexpected water contents (δ(2)H and δ(18)O) in blood plasma of body water may shed light on a novel assessment of renal functions.

  9. Identification of human plasma cells with a lamprey monoclonal antibody

    PubMed Central

    Yu, Cuiling; Liu, Yanling; Chan, Justin Tze Ho; Tong, Jiefei; Li, Zhihua; Shi, Mengyao; Davani, Dariush; Parsons, Marion; Khan, Srijit; Zhan, Wei; Kyu, Shuya; Grunebaum, Eyal; Campisi, Paolo; Propst, Evan J.; Jaye, David L.; Trudel, Suzanne; Moran, Michael F.; Ostrowski, Mario; Herrin, Brantley R.; Lee, F. Eun-Hyung; Sanz, Ignacio; Cooper, Max D.; Ehrhardt, Götz R.A.

    2016-01-01

    Ab-producing plasma cells (PCs) serve as key participants in countering pathogenic challenges as well as being contributors to autoimmune and malignant disorders. Thus far, only a limited number of PC–specific markers have been identified. The characterization of the unique variable lymphocyte receptor (VLR) Abs that are made by evolutionarily distant jawless vertebrates prompted us to investigate whether VLR Abs could detect novel PC antigens that have not been recognized by conventional Abs. Here, we describe a monoclonal lamprey Ab, VLRB MM3, that was raised against primary multiple myeloma cells. VLRB MM3 recognizes a unique epitope of the CD38 ectoenzyme that is present on plasmablasts and PCs from healthy individuals and on most, but not all, multiple myelomas. Binding by the VLRB MM3 Ab coincides with CD38 dimerization and NAD glycohydrolase activity. Our data demonstrate that the lamprey VLRB MM3 Ab is a unique reagent for the identification of plasmablasts and PCs, with potential applications in the diagnosis and therapeutic intervention of PC or autoimmune disorders. PMID:27152361

  10. Quantitation of Human Papillomavirus DNA in Plasma of Oropharyngeal Carcinoma Patients

    SciTech Connect

    Cao Hongbin; Banh, Alice; Kwok, Shirley; Shi Xiaoli; Wu, Simon; Krakow, Trevor; Khong, Brian; Bavan, Brindha; Bala, Rajeev; Pinsky, Benjamin A.; Colevas, Dimitrios; Pourmand, Nader; Koong, Albert C.; Kong, Christina S.; Le, Quynh-Thu

    2012-03-01

    Purpose: To determine whether human papillomavirus (HPV) DNA can be detected in the plasma of patients with HPV-positive oropharyngeal carcinoma (OPC) and to monitor its temporal change during radiotherapy. Methods and Materials: We used polymerase chain reaction to detect HPV DNA in the culture media of HPV-positive SCC90 and VU147T cells and the plasma of SCC90 and HeLa tumor-bearing mice, non-tumor-bearing controls, and those with HPV-negative tumors. We used real-time quantitative polymerase chain reaction to quantify the plasma HPV DNA in 40 HPV-positive OPC, 24 HPV-negative head-and-neck cancer patients and 10 non-cancer volunteers. The tumor HPV status was confirmed by p16{sup INK4a} staining and HPV16/18 polymerase chain reaction or HPV in situ hybridization. A total of 14 patients had serial plasma samples for HPV DNA quantification during radiotherapy. Results: HPV DNA was detectable in the plasma samples of SCC90- and HeLa-bearing mice but not in the controls. It was detected in 65% of the pretreatment plasma samples from HPV-positive OPC patients using E6/7 quantitative polymerase chain reaction. None of the HPV-negative head-and-neck cancer patients or non-cancer controls had detectable HPV DNA. The pretreatment plasma HPV DNA copy number correlated significantly with the nodal metabolic tumor volume (assessed using {sup 18}F-deoxyglucose positron emission tomography). The serial measurements in 14 patients showed a rapid decline in HPV DNA that had become undetectable at radiotherapy completion. In 3 patients, the HPV DNA level had increased to a discernable level at metastasis. Conclusions: Xenograft studies indicated that plasma HPV DNA is released from HPV-positive tumors. Circulating HPV DNA was detectable in most HPV-positive OPC patients. Thus, plasma HPV DNA might be a valuable tool for identifying relapse.

  11. Determination of bupropion using liquid chromatography with fluorescence detection in pharmaceutical preparations, human plasma and human urine.

    PubMed

    Ulu, Sevgi Tatar; Tuncel, Muzaffer

    2012-05-01

    A novel pre-column derivatization reversed-phase high-performance liquid chromatography with fluorescence detection is described for the determination of bupropion in pharmaceutical preparation, human plasma and human urine using mexiletine as internal standard. The proposed method is based on the reaction of 4-chloro-7-nitrobenzofurazan (NBD-Cl) with bupropion to produce a fluorescent derivative. The derivative formed is monitored on a C18 (150 mm × 4.6 mm i.d., 5 µm) column using a mobile phase consisting of methanol-water 75:25 (v/v), at a flow-rate of 1.2 mL/min and detected fluorimetrically at λ(ex) = 458 and λ(em) = 533 nm. The assay was linear over the concentration ranges of 5-500 and 10-500 ng/mL for plasma and urine, respectively. The limits of detection and quantification were calculated to be 0.24 and 0.72 ng/mL for plasma and urine, respectively (inter-day results). The recoveries obtained for plasma and urine were 97.12% ± 0.45 and 96.00% ± 0.45, respectively. The method presents good performance in terms of precision, accuracy, specificity, linearity, detection and quantification limits and robustness. The proposed method is applied to determine bupropion in commercially available tablets. The results were compared with an ultraviolet spectrophotometry method using t- and F-tests. PMID:22454390

  12. Determination of bupropion using liquid chromatography with fluorescence detection in pharmaceutical preparations, human plasma and human urine.

    PubMed

    Ulu, Sevgi Tatar; Tuncel, Muzaffer

    2012-05-01

    A novel pre-column derivatization reversed-phase high-performance liquid chromatography with fluorescence detection is described for the determination of bupropion in pharmaceutical preparation, human plasma and human urine using mexiletine as internal standard. The proposed method is based on the reaction of 4-chloro-7-nitrobenzofurazan (NBD-Cl) with bupropion to produce a fluorescent derivative. The derivative formed is monitored on a C18 (150 mm × 4.6 mm i.d., 5 µm) column using a mobile phase consisting of methanol-water 75:25 (v/v), at a flow-rate of 1.2 mL/min and detected fluorimetrically at λ(ex) = 458 and λ(em) = 533 nm. The assay was linear over the concentration ranges of 5-500 and 10-500 ng/mL for plasma and urine, respectively. The limits of detection and quantification were calculated to be 0.24 and 0.72 ng/mL for plasma and urine, respectively (inter-day results). The recoveries obtained for plasma and urine were 97.12% ± 0.45 and 96.00% ± 0.45, respectively. The method presents good performance in terms of precision, accuracy, specificity, linearity, detection and quantification limits and robustness. The proposed method is applied to determine bupropion in commercially available tablets. The results were compared with an ultraviolet spectrophotometry method using t- and F-tests.

  13. Inhibition of phospholipase A/sub 2/ from human plasma by sodium bisulfite

    SciTech Connect

    Wiggins, C.W.; Franson, R.C.

    1987-05-01

    The anti-oxidant sodium bisulfite has been shown to inhibit acid active(lysosomal), non-Ca/sup + +/-dependent phospholipase A/sub 2/ (PLA/sub 2/), and to interact reversibly with unsaturated fatty acids, altering their chromatographic mobility. The authors examined the effect of bisulfite on neutral active, Ca/sup + +/-dependent PLA/sub 2/ from human plasma. Using (1-/sup 14/C)oleate-labelled autoclaved E. coli as substrate, PLA/sub 2/ activity was inhibited in a dose-dependent manner by bisulfite. Maximal inhibition occurred at 100..mu..M bisulfite. Preincubation of plasma for 0-30 minutes with bisulfite resulted in a time-dependent increase in PLA/sub 2/ inhibition. Preincubation of substrate with bisulfite had no such effect. When the plasma PLA/sub 2/ was purified 25-fold by SP-Sephadex chromatography it was no longer inhibited by bisulfite. The SP-Sephadex wash through fraction, which contained greater than 95% of the applied protein but not PLA/sub 2/ activity, did not inhibit the purified enzyme. When incubated with bisulfite however, the SP-wash through fraction produced dose-dependent inhibition of the purified enzyme. These results indicate that sodium bisulfite inhibits human plasma PLA/sub 2/, in vitro, indirectly by interaction with a factor(s) present in plasma and suggests that anti-oxidants may similarly influence expression of extracellular PLA/sub 2/ in vivo.

  14. Additions to the Human Plasma Proteome via a Tandem MARS Depletion iTRAQ-Based Workflow.

    PubMed

    Cao, Zhiyun; Yende, Sachin; Kellum, John A; Robinson, Renã A S

    2013-01-01

    Robust platforms for determining differentially expressed proteins in biomarker and discovery studies using human plasma are of great interest. While increased depth in proteome coverage is desirable, it is associated with costs of experimental time due to necessary sample fractionation. We evaluated a robust quantitative proteomics workflow for its ability (1) to provide increased depth in plasma proteome coverage and (2) to give statistical insight useful for establishing differentially expressed plasma proteins. The workflow involves dual-stage immunodepletion on a multiple affinity removal system (MARS) column, iTRAQ tagging, offline strong-cation exchange chromatography, and liquid chromatography tandem mass spectrometry (LC-MS/MS). Independent workflow experiments were performed in triplicate on four plasma samples tagged with iTRAQ 4-plex reagents. After stringent criteria were applied to database searched results, 689 proteins with at least two spectral counts (SC) were identified. Depth in proteome coverage was assessed by comparison to the 2010 Human Plasma Proteome Reference Database in which our studies reveal 399 additional proteins which have not been previously reported. Additionally, we report on the technical variation of this quantitative workflow which ranges from ±11 to 30%.

  15. Additions to the Human Plasma Proteome via a Tandem MARS Depletion iTRAQ-Based Workflow

    PubMed Central

    Cao, Zhiyun; Yende, Sachin; Kellum, John A.; Robinson, Renã A. S.

    2013-01-01

    Robust platforms for determining differentially expressed proteins in biomarker and discovery studies using human plasma are of great interest. While increased depth in proteome coverage is desirable, it is associated with costs of experimental time due to necessary sample fractionation. We evaluated a robust quantitative proteomics workflow for its ability (1) to provide increased depth in plasma proteome coverage and (2) to give statistical insight useful for establishing differentially expressed plasma proteins. The workflow involves dual-stage immunodepletion on a multiple affinity removal system (MARS) column, iTRAQ tagging, offline strong-cation exchange chromatography, and liquid chromatography tandem mass spectrometry (LC-MS/MS). Independent workflow experiments were performed in triplicate on four plasma samples tagged with iTRAQ 4-plex reagents. After stringent criteria were applied to database searched results, 689 proteins with at least two spectral counts (SC) were identified. Depth in proteome coverage was assessed by comparison to the 2010 Human Plasma Proteome Reference Database in which our studies reveal 399 additional proteins which have not been previously reported. Additionally, we report on the technical variation of this quantitative workflow which ranges from ±11 to 30%. PMID:23509626

  16. Stimulus-dependent secretion of plasma proteins from human neutrophils.

    PubMed Central

    Borregaard, N; Kjeldsen, L; Rygaard, K; Bastholm, L; Nielsen, M H; Sengeløv, H; Bjerrum, O W; Johnsen, A H

    1992-01-01

    In search for matrix proteins released from secretory vesicles of human neutrophils, a prominent 67-kD protein was identified in the extracellular medium of neutrophils stimulated by the chemotactic peptide, FMLP. The protein was purified to apparent homogeneity and partially sequenced. The sequence of the first 32 NH2-terminal amino acids was identical to the sequence of albumin. mRNA for human albumin could not be detected in bone marrow cells, nor could biosynthetic labeling of albumin be demonstrated in bone marrow cells during incubation with [14C]leucine. Immunofluorescence studies on single cells demonstrated the presence of intracellular albumin in fixed permeabilized neutrophils. Light microscopy of immunogold-silver-stained cryosections visualized albumin in cytoplasmic "granules." The morphology of these was determined by immunoelectron microscopy as vesicles of varying form and size. Subcellular fractionation studies on unstimulated neutrophils demonstrated the presence of albumin in the low density pre-gamma and gamma-regions that contain secretory vesicles, but are devoid of specific granules and azurophil granules. Albumin was readily released from these structures during activation of neutrophils with inflammatory mediators. Immunoblotting demonstrated the presence of immunoglobulin and transferrin along with albumin in exocytosed material from stimulated neutrophils. This indicates that secretory vesicles are unique endocytic vesicles that can be triggered to exocytose by inflammatory stimuli. Images PMID:1378856

  17. The low photo-inactivation rate of bacteria in human plasma II. Inhibition of methylene blue bleaching in plasma and effective bacterial destruction by the addition of dilute acetic acid to human plasma.

    PubMed

    Chen, Jie; Cesario, Thomas C; Li, Runze; Er, Ali O; Rentzepis, Peter M

    2015-10-01

    Methylene blue (MB) and other photo-sensitizer molecules have been recognized as effective means for the inactivation of bacteria and other pathogens owing to their ability to photo-generate reactive oxygen species (ROS) including singlet oxygen. These reactive species react with the membrane of the bacteria causing their destruction. However, the efficiency of MB to destroy bacteria in plasma is very low because the MB 660 nm absorption band, that is responsible for the ROS generation, is bleached. The bleaching of MB, in plasma, is caused by the attachment of a hydrogen atom to the central ring nitrogen of MB, which destroys the ring conjugation and forms Leuco-MB which does not absorb in the 600 nm region. In this paper we show that addition of dilute acetic acid, ∼10(-4) M, to human plasma, prevents H-atom attachment to MB, allowing MB to absorb at 660 nm, generates singlet oxygen and thus inactivates bacteria. The mechanism proposed, for preventing MB bleaching in plasma, is based on the oxidation of cysteine to cystine, by reaction with added dilute acetic acid, thus eliminating the availability of the thiol hydrogen atom which attaches to the MB nitrogen. It is expected that the addition of acetic acid to plasma will be effective in the sterilization of plasma and killing of bacteria in wounds and burns.

  18. Spi-B inhibits human plasma cell differentiation by repressing BLIMP1 and XBP-1 expression.

    PubMed

    Schmidlin, Heike; Diehl, Sean A; Nagasawa, Maho; Scheeren, Ferenc A; Schotte, Remko; Uittenbogaart, Christel H; Spits, Hergen; Blom, Bianca

    2008-09-01

    The terminal differentiation of B cells into antibody-secreting plasma cells is tightly regulated by a complex network of transcription factors. Here we evaluated the role of the Ets factor Spi-B during terminal differentiation of human B cells. All mature tonsil and peripheral blood B-cell subsets expressed Spi-B, with the exception of plasma cells. Overexpression of Spi-B in CD19(+) B cells inhibited, similar to the known inhibitor BCL-6, the expression of plasma cell-associated surface markers and transcription factors as well as immunoglobulin production, ie, in vitro plasma cell differentiation. The arrest in B-cell differentiation enforced by Spi-B was independent of the transactivation domain, but dependent on the Ets-domain. By chromatin immunoprecipitation and assays using an inducible Spi-B construct BLIMP1 and XBP-1 were identified as direct target genes of Spi-B mediated repression. We propose a novel role for Spi-B in maintenance of germinal center and memory B cells by direct repression of major plasma cell factors and thereby plasma cell differentiation.

  19. Simultaneous determination of niacin, niacinamide and nicotinuric acid in human plasma.

    PubMed

    Pfuhl, P; Kärcher, U; Häring, N; Baumeister, A; Tawab, Mona Abdel; Schubert-Zsilavecz, M

    2005-01-01

    A sensitive, specific, accurate, and reproducible HPLC/MS-method for the simultaneous quantitative determination of niacin (NA) and its main metabolites niacinamide (NAM) and nicotinuric acid (NUR) in human plasma using chinolin-3-carboxylic acid as an internal standard was developed and validated according to international guidelines for method validation. All analytes and the internal standard were separated from acidified plasma by solid phase extraction. Afterwards the extracted samples were analyzed by HPLC/MS in the positive electrospray ionization mode (ESI) and selected ion monitoring (SIM). The total run time was 7 min between injections. The assay had a lower limit of quantification of 50.0 ng/mL for each analyte using 1 mL of plasma. The calibration curves were linear in the measured range between 50.0 and 750 ng/mL plasma. The overall precision and accuracy for all concentrations of quality controls and standards was better than 15%. No indications were found for possible instabilities of niacin, niacinamide and nicotinuric acid in plasma at -20 degrees C, in the extraction solvent or after repeated thawing/freezing cycles. In stabilities were observed in whole blood and in plasma at room temperature. The recovery of the extraction method ranged from 86 to 89% for the three analytes.

  20. Acute angiotensin II increases plasma F2-isoprostanes in salt-replete human hypertensives.

    PubMed

    Murphey, Laine J; Morrow, Jason D; Sawathiparnich, Pairunyar; Williams, Gordon H; Vaughan, Douglas E; Brown, Nancy J

    2003-10-01

    Angiotensin (Ang) II induces oxidative stress in vitro and in animal models of hypertension. We tested the hypothesis that Ang II increases oxidative stress in human hypertension, as assessed by plasma F2-isoprostane concentrations. Plasma F2-isoprostanes, hemodynamic and endocrine parameters were measured at baseline and following a 55 min infusion of 3 ng/kg/min Ang II in 13 normotensive and 13 hypertensive volunteers ingesting a high- (200 mmol/d) or low- (10 mmol/d) sodium diet. Mean arterial pressure (MAP) and body mass index were higher in hypertensive subjects. Ang II infusion increased MAP (p<.001) and plasma aldosterone concentrations (p<.001) and decreased plasma renin activity (p<.001) and renal plasma flow (p<.001) to a similar extent in both groups. Plasma F2-isoprostane concentrations were similar at baseline. There was no effect of Ang II on F2-isoprostane concentrations during low-salt intake in either group (normotensive 51.7 +/- 7.1 to 53.7 +/- 6.5 pg/ml and hypertensive 52.2 +/- 8.2 to 56.2 +/- 10.0 pg/ml; mean +/- SE). During high-salt intake, Ang II increased F2-isoprostane concentrations in the hypertensive group (52.3 +/- 7.2 to 63.2 +/- 10.4 pg/ml, p=0.010) but not in the normotensive group (54.2 +/- 4.4 to 58.9 +/- 6.6 pg/ml, p=0.83). Acute Ang II infusion increases oxidative stress in vivo in hypertensive humans. The renin-angiotensin system may contribute to oxidative stress in human cardiovascular disease.

  1. Inhibitors of serotonin reuptake and specific imipramine binding in human blood plasma

    SciTech Connect

    Brusov, O.S.; Fomenko, A.M.; Katasonov, A.B.; Lidemann, R.R.

    1985-12-01

    This paper describes a method of extraction of endogenous inhibitors of specific IMI binding and of 5-HT reuptake, from human blood plasma and the heterogeneity of these compounds is demonstrated. Specific binding was determined as the difference between binding of /sup 3/H-IMI in the absence and in the presence of 50 microM IMI. Under these conditions, specific binding amounted to 70-80% of total binding of /sup 3/H-IMI. It is shown that extract obtained from human blood contains a material which inhibits dose-dependently both 5-HT reuptake and specific binding of /sup 3/H-IMI. Gel-chromatography of extracts of human blood plasma on Biogel P-2 is also shown.

  2. Plasma is the main regulator of Staphylococcus epidermidis biofilms virulence genes transcription in human blood.

    PubMed

    França, Angela; Cerca, Nuno

    2016-03-01

    Staphylococcus epidermidis is frequently associated with the emergence of medical-device-associated bloodstream infections, due to its ability to form biofilms on the surface of vascular catheters. Although these biofilms may be in continuous contact with human blood, how S. epidermidis biofilm cells interact with blood and its cellular and soluble components is poorly understood. Herein, we evaluated biofilm structure, biofilm cells culturability and viability, and the transcription of a panel of genes associated with S. epidermidis biofilms virulence, upon interaction with whole human blood or plasma. Our results showed that although whole human blood caused significant alterations in biofilm structure and in the number of culturable and viable cells, plasma was the main regulator of the transcription of genes with central role in biofilm formation, maturation and immune evasion. These findings highlight the urgent need to intensify studies aiming to evaluate the impact of host soluble factors on S. epidermidis biofilms fitness and persistence. PMID:26712650

  3. Plasma is the main regulator of Staphylococcus epidermidis biofilms virulence genes transcription in human blood.

    PubMed

    França, Angela; Cerca, Nuno

    2016-03-01

    Staphylococcus epidermidis is frequently associated with the emergence of medical-device-associated bloodstream infections, due to its ability to form biofilms on the surface of vascular catheters. Although these biofilms may be in continuous contact with human blood, how S. epidermidis biofilm cells interact with blood and its cellular and soluble components is poorly understood. Herein, we evaluated biofilm structure, biofilm cells culturability and viability, and the transcription of a panel of genes associated with S. epidermidis biofilms virulence, upon interaction with whole human blood or plasma. Our results showed that although whole human blood caused significant alterations in biofilm structure and in the number of culturable and viable cells, plasma was the main regulator of the transcription of genes with central role in biofilm formation, maturation and immune evasion. These findings highlight the urgent need to intensify studies aiming to evaluate the impact of host soluble factors on S. epidermidis biofilms fitness and persistence.

  4. The Redundancy of Peptidoglycan Carboxypeptidases Ensures Robust Cell Shape Maintenance in Escherichia coli

    PubMed Central

    Peters, Katharina; Kannan, Suresh; Rao, Vincenzo A.; Biboy, Jacob; Vollmer, Daniela; Erickson, Stephen W.; Lewis, Richard J.

    2016-01-01

    ABSTRACT Peptidoglycan (PG) is an essential structural component of the bacterial cell wall and maintains the integrity and shape of the cell by forming a continuous layer around the cytoplasmic membrane. The thin PG layer of Escherichia coli resides in the periplasm, a unique compartment whose composition and pH can vary depending on the local environment of the cell. Hence, the growth of the PG layer must be sufficiently robust to allow cell growth and division under different conditions. We have analyzed the PG composition of 28 mutants lacking multiple PG enzymes (penicillin-binding proteins [PBPs]) after growth in acidic or near-neutral-pH media. Statistical analysis of the muropeptide profiles identified dd-carboxypeptidases (DD-CPases) that were more active in cells grown at acidic pH. In particular, the absence of the DD-CPase PBP6b caused a significant increase in the pentapeptide content of PG as well as morphological defects when the cells were grown at acidic pH. Other DD-CPases (PBP4, PBP4b, PBP5, PBP6a, PBP7, and AmpH) and the PG synthase PBP1B made a smaller or null contribution to the pentapeptide-trimming activity at acidic pH. We solved the crystal structure of PBP6b and also demonstrated that the enzyme is more stable and has a lower Km at acidic pH, explaining why PBP6b is more active at low pH. Hence, PBP6b is a specialized DD-CPase that contributes to cell shape maintenance at low pH, and E. coli appears to utilize redundant DD-CPases for normal growth under different conditions. PMID:27329754

  5. Structural features responsible for kinetic thermal stability of a carboxypeptidase from the archaebacterium Sulfolobus solfataricus.

    PubMed Central

    Villa, A; Zecca, L; Fusi, P; Colombo, S; Tedeschi, G; Tortora, P

    1993-01-01

    Investigations were performed on the structural features responsible for kinetic thermal stability of a thermostable carboxypeptidase from the thermoacidophilic archaebacterium Sulfolobus solfataricus which had been purified previously and identified as a zinc metalloprotease [Colombo, D'Auria, Fusi, Zecca, Raia and Tortora (1992) Eur. J. Biochem. 206, 349-357]. Removal of Zn2+ by dialysis led to reversible activity loss, which was promptly restored by addition of 80 microM ZnCl2 to the assay mixture. For the first-order irreversible thermal inactivation the metal-depleted enzyme showed an activation energy value of 205.6 kJ.mol-1, which is considerably lower than that of the holoenzyme (494.4 kJ.mol-1). The values of activation free energies, enthalpies and entropies also dropped with metal removal. Thermal inactivation of the apoenzyme was very quick at 80 degrees C, whereas the holoenzyme was stable at the same temperature. These findings suggest a major stabilizing role for the bivalent cation. Chaotropic salts strongly destabilized the holoenzyme, showing that hydrophobic interactions are involved in maintaining the native conformation of the enzyme. However, the inactivation rate was also increased by sodium sulphate, acetate and chloride, which are not chaotropes, indicating that one or more salt bridges concur in stabilizing the active enzyme. Furthermore, at the extremes of the pH-stability curve, NaCl did not affect the inactivation rate, confirming the stabilizing role of intramolecular ionic bonds, as a pH-dependent decrease in stability is likely to occur from breaking of salt bridges involved in maintaining the native conformation of the protein. PMID:8240298

  6. Structural features responsible for kinetic thermal stability of a carboxypeptidase from the archaebacterium Sulfolobus solfataricus.

    PubMed

    Villa, A; Zecca, L; Fusi, P; Colombo, S; Tedeschi, G; Tortora, P

    1993-11-01

    Investigations were performed on the structural features responsible for kinetic thermal stability of a thermostable carboxypeptidase from the thermoacidophilic archaebacterium Sulfolobus solfataricus which had been purified previously and identified as a zinc metalloprotease [Colombo, D'Auria, Fusi, Zecca, Raia and Tortora (1992) Eur. J. Biochem. 206, 349-357]. Removal of Zn2+ by dialysis led to reversible activity loss, which was promptly restored by addition of 80 microM ZnCl2 to the assay mixture. For the first-order irreversible thermal inactivation the metal-depleted enzyme showed an activation energy value of 205.6 kJ.mol-1, which is considerably lower than that of the holoenzyme (494.4 kJ.mol-1). The values of activation free energies, enthalpies and entropies also dropped with metal removal. Thermal inactivation of the apoenzyme was very quick at 80 degrees C, whereas the holoenzyme was stable at the same temperature. These findings suggest a major stabilizing role for the bivalent cation. Chaotropic salts strongly destabilized the holoenzyme, showing that hydrophobic interactions are involved in maintaining the native conformation of the enzyme. However, the inactivation rate was also increased by sodium sulphate, acetate and chloride, which are not chaotropes, indicating that one or more salt bridges concur in stabilizing the active enzyme. Furthermore, at the extremes of the pH-stability curve, NaCl did not affect the inactivation rate, confirming the stabilizing role of intramolecular ionic bonds, as a pH-dependent decrease in stability is likely to occur from breaking of salt bridges involved in maintaining the native conformation of the protein.

  7. Determination of faropenem in human plasma and urine by liquid chromatography-tandem mass spectrometry.

    PubMed

    Gao, Shouhong; Chen, Wansheng; Tao, Xia; Miao, Haijun; Yang, Shaolin; Wu, Rong

    2008-01-01

    A simple, rapid and sensitive liquid chromatography/electrospray tandem mass spectrometry (LC-MS/MS) quantitative detection method, using cefalexin as internal standard, was developed for the analysis of faropenem in human plasma and urine. After precipitation of the plasma proteins with acetonitrile, the analytes were separated on a C18 reversed-phase column with 0.1% formic acid-methanol (45:55, v/v) and detected by electrospray ionization mass spectrometry in positive multiple reaction monitoring mode. Calibration curves with good linearities (r=0.9991 for plasma sample and r=0.9993 for urine sample) were obtained in the range 5-4000 ng/mL for faropenem. The limit of detection was 5 ng/mL. Recoveries were around 90% for the extraction from human plasma, and good precision and accuracy were achieved. This method is feasible for the evaluation of pharmacokinetic profiles of faropenem in humans, and to our knowledge, it is the first time the pharmacokinetic of faropenem has been elucidated in vivo using LC-MS/MS. PMID:17604362

  8. Human leukocyte antigen-G in the male reproductive system and in seminal plasma.

    PubMed

    Larsen, Margit Hørup; Bzorek, Michael; Pass, Malene B; Larsen, Lise Grupe; Nielsen, Mette Weidinger; Svendsen, Signe Goul; Lindhard, Anette; Hviid, Thomas Vauvert F

    2011-12-01

    One of the non-classical human leukocyte antigen (HLA) class Ib proteins, HLA-G, is believed to exert important immunoregulatory functions, especially during pregnancy. The presence of HLA protein in paternal seminal fluid has been suggested to have an influence on the risk of developing pre-eclampsia. We have investigated whether HLA-G protein is present in human seminal plasma and in different tissue samples of the male reproductive system. Western blot technique and a soluble HLA-G (sHLA-G) assay were used to detect sHLA-G in human seminal plasma samples. Immunohistochemical staining was performed on paraffin-embedded tissue samples. We detected sHLA-G protein in seminal plasma, and HLA-G expression in normal testis and in epididymal tissue of the male reproductive system but not in the seminal vesicle. Furthermore, the results indicated a weak expression of HLA-G in hyperplastic prostatic tissue. In summary, several of the findings reported in this study suggest an immunoregulatory role of HLA-G in the male reproductive system and in seminal plasma.

  9. Determination of total protein in human seminal plasma.

    PubMed

    Hernvann, A; Gonzales, J; Diemert, M C; Galli, J

    1987-06-01

    Methodological problems are encountered in determinating human seminal protein content since results are influenced especially by techniques which are used, and by modifications due to liquefaction process. In order to test these two points, three methods (Folin, Biuret and Meulemans's reactions) were applied to the same seminal samples and results were compared. The differences seem principally due to the high level of glycoproteins. To determine the modifications of total protein content with time after semen collection, each sample was separated in aliquot fractions and proteolysis was blocked at different times for each fraction. There is an increase of the protein concentration during the first fifteen minutes that follows semen collection. The cause of this phenomenon is discussed.

  10. Knockdown of Carboxypeptidase A6 in Zebrafish Larvae Reduces Response to Seizure-Inducing Drugs and Causes Changes in the Level of mRNAs Encoding Signaling Molecules

    PubMed Central

    Lopes, Mark William; Sapio, Matthew R.; Leal, Rodrigo B.; Fricker, Lloyd D.

    2016-01-01

    Carboxypeptidase A6 (CPA6) is an extracellular matrix metallocarboxypeptidase that modulates peptide and protein function by removal of hydrophobic C-terminal amino acids. Mutations in the human CPA6 gene that reduce enzymatic activity in the extracellular matrix are associated with febrile seizures, temporal lobe epilepsy, and juvenile myoclonic epilepsy. The characterization of these human mutations suggests a dominant mode of inheritance by haploinsufficiency through loss of function mutations, however the total number of humans with pathologic mutations in CPA6 identified to date remains small. To better understand the relationship between CPA6 and seizures we investigated the effects of morpholino knockdown of cpa6 mRNA in zebrafish (Danio rerio) larvae. Knockdown of cpa6 mRNA resulted in resistance to the effect of seizure-inducing drugs pentylenetetrazole and pilocarpine on swimming behaviors. Knockdown of cpa6 mRNA also reduced the levels of mRNAs encoding neuropeptide precursors (bdnf, npy, chga, pcsk1nl, tac1, nts, edn1), a neuropeptide processing enzyme (cpe), transcription factor (c-fos), and molecules implicated in glutamatergic signaling (grin1a and slc1a2b). Treatment of zebrafish embryos with 60 mM pilocarpine for 1 hour led to reductions in levels of many of the same mRNAs when measured 1 day after pilocarpine exposure, except for c-fos which was elevated 1 day after pilocarpine treatment. Pilocarpine treatment, like cpa6 knockdown, led to a reduced sensitivity to pentylenetetrazole when tested 1 day after pilocarpine treatment. Taken together, these results add to mounting evidence that peptidergic systems participate in the biological effects of seizure-inducing drugs, and are the first in vivo demonstration of the molecular and behavioral consequences of cpa6 insufficiency. PMID:27050163

  11. Knockdown of Carboxypeptidase A6 in Zebrafish Larvae Reduces Response to Seizure-Inducing Drugs and Causes Changes in the Level of mRNAs Encoding Signaling Molecules.

    PubMed

    Lopes, Mark William; Sapio, Matthew R; Leal, Rodrigo B; Fricker, Lloyd D

    2016-01-01

    Carboxypeptidase A6 (CPA6) is an extracellular matrix metallocarboxypeptidase that modulates peptide and protein function by removal of hydrophobic C-terminal amino acids. Mutations in the human CPA6 gene that reduce enzymatic activity in the extracellular matrix are associated with febrile seizures, temporal lobe epilepsy, and juvenile myoclonic epilepsy. The characterization of these human mutations suggests a dominant mode of inheritance by haploinsufficiency through loss of function mutations, however the total number of humans with pathologic mutations in CPA6 identified to date remains small. To better understand the relationship between CPA6 and seizures we investigated the effects of morpholino knockdown of cpa6 mRNA in zebrafish (Danio rerio) larvae. Knockdown of cpa6 mRNA resulted in resistance to the effect of seizure-inducing drugs pentylenetetrazole and pilocarpine on swimming behaviors. Knockdown of cpa6 mRNA also reduced the levels of mRNAs encoding neuropeptide precursors (bdnf, npy, chga, pcsk1nl, tac1, nts, edn1), a neuropeptide processing enzyme (cpe), transcription factor (c-fos), and molecules implicated in glutamatergic signaling (grin1a and slc1a2b). Treatment of zebrafish embryos with 60 mM pilocarpine for 1 hour led to reductions in levels of many of the same mRNAs when measured 1 day after pilocarpine exposure, except for c-fos which was elevated 1 day after pilocarpine treatment. Pilocarpine treatment, like cpa6 knockdown, led to a reduced sensitivity to pentylenetetrazole when tested 1 day after pilocarpine treatment. Taken together, these results add to mounting evidence that peptidergic systems participate in the biological effects of seizure-inducing drugs, and are the first in vivo demonstration of the molecular and behavioral consequences of cpa6 insufficiency. PMID:27050163

  12. Stereoselective high-performance liquid chromatographic determination of flurbiprofen in human plasma.

    PubMed

    Geisslinger, G; Menzel-Soglowek, S; Schuster, O; Brune, K

    1992-01-01

    An enantioselective high-performance liquid chromatographic assay for the quantitation of the enantiomers of flurbiprofen in human plasma is described. The procedure involved extraction of flurbiprofen from acidified plasma into hexane-diethyl ether (8:2, v/v). Stereoselective separation was achieved with a prepacked alpha 1-acid glycoprotein column without any derivatization procedure. A second assay using a conventional reversed-phase column to determine racemic flurbiprofen is also described. The detection wavelength was set at 246 nm. The limit of quantification was found to be 50 ng/ml for both assays. The method was demonstrated to be sufficiently sensitive for stereoselective pharmacokinetic studies of flurbiprofen.

  13. A Sulfur Amino Acid–Free Meal Increases Plasma Lipids in Humans123

    PubMed Central

    Park, Youngja; Le, Ngoc-Anh; Yu, Tianwei; Strobel, Fred; Gletsu-Miller, Nana; Accardi, Carolyn J.; Lee, Kichun S.; Wu, Shaoxiong; Ziegler, Thomas R.; Jones, Dean P.

    2011-01-01

    The content of sulfur amino acid (SAA) in a meal affects postprandial plasma cysteine concentrations and the redox potential of cysteine/cystine. Because such changes can affect enzyme, transporter, and receptor activities, meal content of SAA could have unrecognized effects on metabolism during the postprandial period. This pilot study used proton NMR (1H-NMR) spectroscopy of human plasma to test the hypothesis that dietary SAA content changes macronutrient metabolism. Healthy participants (18–36 y, 5 males and 3 females) were equilibrated for 3 d to adequate SAA, fed chemically defined meals without SAA for 5 d (depletion), and then fed isoenergetic, isonitrogenous meals containing 56 mg·kg−1·d−1 SAA for 4.5 d (repletion). On the first and last day of consuming the chemically defined meals, a morning meal containing 60% of the daily food intake was given and plasma samples were collected over an 8-h postprandial time course for characterization of metabolic changes by 1H-NMR spectroscopy. SAA-free food increased peak intensity in the plasma 1H-NMR spectra in the postprandial period. Orthogonal signal correction/partial least squares-discriminant analysis showed changes in signals associated with lipids, some amino acids, and lactate, with notable increases in plasma lipid signals (TG, unsaturated lipid, cholesterol). Conventional lipid analyses confirmed higher plasma TG and showed an increase in plasma concentration of the lipoprotein lipase inhibitor, apoC-III. The results show that plasma 1H-NMR spectra can provide useful macronutrient profiling following a meal challenge protocol and that a single meal with imbalanced SAA content alters postprandial lipid metabolism. PMID:21677075

  14. Digitalis-like activity in human plasma: Relation to blood pressure and sodium balance

    SciTech Connect

    Goto, A.; Yamada, K.; Ishii, M.; Sugimoto, T. )

    1990-10-01

    PURPOSE: On the assumption that renal tubular cells are more important as the target cells for a natriuretic factor than blood cells, we used a well-characterized cultured renal tubular cell line, Madin-Darby canine kidney (MDCK), cells to monitor the circulating digitalis-like factor in human plasma and examine its role in the regulation of blood pressure and sodium balance. SUBJECTS AND METHODS: We investigated the effects of plasma on binding of radioactive ouabain to monolayered MDCK cells in order to determine the level of a circulating digitalis-like factor. First, we measured specific 3H-ouabain binding to MDCK cells in the presence of plasma from 71 outpatients (34 normotensive subjects and 37 hypertensive patients) after incubation for 4 hours. Second, we measured specific 3H-ouabain binding after incubation of cells with plasma from 16 hospitalized subjects (eight normotensive subjects and eight hypertensive patients) receiving low and high sodium diets. RESULTS: In Study 1, ouabain binding was lower by 30% with plasma from hypertensive patients than with plasma from normotensive subjects (p less than 0.01). There was a significant negative correlation between individual subject's systolic or mean blood pressure and ouabain binding (r = -0.34, p less than 0.01 or r = -0.29, p less than 0.01). In Study 2, ouabain binding was also significantly reduced by 25% in the presence of plasma from hypertensive subjects as compared with plasma from normotensive subjects irrespective of sodium intake (p less than 0.01). A significant negative correlation was also found for all subjects between either systolic, diastolic, or mean blood pressure and ouabain binding (r = -0.58, p less than 0.01, r = -0.51, p less than 0.01, or r = -0.55, p less than 0.01, respectively).

  15. Human plasma epidermal growth factor/beta-urogastrone is associated with blood platelets.

    PubMed Central

    Oka, Y; Orth, D N

    1983-01-01

    Human epidermal growth factor (hEGF) has previously been isolated from urine and probably is identical to human beta-urogastrone (hUG). Immunoreactive hEGF/UG has been found in the plasma of normal subjects. In this study, using immunoaffinity chromatography to extract hEGF/UG from plasma, we found that immunoreactive hEGF/UG in blood was associated with blood platelets. It was present in platelet-rich, but not platelet-poor plasma and serum, and was found predominantly in the platelet fraction of whole blood. Sephadex G-50 Fine gel-exclusion chromatography of an extract of outdated blood bank platelets revealed two hEGF/UG components, one of which eluted in the void volume, and the other of which coeluted with purified standard hEGF/UG. The former hEGF/UG component was a high-molecular weight form that was cleaved into hEGF/UG by incubation with either mouse EGF/UG-associated arginine esterase or trypsin. It appeared to be identical to the high-molecular weight hEGF/UG previously reported in human urine, except for its apparently equal activities in radioimmunoassay and radioreceptor assay. The latter hEGF/UG component was immunologically, biologically, and physiochemically indistinguishable from highly purified hEGF/UG from human urine and was immunologically different from purified human platelet-derived growth factor. Platelet-associated hEGF/UG may account for the mitogenic activity of serum in cell lines in which platelet-derived growth factor is not active. Since hEGF/UG appears to be liberated from platelets during coagulation, platelet-associated EGF/UG may be involved in normal vascular and tissue repair and in the pathogenesis of atherosclerotic lesions. The discovery that the EGF/UG in plasma is associated with blood platelets raises important new possibilities for its role in human health and disease. PMID:6603475

  16. [The effect of ethanol consumption by dams on the offspring locomotion in the open field test and carboxypeptidase activities in the rat brain and adrenal medulla].

    PubMed

    Mukhina, E S; Saldaev, D A; Vernigora, A N; Gengin, M T

    2005-03-01

    Consumption of dams ethanol increased the posterity locomotion activity in open field test. The increase in female rats was higher then in male ones. Differences in the carboxypeptidase H and PMSF-inhibited carboxypeptidase activities between the brain regions and adrenal medulla of prenatally exposed to ethanol and intact rats were found. The changing of enzyme activities in female rats was higher then in male ones. It is possible that dams ethanol consumption induced profound changes in locomotion mediated, at least partially, by changes in the rate of proteolytic processing of neuropeptide precursors.

  17. [Changes in Kinetics of Chemiluminescence of Plasma as a Measure of Systemic Oxidative Stress in Humans].

    PubMed

    Sozarukova, M M; Polimova, A M; Proskurnina, E V; Vladimirov, Yu A

    2016-01-01

    Oxidative stress is a pathogenetic factor of many diseases. The control of its level is important for early diagnosis and therapy adjustment. In this work, antioxidant status was estimated in blood plasma. In the system of 2,2'-azo-bis(2-amidinopropane)dihydrochloride-luminol a set of chemiluminescence kinetic curve parameters is proposed for oxidative stress level estimation (the latent period τ(lat) and the increasing of analytical signal ΔI(CL)). Uric acid and albumin were shown as the main components that responsible for changes in chemiluminescence kinetic curve of plasma. Serum albumin undergoes oxidative modification in dose-depend manner under the action of UV irradiation, it causes the enhancement of antioxidant properties. Changes in plasma chemiluminescence kinetics are proposed as a measure of oxidative stress in human body. PMID:27192837

  18. Exercise-induced oxidatively damaged DNA in humans: evaluation in plasma or urine?

    PubMed

    Karpouzi, Christina; Nikolaidis, Stefanos; Kabasakalis, Athanasios; Tsalis, George; Mougios, Vassilis

    2016-01-01

    Physical exercise can induce oxidative damage in humans. 8-Hydroxy-2'-deoxyguanosine (8-OHdG) is a widely known biomarker of DNA oxidation, which can be determined in blood and urine. The aim of the present study was to compare these two biological fluids in terms of which is more suitable for the estimation of the oxidative damage of DNA by measuring the concentration of 8-OHdG one hour after maximal exercise by enzyme immunoassay. The concentration of 8-OHdG increased with exercise only in plasma (p < 0.001), and values differed between exercise tests in both plasma and urine (p < 0.05). In conclusion, plasma appears to be more sensitive to exercise-induced 8-OHdG changes than urine and, hence, a more appropriate medium for assessing oxidative damage of DNA, although the poor repeatability of the measurement needs to be addressed in future studies. PMID:26849281

  19. Determination of acenocoumarol in human plasma by capillary gas chromatography with mass-selective detection.

    PubMed

    Pommier, F; Ackermann, R; Sioufi, A; Godbillon, J

    1994-03-18

    A method for the determination of acenocoumarol in human plasma by capillary gas chromatography-mass-selective detection is described. After addition of a structurally related analogue as the internal standard, the compounds are extracted from plasma at acidic pH into toluene, back-extracted with a basic solution and re-extracted from hydrochloric acid solution with toluene, which is then evaporated to dryness. The compounds are converted into their methyl derivatives, which are determined by gas chromatography using a mass-selective detector at m/z 324 for acenocoumarol and m/z 338 for the internal standard. The reproducibility and accuracy of the method were found to be suitable over the acenocoumarol concentrations range 2.2-74 nmol/l. The method could be considered as selective for acenocoumarol in the presence of its major metabolites in plasma.

  20. [Changes in Kinetics of Chemiluminescence of Plasma as a Measure of Systemic Oxidative Stress in Humans].

    PubMed

    Sozarukova, M M; Polimova, A M; Proskurnina, E V; Vladimirov, Yu A

    2016-01-01

    Oxidative stress is a pathogenetic factor of many diseases. The control of its level is important for early diagnosis and therapy adjustment. In this work, antioxidant status was estimated in blood plasma. In the system of 2,2'-azo-bis(2-amidinopropane)dihydrochloride-luminol a set of chemiluminescence kinetic curve parameters is proposed for oxidative stress level estimation (the latent period τ(lat) and the increasing of analytical signal ΔI(CL)). Uric acid and albumin were shown as the main components that responsible for changes in chemiluminescence kinetic curve of plasma. Serum albumin undergoes oxidative modification in dose-depend manner under the action of UV irradiation, it causes the enhancement of antioxidant properties. Changes in plasma chemiluminescence kinetics are proposed as a measure of oxidative stress in human body.

  1. Ascorbic acid protects lipids in human plasma and low-density lipoprotein against oxidative damage

    SciTech Connect

    Frei, B. )

    1991-12-01

    The authors exposed human blood plasma and low-density lipoprotein (LDL) to many different oxidative challenges and followed the temporal consumption of endogenous antioxidants in relation to the initiation of oxidative damage. Under all types of oxidizing conditions, ascorbic acid completely protects lipids in plasma and LDL against detectable peroxidative damage as assessed by a specific and highly sensitive assay for lipid peroxidation. Ascorbic acid proved to be superior to the other water-soluble plasma antioxidants bilirubin, uric acid, and protein thiols as well as to the lipoprotein-associated antioxidants alpha-tocopherol, ubiquinol-10, lycopene, and beta-carotene. Although these antioxidants can lower the rate of detectable lipid peroxidation, they are not able to prevent its initiation. Only ascorbic acid is reactive enough to effectively intercept oxidants in the aqueous phase before they can attack and cause detectable oxidative damage to lipids.

  2. Method for breast cancer diagnosis by phase spectrophotometry of human blood plasma

    NASA Astrophysics Data System (ADS)

    Mintser, Ozar P.; Oliinychenko, B. P.

    2012-01-01

    The possibility of breast cancer diagnostics by means of phase structure measurements of laser radiation transformed by human blood plasma samples. The theoretical fundamentals of polarization filtration method for direct phase shifts measurements of microscopic images are provided. The optical model of polycrystalline networks of blood plasma proteins is suggested. The results of investigating the interrelation between the values of statistical (statistical moments of the 1st-4th order), correlation (correlation area, asymmetry coefficient and autocorrelation function excess) and fractal (dispersion of logarithmic dependencies of power spectra) parameters are presented. They characterize the coordinate distributions of phase shifts between the orthogonal components of the amplitude in the points of laser images of blood plasma smears and pathological changes in the mammary gland tissue. The diagnostic criteria of breast cancer nascency are determined.

  3. Method for breast cancer diagnosis by phase spectrophotometry of human blood plasma

    NASA Astrophysics Data System (ADS)

    Mintser, Ozar P.; Oliinychenko, B. P.

    2011-09-01

    The possibility of breast cancer diagnostics by means of phase structure measurements of laser radiation transformed by human blood plasma samples. The theoretical fundamentals of polarization filtration method for direct phase shifts measurements of microscopic images are provided. The optical model of polycrystalline networks of blood plasma proteins is suggested. The results of investigating the interrelation between the values of statistical (statistical moments of the 1st-4th order), correlation (correlation area, asymmetry coefficient and autocorrelation function excess) and fractal (dispersion of logarithmic dependencies of power spectra) parameters are presented. They characterize the coordinate distributions of phase shifts between the orthogonal components of the amplitude in the points of laser images of blood plasma smears and pathological changes in the mammary gland tissue. The diagnostic criteria of breast cancer nascency are determined.

  4. Plasma native and peptidase-derivable Met-enkephalin responses to restraint stress in rats. Adaptation to repeated restraint.

    PubMed Central

    Pierzchala, K; Van Loon, G R

    1990-01-01

    Met-enkephalin and related proenkephalin A-derived peptides circulate in plasma at picomolar concentration as free, native pentapeptide and at nanomolar concentration in cryptic forms. We have optimized conditions for measurement of immunoreactive Met-enkephalin in plasma and for generation by trypsin and carboxypeptidase B of much greater amounts of total peptidase-derivable Met-enkephalin in plasma of rats, dogs, and humans. Free Met-enkephalin (11 pM) is constituted by native pentapeptide and its sulfoxide. Characterization of plasma total Met-enkephalin derived by peptidic hydrolysis revealed a small amount (38 pM) of Met-enkephalin associated with peptides of molecular mass less than 30,000 D, and probably derived from proenkephalin A, but much larger amounts of Met-enkephalin associated with albumin (1.2 nM) and with a globulin-sized protein (2.8 nM). Thus, plasma protein precursors for peptidase-derivable Met-enkephalin differ structurally and chemically from proenkephalin A. Met-enkephalin generated from plasma by peptidic hydrolysis showed naloxone-reversible bioactivity comparable to synthetic Met-enkephalin. Prolonged exposure of adult, male rats to restraint stress produced biphasic plasma responses, with peaks occurring at 30 s and 30 min in both free native and total peptidase-derivable Met-enkephalin. Repeated daily exposure to this 30-min stress resulted in adaptive loss of responses of both forms to acute restraint. Initial plasma responses of Met-enkephalin paralleled those of epinephrine and norepinephrine, but subsequently showed divergence of response. In conclusion, Met-enkephalin circulates in several forms, some of which may be derived from proteins other than proenkephalin A, and plasma levels of both free native, and peptidase-derivable Met-enkephalin are modulated physiologically. PMID:2312729

  5. Human Plasma Lipidome Is Pleiotropically Associated with Cardiovascular Risk Factors and Death

    PubMed Central

    Mamtani, Manju; Kent, Jack W.; Wong, Gerard; Weir, Jacquelyn M.; Barlow, Christopher K.; Diego, Vincent; Almeida, Marcio; Dyer, Thomas D.; Göring, Harald H.H.; Almasy, Laura; Mahaney, Michael C.; Comuzzie, Anthony G.; Williams-Blangero, Sarah; Meikle, Peter J.; Blangero, John; Curran, Joanne E.

    2014-01-01

    Background Cardiovascular disease (CVD) is the most common cause of death in the United States and is associated with a high economic burden. Prevention of CVD focuses on controlling or improving the lipid profile of patients at risk. The human lipidome is made up of thousands of ubiquitous lipid species. By studying biologically simple canonical lipid species, we investigated whether the lipidome is genetically redundant and whether its genetic influences can provide clinically relevant clues of CVD risk. Methods and Results We performed a genetic study of the human lipidome in 1,212 individuals from 42 extended Mexican American families. High-throughput mass spectrometry enabled rapid capture of precise lipidomic profiles, providing 319 unique species. Using variance-component based heritability analyses and bivariate trait analyses, we detected significant genetic influences on each lipid assayed. Median heritability of the plasma lipid species was 0.37. Hierarchical clustering based on complex genetic correlation patterns identified 12 genetic clusters that characterized the plasma lipidome. These genetic clusters were differentially but consistently associated with risk factors of CVD, including central obesity, obesity, type 2 diabetes, raised serum triglycerides and metabolic syndrome. Also these clusters consistently predicted occurrence of cardiovascular deaths during follow-up. Conclusions The human plasma lipidome is heritable. Shared genetic influences reduce the dimensionality of the human lipidome into clusters that are associated with risk factors of CVD. PMID:25363705

  6. LC-MS-MS simultaneous determination of atorvastatin and ezetimibe in human plasma.

    PubMed

    El-Bagary, Ramzia I; Elkady, Ehab F; El-Sherif, Zeinab Abdelaziz; Kadry, Ahmed M

    2014-09-01

    Atorvastatin and ezetimibe are lipid-lowering drugs prescribed for the treatment of hypercholesterolemia. An LC-MS-MS method has been developed and validated for the simultaneous estimation of atorvastatin and ezetimibe in human plasma using pitavastatin as an internal standard. Liquid-liquid extraction was used for the purification and preconcentration of analytes from human plasma matrix. The chromatographic separation was achieved within 3.0 min by an isocratic mobile phase consisting of 0.2% formic acid in water-acetonitrile (30:70, v/v), flowing through Agilent Eclipse-plus C18, 100 × 4.6 mm, 3.5 µm analytical column, at a flow rate of 0.6 mL min(-1). Multiple reaction monitoring transitions were measured in the positive ion mode for atorvastatin and internal standard, while ezetimibe was measured in negative ion mode. A detailed validation of the method was performed as per US-FDA guidelines and the standard curves were found to be linear in the range of 0.2-30.0 ng mL(-1) with a mean correlation coefficient >0.999 for both drugs. In human plasma, atorvastatin and ezetimibe were stable for at least 36 days at -70 ± 5 °C and 6 h at ambient temperature. After extraction from plasma, the reconstituted samples of atorvastatin and ezetimibe were stable in an autosampler at ambient temperature for 6 h. Also, the cited drugs were stable in plasma samples upon subjecting to three freeze thaw cycles. The method is simple, specific, sensitive, precise, accurate and suitable for bioequivalence and pharmacokinetic studies of this combination.

  7. Directing membrane chromatography to manufacture α1-antitrypsin from human plasma fraction IV.

    PubMed

    Fan, Jinxin; Luo, Jianquan; Song, Weijie; Chen, Xiangrong; Wan, Yinhua

    2015-12-01

    The surging demand for plasma proteins, mainly driven by the growing market and the development of new therapeutic indications, is promoting manufacturers to improve the throughput of plasma proteins. Due to the inherent convective mass transfer, membrane chromatography has been proved to be an efficient approach for extracting a small amount of target proteins from large-volume feed. In this study, α1-antitrypsin (AAT) was extracted from human plasma fraction IV by a two-step membrane chromatography. An anion-exchange membrane chromatography (AEMC) was used to capture the plasma proteins in bind/elute mode, and the obtained effluent was further polished by a hydrophobic interaction membrane chromatography (HIMC) in flow-through mode. Under optimal conditions, the recovery and purity of AAT achieved 87.0% and 0.58 AAT/protein (g/g) by AEMC, respectively. After the precise polishing by HIMC, the purity of AAT was 1.22 AAT/protein (g/g). The comparison results showed that membrane chromatography outperformed column chromatography in both steps because of its high throughput. This two-step membrane chromatography could obtain an AAT recovery of 83.3% and an activity recovery of 91.4%. The outcome of this work not only offers an alternative process for protein purification from plasma, but also provides guidelines for manufacturing product from a large-volume feed with multi-components by membrane chromatography.

  8. Metabolomic profiling of human plasma in pancreatic cancer using pressurized capillary electrochromatography.

    PubMed

    Zhang, Huiping; Wang, Yan; Gu, Xue; Zhou, Junyi; Yan, Chao

    2011-02-01

    The application of pressurized capillary electrochromatography (pCEC) coupled with ultra violet (UV) detection has been investigated for the production of global metabolite profiles from human plasma, and its capabilities of classifying pancreatic cancer patients. The pCEC separation of plasma samples was performed on a RP column with gradient elution. The applied voltage, detection wavelength and type of acid modifiers on separation of plasma samples were optimized with pooled quality control (QC) sample. The stability and the repeatability of the methodology were also determined by the repeat analysis of QC sample. The effects of different scaling methods on the results of orthogonal partial least-squares discrimination analysis (OPLS-DA) based on pCEC-UV data set were also investigated. The results of the current study clearly showed the different phenotypes of metabolites of pancreatic cancer patients and healthy controls based on pCEC-UV plasma profiles. OPLS-DA data are shown to provide a valuable means of convenient classification. This work indicated that pCEC-UV method can be used as a cost-effective and information-rich, while relatively simple and inexpensive approach for plasma profiling on disease metabolomics studies. PMID:21298661

  9. High-throughput sequencing of human plasma RNA by using thermostable group II intron reverse transcriptases.

    PubMed

    Qin, Yidan; Yao, Jun; Wu, Douglas C; Nottingham, Ryan M; Mohr, Sabine; Hunicke-Smith, Scott; Lambowitz, Alan M

    2016-01-01

    Next-generation RNA-sequencing (RNA-seq) has revolutionized transcriptome profiling, gene expression analysis, and RNA-based diagnostics. Here, we developed a new RNA-seq method that exploits thermostable group II intron reverse transcriptases (TGIRTs) and used it to profile human plasma RNAs. TGIRTs have higher thermostability, processivity, and fidelity than conventional reverse transcriptases, plus a novel template-switching activity that can efficiently attach RNA-seq adapters to target RNA sequences without RNA ligation. The new TGIRT-seq method enabled construction of RNA-seq libraries from <1 ng of plasma RNA in <5 h. TGIRT-seq of RNA in 1-mL plasma samples from a healthy individual revealed RNA fragments mapping to a diverse population of protein-coding gene and long ncRNAs, which are enriched in intron and antisense sequences, as well as nearly all known classes of small ncRNAs, some of which have never before been seen in plasma. Surprisingly, many of the small ncRNA species were present as full-length transcripts, suggesting that they are protected from plasma RNases in ribonucleoprotein (RNP) complexes and/or exosomes. This TGIRT-seq method is readily adaptable for profiling of whole-cell, exosomal, and miRNAs, and for related procedures, such as HITS-CLIP and ribosome profiling.

  10. Decrease of plasma and urinary oxidative metabolites of acetaminophen after consumption of watercress by human volunteers.

    PubMed

    Chen, L; Mohr, S N; Yang, C S

    1996-12-01

    To investigate the effect of the consumption of watercress (Nasturtium officinale R. Br.), a cruciferous vegetable, on acetaminophen metabolism, the pharmacokinetics of acetaminophen and its metabolites were studied in a crossover trial of human volunteers. A single oral dose of acetaminophen (1 gm) was given 10 hours after ingestion of watercress homogenates (50 gm). In comparison with acetaminophen only, the ingestion of watercress resulted in a significant reduction in the area under the plasma cysteine acetaminophen (Cys-acetaminophen) concentration-time curve and in the peak plasma Cys-acetaminophen concentration by 28% +/- 3% and by 21% +/- 4% (mean +/- SE; n = 7; p < 0.005), respectively. Correspondingly, the Cys-acetaminophen formation rate constant and Cys-acetaminophen formation fraction were decreased by 55% +/- 9% and 52% +/- 7% (p < 0.01), respectively. Consistent with the results obtained from the plasma, the total urinary excretion of Cys-acetaminophen in 24 hours was also reduced. A decrease of mercapturate acetaminophen, a Cys-acetaminophen metabolite, was also shown in the plasma and urine samples. However, the plasma pharmacokinetic processes and the urinary excretions of acetaminophen, acetaminophen glucuronide, and acetaminophen sulfate were not altered significantly by the watercress treatment. These results suggest that the consumption of watercress causes a decrease in the levels of oxidative metabolites of acetaminophen, probably due to inhibition of oxidative metabolism of this drug.

  11. Simultaneous determination of grepafloxacin, ciprofloxacin, and theophylline in human plasma and urine by HPLC.

    PubMed

    Kamberi, M; Hajime, N; Kamberi, P; Uemura, N; Nakamura, K; Nakano, S

    1999-06-01

    A specific and sensitive reversed-phase high-performance liquid chromatographic (HPLC) method has been developed and validated for the simultaneous determination of grepafloxacin, ciprofloxacin, and theophylline in human plasma and urine. This assay allows these drugs to elute and be resolved in a single chromatogram at 280 nm, using a linear gradient. The procedure involves liquid-liquid extraction. Separation was achieved on a C18 reversed-phase column. The quantification limits were 0.05 mg/L in plasma and 0.5 mg/L in urine for grepafloxacin and ciprofloxacin and 0.5 mg/L in plasma and urine for theophylline. Standard curves were linear (correlation coefficients >0.999) over the ranges 0.05 to 5 mg/L for grepafloxacin and ciprofloxacin in plasma, from 0.5 to 20 mg/L for theophylline in plasma, and from 0.5 to 500 mg/L for the three drugs in urine. The coefficients of variation for the three drugs were less than 10% for within- and between-day analyses. The recoveries averaged 94.5% for theophylline, 93% for ciprofloxacin, 93.7% for grepafloxacin, and 95.1% for the internal standard (IS). The assay can be used for pharmacokinetic studies of these drugs, to investigate the interaction of grepafloxacin and ciprofloxacin with theophylline, or for routine simultaneous monitoring of theophylline, grepafloxacin, and ciprofloxacin.

  12. Safflower oil consumption does not increase plasma conjugated linoleic acid concentrations in humans.

    PubMed

    Herbel, B K; McGuire, M K; McGuire, M A; Shultz, T D

    1998-02-01

    Conjugated linoleic acid (CLA) is a mixture of positional and geometric isomers of linoleic acid (LA) with conjugated double bonds. CLA has anticarcinogenic properties and has been identified in human tissues, dairy products, meats, and certain vegetable oils. A variety of animal products are good sources of CLA, but plant oils contain much less. However, plant oils are a rich source of LA, which may be isomerized to CLA by intestinal microorganisms in humans. To investigate the effect of triacylglycerol-esterified LA consumption on plasma concentrations of esterified CLA in total lipids, a dietary intervention (6 wk) was conducted with six men and six women. During the intervention period a salad dressing containing 21 g safflower oil providing 16 g LA/d was added to the subjects' daily diets. Three-day diet records and fasting blood were obtained initially and during dietary and postdietary intervention periods. Although LA intake increased significantly during the dietary intervention, plasma CLA concentrations were not affected. Plasma total cholesterol and LDL-cholesterol concentrations were significantly lower after addition of safflower oil to the diet. In summary, consumption of triacylglycerol-esterified LA in safflower oil did not increase plasma concentrations of esterified CLA in total lipids.

  13. Microdevice for plasma separation from whole human blood using bio-physical and geometrical effects

    NASA Astrophysics Data System (ADS)

    Tripathi, Siddhartha; Kumar, Y. V. Balavarun; Agrawal, Amit; Prabhakar, Amit; Joshi, Suhas S.

    2016-06-01

    In this research work, we present a simple and efficient passive microfluidic device for plasma separation from pure blood. The microdevice has been fabricated using conventional photolithography technique on a single layer of polydimethylsiloxane, and has been extensively tested on whole blood and enhanced (upto 62%) hematocrit levels of human blood. The microdevice employs elevated dimensions of about 100 μm such elevated dimensions ensure clog-free operation of the microdevice and is relatively easy to fabricate. We show that our microdevice achieves almost 100% separation efficiency on undiluted blood in the flow rate range of 0.3 to 0.5 ml/min. Detailed biological characterization of the plasma obtained from the microdevice is carried out by testing: proteins by ultra-violet spectrophotometric method, hCG (human chorionic gonadotropin) hormone, and conducting random blood glucose test. Additionally, flow cytometry study has also been carried on the separated plasma. These tests attest to the high quality of plasma recovered. The microdevice developed in this work is an outcome of extensive experimental research on understanding the flow behavior and separation phenomenon of blood in microchannels. The microdevice is compact, economical and effective, and is particularly suited in continuous flow operations.

  14. Proteomic profiling of human plasma exosomes identifies PPAR{gamma} as an exosome-associated protein

    SciTech Connect

    Looze, Christopher; Yui, David; Leung, Lester; Ingham, Matthew; Kaler, Maryann; Yao, Xianglan; Wu, Wells W.; Shen Rongfong; Daniels, Mathew P.; Levine, Stewart J.

    2009-01-16

    Exosomes are nanovesicles that are released from cells as a mechanism of cell-free intercellular communication. Only a limited number of proteins have been identified from the plasma exosome proteome. Here, we developed a multi-step fractionation scheme incorporating gel exclusion chromatography, rate zonal centrifugation through continuous sucrose gradients, and high-speed centrifugation to purify exosomes from human plasma. Exosome-associated proteins were separated by SDS-PAGE and 66 proteins were identified by LC-MS/MS, which included both cellular and extracellular proteins. Furthermore, we identified and characterized peroxisome proliferator-activated receptor-{gamma} (PPAR{gamma}), a nuclear receptor that regulates adipocyte differentiation and proliferation, as well as immune and inflammatory cell functions, as a novel component of plasma-derived exosomes. Given the important role of exosomes as intercellular messengers, the discovery of PPAR{gamma} as a component of human plasma exosomes identifies a potential new pathway for the paracrine transfer of nuclear receptors.

  15. New sensitive direct radioimmunoassay for human plasma renin and its clinical application

    SciTech Connect

    Higaki, J.; Ogihara, T.; Imai, N.; Kumahara, Y.; Hontani, S.; Nishiura, M.; Ogawa, H.; Hirose, S.; Murakami, K.

    1984-12-01

    A new sensitive direct radioimmunoassay for human plasma renin has been developed. Renin was purified from Haas' preparation utilizing a pepstatin-C/sub 6/-Sepharose affinity chromatography. Antiserum, prepared by immunizing rabbits with the purified renin, was used for the direct radioimmunoassay at a final dilution of 1:30,000. The antibody was specific for human renal and plasma renin, but did not cross-react with cathepsin D, trypsin, or renins of mouse, dog, and rat. Radioimmunoassay was performed by the double antibody technique using the delayed tracer addition method. In this method, a standard curve was obtained over a range from 0.2 to 8.0 ng/ml. The values from this assay correlated well with total renin activity measured as the generation rate of angiotensin I after trypsin activation, but correlated weakly with active renin activity. This finding disclosed that both active and inactive renin were detected by this method. In normal participants, plasma renin concentration determined by direct radioimmunoassay was increased by standing and furosemide injection. The plasma renin concentration determined by direct radioimmunoassay of patients with essential hypertension was not significantly different from values in normal controls. The values were higher in patients with renovascular hypertension, malignant hypertension and Bartter's syndrome, but lower in patients with primary aldosteronism than in normal controls. 20 references, 7 figures.

  16. Microdevice for plasma separation from whole human blood using bio-physical and geometrical effects

    PubMed Central

    Tripathi, Siddhartha; Kumar, Y. V. BalaVarun; Agrawal, Amit; Prabhakar, Amit; Joshi, Suhas S.

    2016-01-01

    In this research work, we present a simple and efficient passive microfluidic device for plasma separation from pure blood. The microdevice has been fabricated using conventional photolithography technique on a single layer of polydimethylsiloxane, and has been extensively tested on whole blood and enhanced (upto 62%) hematocrit levels of human blood. The microdevice employs elevated dimensions of about 100 μm; such elevated dimensions ensure clog-free operation of the microdevice and is relatively easy to fabricate. We show that our microdevice achieves almost 100% separation efficiency on undiluted blood in the flow rate range of 0.3 to 0.5 ml/min. Detailed biological characterization of the plasma obtained from the microdevice is carried out by testing: proteins by ultra-violet spectrophotometric method, hCG (human chorionic gonadotropin) hormone, and conducting random blood glucose test. Additionally, flow cytometry study has also been carried on the separated plasma. These tests attest to the high quality of plasma recovered. The microdevice developed in this work is an outcome of extensive experimental research on understanding the flow behavior and separation phenomenon of blood in microchannels. The microdevice is compact, economical and effective, and is particularly suited in continuous flow operations. PMID:27279146

  17. The Effect of Drinking on Plasma Vasopressin and Renin in Dehydrated Human Subjects

    NASA Technical Reports Server (NTRS)

    Geelen, G.; Keil, L. C.; Kravik, S. E.; Wade, C. E.; Thrasher, T. N.; Barnes, P. R.; Pyka, G.; Nesvig, C.; Greenleaf, J. E.

    1996-01-01

    Oropharyngeal mechanisms activated by drinking have been shown to induce a rapid decline in plasma vasopressin which preceeds postabsorptive changes in plasma composition in the dehydrated dog. The present study was undertaken to determine what factor(s) inhibit(s) vasopressin secretion after rehydration in water deprived human subjects. Hematocrit (Hct) and hemoglobin (Hb) were determined on the day of the experiment, together with electrolytes and osmolalities which were measured on freshly separated serum. Plasma was immediately frozen and further analyzed by radioimmunoassay for renin activity (PRA), vasopressin (AVP), and aldosterone. The data were analyzed using an analysis of variance for repeated measurements and significant differences between the dehydrated control period and various time points after the start of rehydration were determined using a multiple-range test. began and reached water replete levels 15 minutes after drinking in the absence of any detectable decline in serum sodium or osmolality, we conclude that 427 oropharyngeal factors, alone or combined with gastric distension account for the extremely rapid inhibition of AVP secretion after drinking in the water-deprived human, as has been shown to be the case in dogs. Our findings are also in agreement wiht the recent demonstration that at the onset of drinking in the dehydrated monkey, there is an abrupt fall in plasma AVP concentration associated with a considerable decrease in the firing rate of the supraoptic neurosecretory neurons.

  18. Functional and ultrastructural studies on In-111-Merc labeled concentrated human platelets in plasma

    SciTech Connect

    Thakur, M.L.; Sedar, A.W.; McKenney, S.L.

    1985-05-01

    Human platelets (1 billion) labeled with 200 ..mu..Ci In-111-oxine in non-plasma medium have been reported to have impaired ultrastructure and function. The ultrastructure and function of platelets labeled in plasma with In-111-Merc was examined. Human platelets (2 billion) suspended in 0.5 ml plasma were incubated with 2 ..mu..g Merc and then labeled with 390-1170 ..mu..Ci In-111. Unlabeled platelets and those incubated with Merc and decayed In-111 solution served as controls. Radiation dose received by platelets in each preparation was estimated. Aggregability studies were performed and platelets were prepared for transmission electron microscopy. Three samples from each pellet were thin sectioned and examined at 4000 to 11,000 magnification. Electron micrographs were obtained from fields containing at least 10 platelets. Assuming uniform labeling and complete decay of In-111, radiation dose per platelet ranged from 367 Gy to 1100 Gy. The aggregability of labeled platelets was unaffected (93.5 +- 3.8%) and the velocity averaged 75.5 +- 3.9% of control. Electron micrographs revealed no change in morphology of mitochondria, alpha granules, dense granules, canalicular system, microtubules and particulate glycogen; these demonstrated normal distribution. The authors conclude that despite the high radiation dose, the function and ultrastructure of platelets labeled in plasma with In-111-Merc remain unaltered.

  19. Determination of trimebutine and desmethyl-trimebutine in human plasma by HPLC.

    PubMed

    Lavit, M; Saivin, S; Boudra, H; Michel, F; Martin, A; Cahiez, G; Labaune, J P; Chomard, J M; Houin, G

    2000-07-01

    A simple and sensitive HPLC method has been developed to measure trimebutine (CAS 39133-31-8, maleate: CAS 34140-59-5) and its main metabolite desmethyl-trimebutine in human plasma. The method was validated according to the Washington Consensus Conference on the Validation of Analytical Methods. It involved extraction of the plasma with n-hexane containing 2-pentanol, followed by reversed-phase HPLC using a Partisil ODS2 10 microns column and UV detection at 265 nm. The retention times of the internal standard (procaine), desmethyl-trimebutine and trimebutine were 2.4, 4.3 and 6.5 min, respectively. The standard curves were linear from 20 ng.ml-1 (limit of quantitation) to 5000 ng.ml-1 for both compounds. The coefficient of variation for all the criteria of validation were less than 15%. The extraction recoveries obtained for trimebutine and desmethyl-trimebutine were about 90%. Both compounds were very stable upon storage in plasma. The method was tested by measuring the plasma concentrations following oral administration to humans during a bioequivalence study and was shown suitable for pharmacokinetic studies. PMID:10965422

  20. Comprehensive Analysis of Low-Molecular-Weight Human Plasma Proteome Using Top-Down Mass Spectrometry.

    PubMed

    Cheon, Dong Huey; Nam, Eun Ji; Park, Kyu Hyung; Woo, Se Joon; Lee, Hye Jin; Kim, Hee Cheol; Yang, Eun Gyeong; Lee, Cheolju; Lee, Ji Eun

    2016-01-01

    While human plasma serves as a great source for disease diagnosis, low-molecular-weight (LMW) proteome (<30 kDa) has been shown to contain a rich source of diagnostic biomarkers. Here we employ top-down mass spectrometry to analyze the LMW proteoforms present in four types of human plasma samples pooled from three healthy controls (HCs) without immunoaffinity depletion and with depletion of the top two, six, and seven high-abundance proteins. The LMW proteoforms were first fractionated based on molecular weight using gel-eluted liquid fraction entrapment electrophoresis (GELFrEE). Then, the GELFrEE fractions containing up to 30 kDa were subjected to nanocapillary-LC-MS/MS, and the high-resolution MS and MS/MS data were processed using ProSightPC 3.0. As a result, a total of 442 LMW proteins and cleaved products, including those with post-translational modifications and single amino acid variations, were identified. From additional comparative analysis of plasma samples without immunoaffinity depletion between HCs and colorectal cancer (CRC) patients via top-down approach, tens of LMW proteoforms, including platelet factor 4, were found to show >1.5-fold changes between the plasma samples of HCs and CRC patients, and six of the LMW proteins were verified by Western blot analysis.

  1. Plasma from human volunteers subjected to remote ischemic preconditioning protects human endothelial cells from hypoxia-induced cell damage.

    PubMed

    Weber, Nina C; Riedemann, Isabelle; Smit, Kirsten F; Zitta, Karina; van de Vondervoort, Djai; Zuurbier, Coert J; Hollmann, Markus W; Preckel, Benedikt; Albrecht, Martin

    2015-03-01

    Short repeated cycles of peripheral ischemia/reperfusion (I/R) can protect distant organs from subsequent prolonged I/R injury; a phenomenon known as remote ischemic preconditioning (RIPC). A RIPC-mediated release of humoral factors might play a key role in this protection and vascular endothelial cells are potential targets for these secreted factors. In the present study, RIPC-plasma obtained from healthy male volunteers was tested for its ability to protect human umbilical endothelial cells (HUVEC) from hypoxia-induced cell damage. 10 healthy male volunteers were subjected to a RIPC-protocol consisting of 4 × 5 min inflation/deflation of a blood pressure cuff located at the upper arm. Plasma was collected before (T0; control), directly after (T1) and 1 h after (T2) the RIPC procedure. HUVEC were subjected to 24 h hypoxia damage and simultaneously incubated with 5% of the respective RIPC-plasma. Cell damage was evaluated by lactate dehydrogenase (LDH)-measurements. Western blot experiments of hypoxia inducible factor 1 alpha (HIF1alpha), phosphorylated signal transducer and activator of transcription 5 (STAT5), protein kinase B (AKT) and extracellular signal-related kinase 1/2 (ERK-1/2) were performed. Furthermore, the concentrations of hVEGF were evaluated in the RIPC-plasma by sandwich ELISA. Hypoxia-induced cell damage was significantly reduced by plasma T1 (p = 0.02 vs T0). The protective effect of plasma T1 was accompanied by an augmentation of the intracellular HIF1alpha (p = 0.01 vs T0) and increased phosphorylation of ERK-1/2 (p = 0.03 vs T0). Phosphorylation of AKT and STAT5 remained unchanged. Analysis of the protective RIPC-plasma T1 showed significantly reduced levels of hVEGF (p = 0.01 vs T0). RIPC plasma protects endothelial cells from hypoxia-induced cell damage and humoral mediators as well as intracellular HIF1alpha may be involved.

  2. Measurement of factor v activity in human plasma using a microplate coagulation assay.

    PubMed

    Tilley, Derek; Levit, Irina; Samis, John A

    2012-09-09

    In response to injury, blood coagulation is activated and results in generation of the clotting protease, thrombin. Thrombin cleaves fibrinogen to fibrin which forms an insoluble clot that stops hemorrhage. Factor V (FV) in its activated form, FVa, is a critical cofactor for the protease FXa and accelerator of thrombin generation during fibrin clot formation as part of prothrombinase (1, 2). Manual FV assays have been described (3, 4), but they are time consuming and subjective. Automated FV assays have been reported (5-7), but the analyzer and reagents are expensive and generally provide only the clot time, not the rate and extent of fibrin formation. The microplate platform is preferred for measuring enzyme-catalyzed events because of convenience, time, cost, small volume, continuous monitoring, and high-throughput (8, 9). Microplate assays have been reported for clot lysis (10), platelet aggregation (11), and coagulation Factors (12), but not for FV activity in human plasma. The goal of the method was to develop a microplate assay that measures FV activity during fibrin formation in human plasma. This novel microplate method outlines a simple, inexpensive, and rapid assay of FV activity in human plasma. The assay utilizes a kinetic microplate reader to monitor the absorbance change at 405 nm during fibrin formation in human plasma (Figure 1) (13). The assay accurately measures the time, initial rate, and extent of fibrin clot formation. It requires only μl quantities of plasma, is complete in 6 min, has high-throughput, is sensitive to 24-80 pM FV, and measures the amount of unintentionally activated (1-stage activity) and thrombin-activated FV (2-stage activity) to obtain a complete assessment of its total functional activity (2-stage activity - 1-stage activity). Disseminated intravascular coagulation (DIC) is an acquired coagulopathy that most often develops from pre-existing infections (14). DIC is associated with a poor prognosis and increases mortality

  3. Synthesis and evaluation of an inhibitor of carboxypeptidase A with a K sub i value in the femtomolar range

    SciTech Connect

    Kaplan, A.P.; Bartlett, P.A. )

    1991-08-20

    Comparative studies among a series of tripeptide phosphonate inhibitors of the zinc peptidase carboxypeptidase A indicate that incorporation of the phosphonic acid analogue of valine at the P{sub 1} position results in significantly higher affinity than the glycine, alanine, or phenylalanine analogues. When applied to the tripeptide analogue Cbz-Phe-Val{sup p}-(O)Phe(ZFV{sup P}(O)F), determination of the inhibition constant K{sub i} was complicated by the very slow rate of dissociation. The rate of exchange of ({sup 3}H)ZFV{sup P}(O)F with enzyme-bound ({sup 14}C)ZFV{sup P}(O)F was followed for periods of 3-4 months to measure dissociation rate constants in the range of (1.7-4.4) X 10{sup {minus}9} s{sup {minus}1}, corresponding to half of 5-13 years. Although the on- and off-rate constants differ for different carboxypeptidase isozymes, their ratios, corresponding to the inhibition constants K{sub i}, are consistently in the range of 10-27 fM. Both the inhibition constants and the dissociation rate constants appear to be the lowest values yet determined for an enzyme-small inhibitor interaction.

  4. High-performance liquid chromatographic assay for cinnarizine in human plasma.

    PubMed

    Nowacka-Krukowska, Hanna; Rakowska, Monika; Neubart, Kinga; Kobylińska, Maria

    2007-01-01

    The high performance liquid chromatography for the determination of cinnarizine in human plasma is described. The procedure involves liquid-liquid extraction followed by reversed phase high-performance chromatographic analysis with fluorometric detection. The method was validated for accuracy, precision, specificity, linearity, sensitivity, recovery, and stability. No endogenous compounds were found to interfere. The absolute extraction recovery of cinnarizine and clocinizine (internal standard) from plasma samples were 97% and 89%, respectively. The linearity was assessed in the range 1-100 ng/mL. The intra-day and inter-day relative standard deviations were less than 10%, and the accuracy of the assay expressed by bias was in the range 0.14-2.37%. The method was proved to be suitable for human pharmacokinetic studies following single oral dose. PMID:18540159

  5. Radioimmunoassay for an inhibin-like peptide from human seminal plasma

    SciTech Connect

    Hammonds, R.G. Jr.; Li, C.H.; Yamashiro, D.; Cabrera, C.M.; Westphal, M.

    1985-01-01

    Antisera raised in rabbits to a synthetic peptide consisting of 31 amino acids with a sequence identical to inhibin-like peptide (ILP) isolated from human seminal plasma afford a highly specific and sensitive radioimmunoassay. Synthetic ILP completely displaces antiserum binding of radioiodinated (Tyr4)-ILP, with half maximal displacement at 36 fmoles ILP/tube. ILP, (Tyr4)-ILP and ILP-(9-31) had essentially equal potency, while ILP-(1-25), ILP-(1-23) and ILP-(1-16) had reduced potency. No cross reactivity was found among a variety of peptide hormones and proteins. Human seminal plasma displaces 50% of (/sup 125/I-Tyr4)-ILP at dilutions equivalent to 50-250 pl/tube, corresponding to immunoreactive ILP concentration of 0.5-2.5 mg/ml.

  6. Proteomic Profiling of Nonenzymatically Glycated Proteins in Human Plasma and Erythrocyte Membrane

    SciTech Connect

    Zhang, Qibin; Tang, Ning; Schepmoes, Athena A.; Phillips, Lawrence S.; Smith, Richard D.; Metz, Thomas O.

    2008-05-01

    Non-enzymatic glycation of peptides and proteins by D-glucose has important implications in the pathogenesis of diabetes mellitus, particularly in the development of diabetic complications. In this report, a thorough proteomic profiling of glycated proteins was attempted by using phenylboronate affinity chromatography to enrich glycated proteins and glycated, tryptic peptides from human plasma and erythrocyte membranes. Enriched peptides were subsequently analyzed by liquid chromatography coupled with electron transfer dissociation tandem mass spectrometry, and 76 and 31 proteins were confidently identified as glycated from human plasma and erythrocyte membrane, respectively. It was observed that most of the glycated proteins can be identified in samples from individuals with normal glucose tolerance, although samples from individuals with impaired glucose tolerance and type 2 diabetes mellitus have slightly higher numbers of glycated proteins and more glycation sites identified.

  7. Dye-attached magnetic poly(hydroxyethyl methacrylate) nanospheres for albumin depletion from human plasma.

    PubMed

    Gökay, Öznur; Karakoç, Veyis; Andaç, Müge; Türkmen, Deniz; Denizli, Adil

    2015-02-01

    The selective binding of albumin on dye-affinity nanospheres was combined with magnetic properties as an alternative approach for albumin depletion from human plasma. Magnetic poly(hydroxyethyl methacrylate) (mPHEMA) nanospheres were synthesized using mini-emulsion polymerization method in the presence of magnetite powder. The specific surface area of the mPHEMA nanospheres was found to be 1302 m(2)/g. Subsequent to Cibacron Blue F3GA (CB) immobilization onto mPHEMA nanospheres, a serial characterization processing was implemented. The quantity of immobilized CB was calculated as 800 μmol/g. Ultimately, albumin adsorption performance of the CB-attached mPHEMA nanospheres from both aqueous dissolving medium and human plasma were explored.

  8. Measurement of Ether Phospholipids in Human Plasma with HPLC-ELSD and LC/ESI-MS After Hydrolysis of Plasma with Phospholipase A1.

    PubMed

    Mawatari, Shiro; Hazeyama, Seira; Fujino, Takehiko

    2016-08-01

    Ethanolamine ether phospholipid (eEtnGpl) and choline ether phospholipid (eChoGpl) are present in human plasma or serum, but the relative concentration of the ether phospholipids in plasma is very low as compared to those in other tissues. Nowadays, measurement of ether phospholipids in plasma depends on tandem mass spectrometry (LC/MS/MS), but a system for LC/MS/MS is generally too expensive for usual clinical laboratories. Treatment of plasma with phospholipase A1 (PLA1) causes complete hydrolysis of diacylphospholipids, but ether phospholipids remain intact. After the treatment of plasma with PLA1, both eEtnGpl and eChoGpl are detected as independent peaks by high-performance liquid chromatography with evaporative light scattering detection (HPLC-ELSD). The same sample used for HPLC-ELSD can be applied to detect eEtnGpl and eChoGpl with electrospray ionization mass spectrometry. Presence of alkylacylphospholipids in both eChoGpl and eEtnGpl in human plasma was indicated by sequential hydrolysis of plasma with PLA1 and hydrochloric acid. PMID:27386871

  9. Simultaneous liquid chromatography-mass spectrometry quantification of cefixime and clavulanic acid in human plasma.

    PubMed

    Dubala, Anil; Nagarajan, Janaki Sankarachari Krishnan; Vimal, Chandran Sathish; George, Renjith

    2015-01-01

    A simple and specific liquid chromatography-atmospheric pressure chemical ionization-mass spectrometry (LC-APCI-MS) assay method has been developed and fully validated for the simultaneous quantification of cefixime (CX) and clavulanic acid (CA) in human plasma. Analytes and internal standard were extracted from human plasma by a solid phase extraction technique using a Sam prep (3 mL, 100 mg) extraction cartridge. The extracted samples were chromatographed on a reverse phase C18 column using a mixture of methanol : acetonitrile : 2 mM ammonium acetate (pH 3.5) (25 : 25 : 50, v/v/v) as the mobile phase at a flow rate of 0.8 mL/min. Quantification of the analytes were carried out using single quadrupole LC-APCI-MS through selected ion monitoring at m/z 452 and 198, respectively, for CX and CA. The assay was linear over the concentration range of 0.05-10.0 and 0.1-10.0 μg/mL, respectively, for CX and CA. The mean plasma extraction recoveries of the CX and CA were found to be 95.20-96.27% and 94.67-95.58%, respectively. The method was successfully applied for the determination of pharmacokinetics of CX and CA after oral administration of single dosage CX/CA (200/125 mg) pill to the humans (n = 12).

  10. Plasma proteomics, the Human Proteome Project, and cancer-associated alternative splice variant proteins.

    PubMed

    Omenn, Gilbert S

    2014-05-01

    This article addresses three inter-related subjects: the development of the Human Plasma Proteome Peptide Atlas, the launch of the Human Proteome Project, and the emergence of alternative splice variant transcripts and proteins as important features of evolution and pathogenesis. The current Plasma Peptide Atlas provides evidence on which peptides have been detected for every protein confidently identified in plasma; there are links to their spectra and their estimated abundance, facilitating the planning of targeted proteomics for biomarker studies. The Human Proteome Project (HPP) combines a chromosome-centric C-HPP with a biology and disease-driven B/D-HPP, upon a foundation of mass spectrometry, antibody, and knowledgebase resource pillars. The HPP aims to identify the approximately 7000 "missing proteins" and to characterize all proteins and their many isoforms. Success will enable the larger research community to utilize newly-available peptides, spectra, informative MS transitions, and databases for targeted analyses of priority proteins for each organ and disease. Among the isoforms of proteins, splice variants have the special feature of greatly enlarging protein diversity without enlarging the genome; evidence is accumulating of striking differential expression of splice variants in cancers. In this era of RNA-sequencing and advanced mass spectrometry, it is no longer sufficient to speak simply of increased or decreased expression of genes or proteins without carefully examining the splice variants in the protein mixture produced from each multi-exon gene. This article is part of a Special Issue entitled: Biomarkers: A Proteomic Challenge.

  11. Identification of phosphorylated butyrylcholinesterase in human plasma using immunoaffinity purification and mass spectrometry

    SciTech Connect

    Aryal, Uma K.; Lin, Chiann Tso; Kim, Jong Seo; Heibeck, Tyler H.; Wang, Jun; Qian, Weijun; Lin, Yuehe

    2012-04-20

    Paraoxon (diethyl 4-nitrophenyl phosphate) is an active metabolite of the common insecticide parathion and is acutely toxic due to the inhibition of cholinesterase (ChE) activity in the nervous systems. The Inhibition of butyrylcholinesterase (BChE) activity by paraoxon is due to the formation of phosphorylated BChE adduct, and the detection of the phosphorylated BChE adduct in human plasma can serve as an exposure biomarker of organophosphate pesticides and nerve agents. In this study, we performed immunoaffinity purification and liquid chromatography tandem mass spectrometry (LC-MS/MS) analysis for identifying phosphorylated BChE in human plasma treated by paraoxon. BChE was captured by biotinylated anti-BChE polyclonal antibodies conjugated to streptavidin magnetic beads. Western blot analysis showed that the antibody was effective to recognize both native and modified BChE with high specificity. The exact phosphorylation site of BChE was confirmed on Serine 198 by MS/MS with a 108 Da modification mass and accurately measured parent ion masses. The phosphorylated BChE peptide was also successfully detected in the immunoaffinity purified sample from paraoxon treated human plasma. Thus, immunoaffinity purification combined with mass spectrometry represents a viable approach for the detection of paraoxon-modified BChE and other forms of modified BChE as exposure biomarkers of organophosphates and nerve agents.

  12. Quantification of doxazosin in human plasma using hydrophilic interaction liquid chromatography with tandem mass spectrometry.

    PubMed

    Ji, Hye Young; Park, Eun Jeong; Lee, Kang Choon; Lee, Hye Suk

    2008-05-01

    Hydrophilic interaction LC with MS/MS (HILIC-MS/MS) was described as a rapid, sensitive, and selective method for the quantification of doxazosin in human plasma. Doxazosin and cisapride (internal standard) were extracted from human plasma with ethyl acetate at alkaline pH and analyzed on an Atlantis HILIC Silica column with the mobile phase of ACN/ammonium formate (100 mM, pH 4.5) (93:7 v/v). The analytes were detected using an ESI MS/MS in the selective-reaction-monitoring mode. The standard curve was linear (r = 0.9994) over the concentration range of 0.2-50 ng/mL. The LOQ for doxazosin was 0.2 ng/mL using 100 microL plasma sample. The CV and relative error for intra- and interassay at four QC levels were 3.7-8.7% and 0.0-9.8%, respectively. The matrix effect for doxazosin and cisapride were practically absent. The recoveries of doxazosin and cisapride were 67.4 and 61.7%, respectively. This method was successfully applied to the pharmacokinetic study of doxazosin in humans.

  13. Capsular polysaccharide of Group B Streptococcus mediates biofilm formation in the presence of human plasma.

    PubMed

    Xia, Fan Di; Mallet, Adeline; Caliot, Elise; Gao, Cherry; Trieu-Cuot, Patrick; Dramsi, Shaynoor

    2015-01-01

    Group B Streptococcus (GBS) is an asymptomatic colonizer of human mucosal surfaces that is responsible for sepsis and meningitis in neonates. Bacterial persistence and pathogenesis often involves biofilm formation. We previously showed that biofilm formation in medium supplemented with glucose is mediated by the PI-2a pilus. Here, biofilm formation was tested in cell culture medium supplemented with human plasma. GBS strains were able to form biofilms in these conditions unlike Group A Streptococcus (GAS) or Staphylococcus aureus. Analysis of mutants impaired for various surface components revealed that the GBS capsule is a key component in this process.

  14. Effect of hypertonic saline infusion on the level of immunoreactive dynorphin in extracted human plasma.

    PubMed

    Margioris, A N; Brockmann, G; Kalogeras, K T; Fjellestad-Paulsen, A; Stratakis, C A; Vamvakopoulos, N; Chrousos, G P

    1990-08-01

    Dynorphin-A and its related peptides are derived from prodynorphin, one of the three known endogenous opioid precursors. The prodynorphin gene is expressed in the vasopressinergic magnocellular neurons of the hypothalamus, while its peptide products are present in the vasopressin (AVP) neurosecretory vesicles of the neurohypophysis. The concentration of immunoreactive (IR) dynorphin is orders of magnitude higher in the neurohypophysis than in any other tissue, suggesting that perhaps the prodynorphin-derived peptides are secreted from the hypothalamic-neurohypophyseal unit into the general circulation. Experiments in rats have shown that osmotic stimuli increase both AVP and prodynorphin in the hypothalamus. To determine whether human hypothalamic prodynorphin is also under osmotic regulation, we measured plasma IR-dynorphin, plasma IR-AVP, and serum sodium immediately before and during the infusion of normal or hypertonic saline in normal human volunteers. Because of the unusual susceptibility of the prodynorphin-derived peptides to cleavage by endopeptidases, we also developed an appropriate plasma dynorphin extraction technique. We found that the IR-dynorphin present in human plasma was composed of 6K- and 4K-sized peptides and that no larger than 6K or smaller than 4K dynorphins were present. The infusion of normal saline did not have any significant effect on plasma IR-dynorphin, while 3% hypertonic saline increased its plasma levels. Thus, the mean IR-dynorphin level in the plasma of the volunteers infused with normal saline was 40.3 +/- 6.4 fmol/mL (mean +/- SE; n = 6) at zero time; after 30 min of infusion, plasma IR-dynorphin was 36.0 +/- 6.3, after 60 min it was 29.9 +/- 5, after 90 min it was 36.0 +/- 4.7, after 120 min it was 36.8 +/- 3.2, and after 150 min it was 36.0 +/- 6.1. The plasma IR-dynorphin level in the volunteers infused with hypertonic saline was 31.7 +/- 3.5 fmol/mL (mean +/- SE; n = 10) at zero time. After 30 min of infusion it increased

  15. Crystal structure of the complex of carboxypeptidase A with a strongly bound phosphonate in a new crystalline form: Comparison with structures of other complexes

    SciTech Connect

    Kim, Hidong; Lipscomb, W.N. )

    1990-06-12

    O-(((1R)-((N-(Phenylmethoxycarbonyl)-L-alanyl)amino)ethyl)hydroxyphosphinyl)-L-3-phenyllacetate (ZZA{sup P}(O)F), an analogue of (benzyloxycarbonyl)-Ala-Ala-Phe or (benzyloxycarbonyl)-Ala-Ala-phenyllactate, binds to carboxypeptidase A with great affinity. Similar phosphonates have been shown to be transition-state analogues of the CPA-catalyzed hydrolysis. In the present study, the structure of the complex of phosphonate with carboxypeptidase A has been determined by X-ray crystallography to a resolution of 2.0 {angstrom}. The structure of the complex was solved by molecular replacement. Refinement of the structure against 20,776 unique reflections between 10.0 and 2.0 {angstrom} yields a crystallographic residual of 0.193, including 140 water molecules. The two phosphinyl oxygens of the inhibitor bind to the active-site zinc at 2.2 {angstrom} on the electrophilic (Arg-127) side and 3.1 {angstrom} on the nucleophilic (Glu-270) side. Various features of the binding mode of this phosphonate inhibitor are consistent with the hypothesis that carboxypeptidase A catalyzed hydrolysis proceeds through a general-base mechanism in which the carbonyl carbon of the substrate is attached by Zn-hydroxyl (or Zn-water). This complex structure is compared with previous structures of carboxypeptidase A, including the complexes with the potato inhibitor, a hydrated keto methylene substrate analogue, and a phosphonamidate inhibitor. Comparisons are also made with the complexes of thermolysin with some phosphonamidate inhibitors.

  16. 96-Well Plate Colorimetric Assay for K(sub i) Determination of (plusmn)-2-Benzylsuccinic Acid, an Inhibitor of Carboxypeptidase A

    ERIC Educational Resources Information Center

    Wentland, Mark P.; Raza, Shaan; Yingtong Gao

    2004-01-01

    An appropriate assay to determine the inhibition potency of carboxypeptidase A (CPA) in 96-well format to illustrate how high throughput screening is used in modern drug discovery to identify bioactive molecules is developed. Efforts in developing a colorimetric 96-well plate assay for determination of the K(sub i) for inhibition of CPA by…

  17. Variations in riboflavin binding by human plasma: identification of immunoglobulins as the major proteins responsible

    SciTech Connect

    Innis, W.S.; McCormick, D.B.; Merrill, A.H. Jr.

    1985-10-01

    Riboflavin binding by plasma proteins from healthy human subjects was examined by equilibrium dialysis using a physiological concentration of (2-14C)riboflavin (0.04 microM). Binding ranged from 0.080 to 0.917 pmole of riboflavin/mg of protein (with a mean +/- SD of 0.274 +/- 0.206), which corresponded to 4.14 to 49.4 pmole/ml of plasma (15.5 +/- 11.0) (N = 34). Males and females yielded similar results. Upon fractionation of plasma by gel filtration, the major riboflavin-binding components eluted with albumin and gamma-globulins. Albumin was purified and found to bind riboflavin only very weakly (Kd = 3.8 to 10.4 mM), although FMN and photochemical degradation products (e.g., lumiflavine and lumichrome) were more tightly bound. Binding in the gamma-globulin fraction was attributed to IgG and IGA because the binding protein(s) and immunoglobulins copurified using various methods were removed by treatment of plasma with protein A-agarose, and were coincident upon immunoelectrophoresis followed by autoradiography to detect (2-14C)riboflavin. Differences among the plasma samples correlated with the binding recovered with the immunoglobulins. Binding was not directly related to the total IgG or IgA levels of subjects. Hence, it appears that the binding is due to a subfraction of these proteins. These findings suggest that riboflavin-binding immunoglobulins are a major cause of variations in riboflavin binding in human circulation, and may therefore affect the utilization of this micronutrient.

  18. Enantioselective analysis of 4-hydroxycyclophosphamide in human plasma with application to a clinical pharmacokinetic study.

    PubMed

    de Castro, Francine Attié; Scatena, Gabriel dos Santos; Rocha, Otávio Pelegrino; Marques, Maria Paula; Cass, Quézia Bezerra; Simões, Belinda Pinto; Lanchote, Vera Lucia

    2016-02-01

    Cyclophosphamide (CY) is one of the most common immunosuppressive agents used in autologous hematopoietic stem cell transplantation. CY is a prodrug and is metabolized to active 4-hydroxycyclophosphamide (HCY). Many authors have suggested an association between enantioselectivity in CY metabolism and treatment efficacy and/or complications. This study describes the development and validation of an analytical method of HCY enantiomers in human plasma by high-performance liquid chromatography-tandem mass spectrometry (LC-MS/MS) that can be applied to pharmacokinetic studies, filling this gap in the literature. HCY enantiomers previously derivatized with phenylhydrazine were extracted from 200-μL plasma aliquots spiked with antipyrine as internal standard and a mixture of hexane and dichloromethane (80:20, v/v) was used as the extraction solvent. The derivatized HCY enantiomers were resolved on a Chiracel(®) OD-R column using water:acetonitrile:formic acid (55:45:0.2, v/v) as the mobile phase. No matrix effect was observed and the analysis of HCY enantiomers was linear for plasma concentrations of 5-5000ng of each enantiomer/mL plasma. The coefficients of variation and inaccuracy calculated in precision and accuracy assessments were less than 15%. HCY was stable in human plasma after three successive freeze/thaw cycles, during 3h at room temperature, and in the autosampler at 4°C for 24h after processing, with deviation values less than 15%. The method was applied to evaluate the kinetic disposition of HCY in a patient with multiple sclerosis who was pretreated with intravenous racemic CY for stem cell transplantation. The clinical study showed enantioselectivity in the pharmacokinetics of HCY.

  19. Saliva vs. plasma bioequivalence of metformin in humans: validation of class II drugs of the salivary excretion classification system.

    PubMed

    Idkaidek, N; Arafat, T

    2014-11-01

    To study saliva and plasma bioequivalence of metformin in humans, and to investigate the robustness of using saliva instead of plasma as surrogate for bioequivalence of class II drugs according to the salivary excretion classification system (SECS).Plasma and saliva samples were collected for 12 h after 500 mg oral dosing of metformin to 16 healthy humans. Plasma and saliva pharmacokinetic parameters, 90% confidence intervals and intra-subject variability values were calculated using Kinetica V5. Descriptive statistics and dimensional analysis were calculated by Excel. SimCYP program V13 was used for estimation of effective intestinal permeability.Metformin was subjected to salivary excretion since it falls into class II (Low permeability/High fraction unbound to plasma proteins), with correlation coefficients of 0.95-0.99 between plasma and saliva concentrations. Saliva/plasma concentration ratios were 0.29-0.39. The 90% confidence limits of all parameters failed in both saliva and plasma. Intra-subject variability values in saliva were higher than plasma leading to need for higher number of subjects to be used in saliva.Saliva instead of plasma can be used as surrogate for bioequivalence of class II drugs according to SECS when adequate sample size is used. Future work is planned to demonstrate SECS robustness in drugs that fall into class III.

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

    PubMed

    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.

  1. 1H NMR studies of reactions of copper complexes with human blood plasma and urine.

    PubMed

    Bligh, S W; Boyle, H A; McEwen, A B; Sadler, P J; Woodham, R H

    1992-01-22

    Reactions of the copper complexes Cu(II)Cl2, [Cu(II)(EDTA)]2-, [Cu(II)2(DIPS)4] and [Cu(I)(DMP)2]+ (where DIPS is 3,5-diisopropylsalicylate and DMP is 2,9-dimethylphenanthroline) with human blood plasma and urine have been studied by 500 MHz 1H NMR spectroscopy, and CD spectroscopy has been used to monitor the transfer of Cu(II) onto albumin in plasma. The rate of transfer of Cu(II) from [Cu(II)(EDTA)]2- onto albumin as measured by CD (T1/2 26 min, 0.5 mM Cu, 21 degrees), was similar to the rate of Cu(II) binding to amino acids and citrate, and to the rate of formation of [Ca(II)(EDTA)]2- in plasma. Reactions of Cu(II)Cl2 and [Cu(II)2(DIPS)4] in plasma followed a similar course, but were more rapid. The latter complex also appeared to give rise to the displacement of lactate from protein binding. Reactions of copper complexes in plasma therefore involve a range of low Mr ligands as well as albumin, and the ligands play a major role in determining the kinetics of the reactions. These factors, as well as the partitioning of both complexes and displaced ligands into lipoproteins, are likely to play important roles in the molecular pharmacology of copper-containing drugs. In urine, His and formate were involved in EDTA and DIPS displacement from their respective copper complexes, and peaks for free DIPS and [Ca(II)(EDTA)]2- were observed. The complex (Cu(I)(DMP)2]+ appeared to be relatively stable in both plasma and urine. PMID:1739401

  2. The effect of the plasma needle on the human keratinocytes related to the wound healing process

    NASA Astrophysics Data System (ADS)

    Korolov, Ihor; Fazekas, Barbara; Széll, Márta; Kemény, Lajos; Kutasi, Kinga

    2016-01-01

    In the present study we aim to verify the influence of a non-thermal atmospheric pressure plasma on the wound healing process. In this process the major contributors are the keratinocytes, which migrate to fill in the gap created by the wound. Therefore, we performed the direct treatment of HPV-immortalized human keratinocytes, protected by a layer of phosphate buffered saline (PBS) solution, with the glow discharge generated in flowing helium by a plasma needle. To mimick a wound, a 4 mm scratch was performed on the cell culture (scratch assay). We conducted two types of experiments: (i) cell proliferation and (ii) wound-healing model experiments. The plasma needle configuration, the plasma treatment conditions and the thickness of the protecting PBS layer were set based on viability experiments. The proliferation studies showed that short, 5-10 s, and low power treatments, such as 18 W and 20 W input power, could positively influence the cell proliferation when keratinocytes were protected by PBS. On the other hand, the plasma treatment of cell medium covered keratinocytes resulted in the decrease of proliferation. The wound-healing model (scratch assay) studies showed, that there was a maximum in the wound reduction as a function of the input power and treatment time, namely, at 18 W and 5 s. Furthermore, the wound reduction strongly depended on the treated cell—PBS interaction time. To mimic an infected wound, the scratch assay was covered with a 1× {{10}9} cfu ml-1 Propionibacterium acnes suspension. The plasma treatment of this infected assay resulted in closing of the scratch, while in the non-treated assay the wound did not close at all.

  3. Cellular and transcriptomic analysis of human mesenchymal stem cell response to plasma-activated hydroxyapatite coating.

    PubMed

    Tan, Fei; O'Neill, Feidhlim; Naciri, Mariam; Dowling, Denis; Al-Rubeai, Mohamed

    2012-04-01

    Atmospheric pressure plasma has recently emerged as a technique with a promising future in the medical field. In this work we used the technique as a post-deposition modification process as a means to activate hydroxyapatite (HA) coatings. Contact angle goniometry, optical profilometry, scanning electron microscopy morphology imaging and X-ray photoelectron spectroscopy analysis demonstrate that surface wettability is improved after treatment, without inducing any concomitant damage to the coating. The protein adsorption pattern has been found to be preferable for MSC, and this may result in greater cell attachment and adhesion to plasma-activated HA than to untreated samples. Cell cycle distribution analysis using flow cytometry reveals a faster transition from G(1) to S phase, thus leading to a faster cell proliferation rate on plasma-activated HA. This indicates that the improvement in surface wettability independently enhances cell attachment and cell proliferation, which is possibly mediated by FAK phosphorylation. Pathway-specific polymerase chain reaction arrays revealed that wettability has a substantial influence on gene expression during osteogenic differentiation of human MSC. Plasma-activated HA tends to enhance this process by systemically deregulating multiple genes. In addition, the majority of these deregulated genes had been appropriately translated, as confirmed by ELISA protein quantification. Lastly, alizarin red staining showed that plasma-activated HA is capable of improving mineralization for up to 3 weeks of in vitro culture. It was concluded from this study that atmospheric pressure plasma is a potent tool for modifying the biological function of a material without causing thermal damage, such that adhesion molecules and drugs might be deposited on the original coating to improve performance.

  4. Site-specific O-Glycosylation Analysis of Human Blood Plasma Proteins*

    PubMed Central

    Hoffmann, Marcus; Marx, Kristina; Reichl, Udo; Wuhrer, Manfred; Rapp, Erdmann

    2016-01-01

    Site-specific glycosylation analysis is key to investigate structure-function relationships of glycoproteins, e.g. in the context of antigenicity and disease progression. The analysis, though, is quite challenging and time consuming, in particular for O-glycosylated proteins. In consequence, despite their clinical and biopharmaceutical importance, many human blood plasma glycoproteins have not been characterized comprehensively with respect to their O-glycosylation. Here, we report on the site-specific O-glycosylation analysis of human blood plasma glycoproteins. To this end pooled human blood plasma of healthy donors was proteolytically digested using a broad-specific enzyme (Proteinase K), followed by a precipitation step, as well as a glycopeptide enrichment and fractionation step via hydrophilic interaction liquid chromatography, the latter being optimized for intact O-glycopeptides carrying short mucin-type core-1 and -2 O-glycans, which represent the vast majority of O-glycans on human blood plasma proteins. Enriched O-glycopeptide fractions were subjected to mass spectrometric analysis using reversed-phase liquid chromatography coupled online to an ion trap mass spectrometer operated in positive-ion mode. Peptide identity and glycan composition were derived from low-energy collision-induced dissociation fragment spectra acquired in multistage mode. To pinpoint the O-glycosylation sites glycopeptides were fragmented using electron transfer dissociation. Spectra were annotated by database searches as well as manually. Overall, 31 O-glycosylation sites and regions belonging to 22 proteins were identified, the majority being acute-phase proteins. Strikingly, also 11 novel O-glycosylation sites and regions were identified. In total 23 O-glycosylation sites could be pinpointed. Interestingly, the use of Proteinase K proved to be particularly beneficial in this context. The identified O-glycan compositions most probably correspond to mono- and disialylated core-1

  5. Site-specific O-Glycosylation Analysis of Human Blood Plasma Proteins.

    PubMed

    Hoffmann, Marcus; Marx, Kristina; Reichl, Udo; Wuhrer, Manfred; Rapp, Erdmann

    2016-02-01

    Site-specific glycosylation analysis is key to investigate structure-function relationships of glycoproteins, e.g. in the context of antigenicity and disease progression. The analysis, though, is quite challenging and time consuming, in particular for O-glycosylated proteins. In consequence, despite their clinical and biopharmaceutical importance, many human blood plasma glycoproteins have not been characterized comprehensively with respect to their O-glycosylation. Here, we report on the site-specific O-glycosylation analysis of human blood plasma glycoproteins. To this end pooled human blood plasma of healthy donors was proteolytically digested using a broad-specific enzyme (Proteinase K), followed by a precipitation step, as well as a glycopeptide enrichment and fractionation step via hydrophilic interaction liquid chromatography, the latter being optimized for intact O-glycopeptides carrying short mucin-type core-1 and -2 O-glycans, which represent the vast majority of O-glycans on human blood plasma proteins. Enriched O-glycopeptide fractions were subjected to mass spectrometric analysis using reversed-phase liquid chromatography coupled online to an ion trap mass spectrometer operated in positive-ion mode. Peptide identity and glycan composition were derived from low-energy collision-induced dissociation fragment spectra acquired in multistage mode. To pinpoint the O-glycosylation sites glycopeptides were fragmented using electron transfer dissociation. Spectra were annotated by database searches as well as manually. Overall, 31 O-glycosylation sites and regions belonging to 22 proteins were identified, the majority being acute-phase proteins. Strikingly, also 11 novel O-glycosylation sites and regions were identified. In total 23 O-glycosylation sites could be pinpointed. Interestingly, the use of Proteinase K proved to be particularly beneficial in this context. The identified O-glycan compositions most probably correspond to mono- and disialylated core-1

  6. Immunoradiometric quantitation of tissue plasminogen activator-related antigen in human plasma: crypticity phenomenon and relationship to plasma fibrinolysis

    SciTech Connect

    Wun, T.C.; Capuano, A.

    1987-05-01

    A two-site immunoradiometric assay for tissue plasminogen activator (tPA) antigen has been developed using immunoaffinity purified antibody. Various treatments enhanced the detection of tPA antigen in the plasma samples. Maximum detection was obtained by acidification of plasma to pH 4.8 to 6.5 or addition of 0.5 mol/L of L-lysine or L-arginine. Acidification or addition of lysine to plasma is also required for maximum immunoadsorption of plasma tPA antigen on anti-tPA-Ig-sepharose. These results indicate that plasma tPA antigen is partially cryptic to antibody in untreated plasma. The plasma tPA antigen isolated by immunoadsorption of either untreated plasma or acidified plasma on anti-tPA-Ig-sepharose consists mainly of a 100-kd plasminogen activator species as determined by fibrin-agar zymography. The 100-kd activity is possibly a tPA:inhibitor complex. A standardized sample preparation method was conveniently adopted by mixing 3 vol of plasma and 1 vol of 2 mol/L of L-lysine for the assay. Reconstitution and recovery studies showed that the method is specific and permits full detection of both free tPA and tPA:inhibitor complex. The validity of the assay is further supported by the finding that the spontaneous plasma fibrinolysis previously demonstrated to be dependent on plasma tPA antigen is correlated with tPA antigen content. Using the standardized assay, we found that tPA antigen concentrations in 16 blood bank plasmas are equivalent to 3.7 to 20 ng of 60 kd tPA/mL. In all the plasma tested, more than half of the antigen is undetected unless the plasma is treated as described above.

  7. High-Performance Liquid Chromatographic Determination of Rivastigmine in Human Plasma for Application in Pharmacokinetic Studies

    PubMed Central

    Amini, Hossein; Ahmadiani, Abolhassan

    2010-01-01

    A simple and reproducible HPLC method with spectrophotometric detection was developed for determination of rivastigmine in human plasma. Liquid-liquid extraction of rivastigmine and donepezil (as internal standard) from plasma samples was performed with 1-butanol/n-hexane (2:98 v/v) in alkaline condition followed by back-extraction into diluted acetic acid. Chromatography was carried out using a Silica column (250 mm × 4.6 mm, 5 μm) under isocratic elution with acetonitrile-50 mM aqueous sodium dihydrogen phosphate (17: 83 v/v, pH 3.1. Analyses were run at a flow-rate of 1.3 mL/min at of 50°C. The recovery was 90.8% and 95.7% for rivastigmine and the internal standard donepezil, respectively. The precision of the method was 2.6% to 9.1% over the concentration range of 0.5-16 ng/mL for rivastigmine in plasma with a linearity greater than 0.999. The method was specific and sensitive, with a quantification limit of 0.5 ng/mL and a detection limit of 0.2 ng/mL in plasma. The method was used for a bioequivalence study in healthy subjects. PMID:24363716

  8. Enhanced Biological Behavior of In Vitro Human Gingival Fibroblasts on Cold Plasma-Treated Zirconia

    PubMed Central

    Zheng, Miao; Yang, Yang; Liu, Xiao-Qiang; Liu, Ming-Yue; Zhang, Xiao-Fei; Wang, Xin; Li, He-Ping; Tan, Jian-Guo

    2015-01-01

    Objective To evaluate whether atmospheric-pressure dielectric-barrier-discharge plasma treatment of zirconia enhances its biocompatibility with human gingival fibroblasts. Materials and Methods The zirconia disks were divided into four groups and treated using helium atmospheric-pressure dielectric-barrier-discharge plasmas for 30, 60 or 90 s or left untreated. The surface morphology, wettability and chemical elements were analyzed. Fibroblasts density, morphology, morphometry and attachment-related genes expression were measured at different time points from 3 to 72 h. Results After plasma treatment, the surface morphology and roughness remained the same, while the contact angle decreased from 78.31° to 43.71°, and the surface C/O ratio decreased from 3.17 to 0.89. The surficial areas and perimeters of HGFs were increased two-fold in the treated groups at 3 h. Fibroblasts density increased on treated disks at all time points, especially the ones treated for 60 s. Attachment-related genes in the groups treated for 30 and 60 s were significantly higher at 3 and 24 h. Conclusion The helium atmospheric-pressure dielectric-barrier-discharge plasma treatment enhances the biological behavior of fibroblasts on zirconia by increasing the expression of attachment-related genes within 24 h and promoting the cell density during longer culture times. Wettability of zirconia, an important physicochemical property, has a vital influence on the cell behaviors. PMID:26461253

  9. In vitro antioxidative activity of (-)-epicatechin glucuronide metabolites present in human and rat plasma.

    PubMed

    Natsume, Midori; Osakabe, Naomi; Yasuda, Akiko; Baba, Seigo; Tokunaga, Takashi; Kondo, Kazuo; Osawa, Toshihiko; Terao, Junji

    2004-12-01

    Recently we identified four conjugated glucuronide metabolites of epicatechin, (-)-epicatechin-3'-O-glucuronide (E3'G), 4'-O-methyl-(-)-epicatechin-3'-O-glucuronide (4'ME3'G), (-)-epicatechin-7-O-glucuronide (E7G) and 3'-O-methyl-(-)-epicatechin-7-O-glucuronide (3'ME7G) from plasma and urine. E3'G and 4'ME3'G were isolated from human urine, while E7G and 3'ME7G were isolated from rats that had received oral administration of (-)-epicatechin (Natsume et al. (2003), Free Radic. Biol. Med. 34,840-849). It has been suggested that these metabolites possess considerable in vivo activity, and therefore we carried out a study to compare the antioxidant activities of the metabolites with that of the parent compound. This was achieved by measuring superoxide scavenging activity, reduction of plasma TBARS production and reduced susceptibility of low-density-lipoprotein (LDL) to oxidation. (-)-Epicatechin was found to have more potent antioxidant activity than the conjugated glucuronide metabolites. Both (-)-epicatechin and E7G had marked antioxidative properties with respect to superoxide radical scavenging activity, plasma oxidation induced by 2,2'-azobis-(2-aminopropane) dihydrochloride (AAPH) and LDL oxidation induced by copper ions or 2,2'-azobis(4-methoxy-2,4-dimethylvaleronitrile) (MeO-AMVN). In contrast, the other metabolites had light antioxidative activities over the range of physiological concentrations found in plasma.

  10. Identification of DNA-binding proteins on human umbilical vein endothelial cell plasma membrane.

    PubMed Central

    Chan, T M; Frampton, G; Cameron, J S

    1993-01-01

    The binding of anti-DNA antibodies to the endothelial cell is mediated through DNA, which forms a bridge between the immunoglobulin and the plasma membrane. We have shown that 32P-labelled DNA bound to the plasma membrane of human umbilical vein endothelial cells (HUVEC) by a saturable process, which could be competitively inhibited by non-radiolabelled DNA. In addition, DNA-binding was enhanced in HUVEC that had been treated with IL-1 alpha or tumour necrosis factor-alpha (TNF-alpha). DNA-binding proteins of mol. wt 46,000, 92,000, and 84,000 were identified by the binding of 32P-labelled DNA to plasma membrane proteins separated on SDS-PAGE. DNA-binding proteins of mol. wt 46,000 and 84,000 were also present in the cytosol and nucleus. Murine anti-DNA MoAb410 bound to a single band, at mol. wt 46,000, of plasma membrane protein, in the presence of DNA. Our results showed that DNA-binding proteins are present in different cellular fractions of endothelial cells. DNA-binding proteins on the cell membrane could participate in the in situ formation of immune deposits; and their presence in the cell nucleus suggests a potential role in the modulation of cell function. Images Fig. 3 Fig. 4 PMID:8419070

  11. Influence of a high-altitude hypoxic environment on human plasma microRNA profiles

    PubMed Central

    Yan, Yan; Shi, Yonghui; Wang, Cheng; Guo, Pengtao; Wang, Junjun; Zhang, Chen-Yu; Zhang, Chunni

    2015-01-01

    Circulating microRNAs (miRNAs) are promising disease biomarkers. However, the influence of high-altitude hypoxic environments on plasma miRNA profiles remains unknown. This study included a total of 509 plasma samples from 278 native Tibetans and 80 newly arrived migrant Han Chinese (Tibet Han) residing at 3560 m and 151 Han Chinese residing at 8.9 m (Nanjing Han). The levels of 754 miRNAs were initially determined using a TaqMan Low Density Array (TLDA) in two pooled samples from 50 Tibet Han and 50 Nanjing Han individuals. Some markedly altered miRNAs in Tibet Han were subsequently measured in all 509 plasma samples by individual qRT-PCR. Compared with the Nanjing Han, 172 miRNAs were differentially expressed in the Tibet Han (105 upregulated and 67 downregulated). The correlation coefficient for the two groups was 0.72. Several upregulated miRNAs were randomly selected for analysis by qRT-PCR, and the results were consistent with those identified by TLDA. These miRNAs were also significantly increased in the Tibetans compared with the Nanjing Han. Furthermore, these altered miRNAs showed strong positive correlations with red blood cell counts and hemoglobin values. These data are the first to provide clear evidence that a high-altitude hypoxic environment significantly affects human plasma miRNA profiles. PMID:26468998

  12. Strawberry consumption improves plasma antioxidant status and erythrocyte resistance to oxidative haemolysis in humans.

    PubMed

    Tulipani, Sara; Alvarez-Suarez, Josè M; Busco, Franco; Bompadre, Stefano; Quiles, Josè L; Mezzetti, Bruno; Battino, Maurizio

    2011-09-01

    Significant increases in the plasma total antioxidant capacity (TAC) have already been reported after acute intake of strawberries. In addition, antihaemolitic effects of strawberry extracts have been recently demonstrated in vitro, revealing that part of the antioxidant properties of strawberry bioactive compounds could lie in their localisation within cell membranes. However, there is a lack of research evidence from in vivo protracted strawberry consumption studies. We carried out a 16-day pilot study where 12 healthy subjects ingested 500g of antioxidants-rich strawberries daily, and we evaluated the potential effects of fruit consumption on biomarkers of plasma and cellular antioxidant status. A significant increase in fasting plasma TAC and in serum vitamin C concentrations were progressively observed during the period of strawberry supplementation. An enhanced resistance to haemolysis was also observed in both AAPH-treated and untreated erythrocytes, collected during and after the period of strawberry consumption. The results obtained in this work suggest that regular consumption of antioxidant-rich strawberries may exert an improvement on the plasma antioxidant status and an increase on the antihaemolitic defenses of human erythrocytes. PMID:25214346

  13. Recovery of Drug Delivery Nanoparticles from Human Plasma Using an Electrokinetic Platform Technology.

    PubMed

    Ibsen, Stuart; Sonnenberg, Avery; Schutt, Carolyn; Mukthavaram, Rajesh; Yeh, Yasan; Ortac, Inanc; Manouchehri, Sareh; Kesari, Santosh; Esener, Sadik; Heller, Michael J

    2015-10-01

    The effect of complex biological fluids on the surface and structure of nanoparticles is a rapidly expanding field of study. One of the challenges holding back this research is the difficulty of recovering therapeutic nanoparticles from biological samples due to their small size, low density, and stealth surface coatings. Here, the first demonstration of the recovery and analysis of drug delivery nanoparticles from undiluted human plasma samples through the use of a new electrokinetic platform technology is presented. The particles are recovered from plasma through a dielectrophoresis separation force that is created by innate differences in the dielectric properties between the unaltered nanoparticles and the surrounding plasma. It is shown that this can be applied to a wide range of drug delivery nanoparticles of different morphologies and materials, including low-density nanoliposomes. These recovered particles can then be analyzed using different methods including scanning electron microscopy to monitor surface and structural changes that result from plasma exposure. This new recovery technique can be broadly applied to the recovery of nanoparticles from high conductance fluids in a wide range of applications.

  14. Recovery of Drug Delivery Nanoparticles from Human Plasma Using an Electrokinetic Platform Technology.

    PubMed

    Ibsen, Stuart; Sonnenberg, Avery; Schutt, Carolyn; Mukthavaram, Rajesh; Yeh, Yasan; Ortac, Inanc; Manouchehri, Sareh; Kesari, Santosh; Esener, Sadik; Heller, Michael J

    2015-10-01

    The effect of complex biological fluids on the surface and structure of nanoparticles is a rapidly expanding field of study. One of the challenges holding back this research is the difficulty of recovering therapeutic nanoparticles from biological samples due to their small size, low density, and stealth surface coatings. Here, the first demonstration of the recovery and analysis of drug delivery nanoparticles from undiluted human plasma samples through the use of a new electrokinetic platform technology is presented. The particles are recovered from plasma through a dielectrophoresis separation force that is created by innate differences in the dielectric properties between the unaltered nanoparticles and the surrounding plasma. It is shown that this can be applied to a wide range of drug delivery nanoparticles of different morphologies and materials, including low-density nanoliposomes. These recovered particles can then be analyzed using different methods including scanning electron microscopy to monitor surface and structural changes that result from plasma exposure. This new recovery technique can be broadly applied to the recovery of nanoparticles from high conductance fluids in a wide range of applications. PMID:26274918

  15. Production of Cloned Miniature Pigs Expressing High Levels of Human Apolipoprotein(a) in Plasma.

    PubMed

    Ozawa, Masayuki; Himaki, Takehiro; Ookutsu, Shoji; Mizobe, Yamato; Ogawa, Junki; Miyoshi, Kazuchika; Yabuki, Akira; Fan, Jianglin; Yoshida, Mitsutoshi

    2015-01-01

    High lipoprotein(a) [Lp(a)] levels are a major risk factor for the development of atherosclerosis. However, because apolipoprotein(a) [apo(a)], the unique component of Lp(a), is found only in primates and humans, the study of human Lp(a) has been hampered due to the lack of appropriate animal models. Using somatic cell nuclear transfer (SCNT) techniques, we produced transgenic miniature pigs expressing human apo(a) in the plasma. First, we placed the hemagglutinin (HA)-tagged cDNA of human apo(a) under the control of the β-actin promoter and cytomegalovirus enhancer, and then introduced this construct into kidney epithelial cells. Immunostaining of cells with anti-HA antibody allowed identification of cells stably expressing apo(a); one of the positive clones was used to provide donor cells for SCNT, yielding blastocysts that expressed apo(a). Immunohistochemical analysis of tissue sections and RT-PCR analysis of total RNA from organs of cloned piglet revealed that apo(a) is expressed in various tissues/organs including heart, liver, kidney, and intestine. More importantly, a transgenic line exhibited a high level (>400 mg/dL) of Lp(a) in plasma, and the transgenic apo(a) gene was transmitted to the offspring. Thus, we generated a human apo(a)-transgenic miniature pig that can be used as a model system to study advanced atherosclerosis related to human disease. The anatomical and physiological similarities between the swine and human cardiovascular systems will make this pig model a valuable source of information on the role of apo(a) in the formation of atherosclerosis, as well as the mechanisms underlying vascular health and disease. PMID:26147378

  16. Production of Cloned Miniature Pigs Expressing High Levels of Human Apolipoprotein(a) in Plasma

    PubMed Central

    Ozawa, Masayuki; Himaki, Takehiro; Ookutsu, Shoji; Mizobe, Yamato; Ogawa, Junki; Miyoshi, Kazuchika; Yabuki, Akira; Fan, Jianglin; Yoshida, Mitsutoshi

    2015-01-01

    High lipoprotein(a) [Lp(a)] levels are a major risk factor for the development of atherosclerosis. However, because apolipoprotein(a) [apo(a)], the unique component of Lp(a), is found only in primates and humans, the study of human Lp(a) has been hampered due to the lack of appropriate animal models. Using somatic cell nuclear transfer (SCNT) techniques, we produced transgenic miniature pigs expressing human apo(a) in the plasma. First, we placed the hemagglutinin (HA)-tagged cDNA of human apo(a) under the control of the β-actin promoter and cytomegalovirus enhancer, and then introduced this construct into kidney epithelial cells. Immunostaining of cells with anti-HA antibody allowed identification of cells stably expressing apo(a); one of the positive clones was used to provide donor cells for SCNT, yielding blastocysts that expressed apo(a). Immunohistochemical analysis of tissue sections and RT-PCR analysis of total RNA from organs of cloned piglet revealed that apo(a) is expressed in various tissues/organs including heart, liver, kidney, and intestine. More importantly, a transgenic line exhibited a high level (>400 mg/dL) of Lp(a) in plasma, and the transgenic apo(a) gene was transmitted to the offspring. Thus, we generated a human apo(a)–transgenic miniature pig that can be used as a model system to study advanced atherosclerosis related to human disease. The anatomical and physiological similarities between the swine and human cardiovascular systems will make this pig model a valuable source of information on the role of apo(a) in the formation of atherosclerosis, as well as the mechanisms underlying vascular health and disease. PMID:26147378

  17. Identification and expression of NEU3, a novel human sialidase associated to the plasma membrane.

    PubMed Central

    Monti, E; Bassi, M T; Papini, N; Riboni, M; Manzoni, M; Venerando, B; Croci, G; Preti, A; Ballabio, A; Tettamanti, G; Borsani, G

    2000-01-01

    Several mammalian sialidases have been described so far, suggesting the existence of numerous polypeptides with different tissue distributions, subcellular localizations and substrate specificities. Among these enzymes, plasma-membrane-associated sialidase(s) have a pivotal role in modulating the ganglioside content of the lipid bilayer, suggesting their involvement in the complex mechanisms governing cell-surface biological functions. Here we describe the identification and expression of a human plasma-membrane-associated sialidase, NEU3, isolated starting from an expressed sequence tag (EST) clone. The cDNA for this sialidase encodes a 428-residue protein containing a putative transmembrane helix, a YRIP (single-letter amino acid codes) motif and three Asp boxes characteristic of sialidases. The polypeptide shows high sequence identity (78%) with the membrane-associated sialidase recently purified and cloned from Bos taurus. Northern blot analysis showed a wide pattern of expression of the gene, in both adult and fetal human tissues. Transient expression in COS7 cells permitted the detection of a sialidase activity with high activity towards ganglioside substrates at a pH optimum of 3.8. Immunofluorescence staining of the transfected COS7 cells demonstrated the protein's localization in the plasma membrane. PMID:10861246

  18. High-performance liquid chromatography method for analyzing the antiretroviral agent efavirenz in human plasma.

    PubMed

    Villani, P; Pregnolato, M; Banfo, S; Rettani, M; Burroni, D; Seminari, E; Maserati, R; Regazzi, M B

    1999-06-01

    Efavirenz (EFV, DMP-266) is a new antiretroviral agent belonging to the class of nonnucleoside reverse transcriptase inhibitors. It has recently been approved by the Food and Drug Administration in management of human immunodeficiency virus (HIV). Preliminary pharmacokinetic studies on EFV in healthy volunteers show that the drug may influence the metabolism of protease inhibitors. For the determination of EFV in human plasma, a validated and specific reverse-phase high-performance liquid chromatography (HPLC) method, with UV detection, was developed. We used 100 microL plasma sample for a liquid-liquid extraction with diethyl ether after basification. The mobile phase was a mixture of acetonitrile and water, pumped at a flow rate of 1.2 mL/min. Ultraviolet detection was carried out at a wavelength of 247 nm. Retention times for EFV and internal standard (IS) were 5.3 and 4.5 minutes, respectively, and there was no chromatographic interference from other commonly administered drugs. The limit of detection was 100 ng/mL. The described assay is a rapid and accurate method for measurement of EFV in plasma: the easy preparation and small sample size makes this assay highly suitable for pharmacokinetic studies and routine clinical analysis in patients with HIV. In addition, the reproducibility of the method is only moderately increased by including IS, so analyzing without IS may be an alternative.

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

    PubMed Central

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

    2013-01-01

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

  20. Mass spectrometric characterization of limited proteolysis activity in human plasma samples under mild acidic conditions.

    PubMed

    Yang, Jingzhi; Röwer, Claudia; Koy, Cornelia; Ruß, Manuela; Rüger, Christopher P; Zimmermann, Ralf; von Fritschen, Uwe; Bredell, Marius; Finke, Juliane C; Glocker, Michael O

    2015-11-01

    We developed a limited proteolysis assay for estimating dynamics in plasma-borne protease activities using MALDI ToF MS analysis as readout. A highly specific limited proteolysis activity was elicited in human plasma by shifting the pH to 6. Mass spectrometry showed that two singly charged ion signals at m/z 2753.44 and m/z 2937.56 significantly increased in abundance under mild acidic conditions as a function of incubation time. For proving that a provoked proteolytic activity in mild acidic solution caused the appearance of the observed peptides, control measurements were performed (i) with pepstatin as protease inhibitor, (ii) with heat-denatured samples, (iii) at pH 1.7, and (iv) at pH 7.5. Mass spectrometric fragmentation analysis showed that the observed peptides encompass the amino acid sequences 1-24 and 1-26 from the N-terminus of human serum albumin. Investigations on peptidase specificities suggest that the two best candidates for the observed serum albumin cleavages are cathepsin D and E. Reproducibility, robustness, and sensitivity prove the potential of the developed limited proteolysis assay to become of clinical importance for estimating dynamics of plasma-borne proteases with respect to associated pathophysiological tissue conditions.

  1. Hippophae rhamnoides L. Fruits Reduce the Oxidative Stress in Human Blood Platelets and Plasma.

    PubMed

    Olas, Beata; Kontek, Bogdan; Malinowska, Paulina; Żuchowski, Jerzy; Stochmal, Anna

    2016-01-01

    Effects of the phenolic fraction from Hippophae rhamnoides fruits on the production of thiobarbituric acid reactive substances (TBARS, a marker of lipid peroxidation) and the generation of superoxide anion (O2 (-∙)) in human blood platelets (resting platelets and platelets stimulated by a strong physiological agonist, thrombin) were studied in vitro. We also examined antioxidant properties of this fraction against human plasma lipid peroxidation and protein carbonylation induced by a strong biological oxidant, hydrogen peroxide (H2O2) or H2O2/Fe (a donor of hydroxyl radicals). The tested fraction of H. rhamnoides (0.5- 50 µg/mL; the incubation time: 15 and 60 min) inhibited lipid peroxidation induced by H2O2 or H2O2/Fe. The H. rhamnoides phenolic fraction inhibited not only plasma lipid peroxidation, but also plasma protein carbonylation stimulated by H2O2 or H2O2/Fe. Moreover, the level of O2 (-∙) in platelets significantly decreased. In comparative experiments, the H. rhamnoides fraction was a more effective antioxidant than aronia extract or grape seed extract (at the highest tested concentration, 50 µg/mL). The obtained results suggest that H. rhamnoides fruits may be a new, promising source of natural compounds with antioxidant and antiplatelet activity beneficial not only for healthy people, but also for those with oxidative stress-associated diseases. PMID:26933473

  2. Determination of Flurbiprofen in Human Plasma by High-Performance Liquid Chromatography.

    PubMed

    Yilmaz, Bilal; Erdem, Ali Fuat

    2015-10-01

    A simple high-performance liquid chromatography method has been developed for determination of flurbiprofen in human plasma. The method was validated on an Ace C18 column using UV detection. The mobile phase was acetonitrile-0.05 M potassium dihydrogen phosphate solution (60:40, v/v) adjusted to pH 3.5 with phosphoric acid. The calibration curve was linear between the concentration range of 0.10-5.0 μg/mL. Intra- and inter-day precision values for flurbiprofen in plasma were <4.47, and accuracy (relative error) was better than 3.67%. The extraction recoveries of flurbiprofen from human plasma were between 93.0 and 98.9%. The limits of detection and quantification of flurbiprofen were 0.03 and 0.10 μg/mL, respectively. In addition, this assay was applied to determine the pharmacokinetic parameters of flurbiprofen in six healthy Turkish volunteers who had been given 100 mg flurbiprofen.

  3. Hippophae rhamnoides L. Fruits Reduce the Oxidative Stress in Human Blood Platelets and Plasma

    PubMed Central

    Olas, Beata; Kontek, Bogdan; Malinowska, Paulina; Żuchowski, Jerzy; Stochmal, Anna

    2016-01-01

    Effects of the phenolic fraction from Hippophae rhamnoides fruits on the production of thiobarbituric acid reactive substances (TBARS, a marker of lipid peroxidation) and the generation of superoxide anion (O2−∙) in human blood platelets (resting platelets and platelets stimulated by a strong physiological agonist, thrombin) were studied in vitro. We also examined antioxidant properties of this fraction against human plasma lipid peroxidation and protein carbonylation induced by a strong biological oxidant, hydrogen peroxide (H2O2) or H2O2/Fe (a donor of hydroxyl radicals). The tested fraction of H. rhamnoides (0.5– 50 µg/mL; the incubation time: 15 and 60 min) inhibited lipid peroxidation induced by H2O2 or H2O2/Fe. The H. rhamnoides phenolic fraction inhibited not only plasma lipid peroxidation, but also plasma protein carbonylation stimulated by H2O2 or H2O2/Fe. Moreover, the level of O2−∙ in platelets significantly decreased. In comparative experiments, the H. rhamnoides fraction was a more effective antioxidant than aronia extract or grape seed extract (at the highest tested concentration, 50 µg/mL). The obtained results suggest that H. rhamnoides fruits may be a new, promising source of natural compounds with antioxidant and antiplatelet activity beneficial not only for healthy people, but also for those with oxidative stress-associated diseases. PMID:26933473

  4. Hippophae rhamnoides L. Fruits Reduce the Oxidative Stress in Human Blood Platelets and Plasma.

    PubMed

    Olas, Beata; Kontek, Bogdan; Malinowska, Paulina; Żuchowski, Jerzy; Stochmal, Anna

    2016-01-01

    Effects of the phenolic fraction from Hippophae rhamnoides fruits on the production of thiobarbituric acid reactive substances (TBARS, a marker of lipid peroxidation) and the generation of superoxide anion (O2 (-∙)) in human blood platelets (resting platelets and platelets stimulated by a strong physiological agonist, thrombin) were studied in vitro. We also examined antioxidant properties of this fraction against human plasma lipid peroxidation and protein carbonylation induced by a strong biological oxidant, hydrogen peroxide (H2O2) or H2O2/Fe (a donor of hydroxyl radicals). The tested fraction of H. rhamnoides (0.5- 50 µg/mL; the incubation time: 15 and 60 min) inhibited lipid peroxidation induced by H2O2 or H2O2/Fe. The H. rhamnoides phenolic fraction inhibited not only plasma lipid peroxidation, but also plasma protein carbonylation stimulated by H2O2 or H2O2/Fe. Moreover, the level of O2 (-∙) in platelets significantly decreased. In comparative experiments, the H. rhamnoides fraction was a more effective antioxidant than aronia extract or grape seed extract (at the highest tested concentration, 50 µg/mL). The obtained results suggest that H. rhamnoides fruits may be a new, promising source of natural compounds with antioxidant and antiplatelet activity beneficial not only for healthy people, but also for those with oxidative stress-associated diseases.

  5. Human plasma protein adsorption onto dextranized surfaces: a two-dimensional electrophoresis and mass spectrometry study.

    PubMed

    Tsai, Irene Y; Tomczyk, Nancy; Eckmann, Joshua I; Composto, Russell J; Eckmann, David M

    2011-05-01

    Protein adsorption is fundamental to thrombosis and to the design of biocompatible materials. We report a two-dimensional electrophoresis and mass spectrometry study to characterize multiple human plasma proteins adsorbed onto four different types of model surfaces: silicon oxide, dextranized silicon, polyurethane and dextranized polyurethane. Dextran was grafted onto the surfaces of silicon and polyurethane to mimic the blood-contacting endothelial cell glycocalyx surface. Surface topography and hydrophobicity/hydrophilicity were determined and analyzed using atomic force microscopy and water contact angle measurements, respectively. Using two-dimensional electrophoresis, we show that, relative to the unmodified surfaces, dextranization significantly inhibits the adsorption of several human plasma proteins including IGHG1 protein, fibrinogen, haptoglobin, Apo A-IV, Apo A-I, immunoglobulin, serum retinal-binding protein and truncated serum albumin. We further demonstrate the selectivity of plasma protein adsorbed onto the different functionalized surfaces and the potential to control and manipulate proteins adsorption on the surfaces of medical devices, implants and microfluidic devices. This result shows that adsorption experiments using a single protein or a binary mixture of proteins are consistent with competitive protein adsorption studies. In summary, these studies indicate that coating blood-contacting biomedical applications with dextran is an effective route to reduce thrombo-inflammatory responses and to surface-direct biological activities. PMID:21277175

  6. Effects of a Nonthermal Atmospheric Pressure Plasma Jet on Human Gingival Fibroblasts for Biomedical Application

    PubMed Central

    2016-01-01

    Nonthermal atmospheric pressure plasma jets (APPJ) have been developed and applied in biomedical research as a cancer treatment or bacterial sterilization. However, the drawback of APPJ on normal oral cells during plasma treatment and underlying cell death mechanisms have not been studied and clearly explained, although there is known to be an influence from reactive oxygen species (ROS). Hence, this study investigates whether and how a nonthermal atmospheric pressure air plasma jet kills human normal gingival cells using immortalized human gingival fibroblasts (hTERT-hNOF cells). In this study, a set of physicochemical or biological methods were used to illuminate the killing mechanisms. It was found that ROS were induced intracellularly without a breakdown of the cell wall and apoptosis was involved in cell death when an air APPJ treatment was performed on the cells directly without media; the air treatment only supported a detachment of the cells without increase of ROS. It was also revealed that a correlation between intracellular ROS concentration and cells viability existed. These results indicated that the direct air APPJ treatment possibly raises safety issue to normal tissue and thereby APPJ application in biomedical field needs more in vitro and in vivo study to optimize it.

  7. A new simultaneous derivatization and microextration method for the determination of memantine hydrochloride in human plasma.

    PubMed

    Jing, Shao-Jun; Li, Qing-Lian; Jiang, Ye

    2016-01-01

    A simple and sensitive method for simultaneous derivatization and hollow fiber liquid phase microextraction (HF-LPME) followed by high performance liquid chromatography-fluorescence detection (HPLC-FL) to determine memantine hydrochloride (MT) in human plasma was developed. The derivatization and microextraction was combined to a single step to ensure the precision. What is more, the derivatization reaction accelerated the mass transfer during the process of microextraction. The hollow fiber was filled with cyclohexane and dansyl chloride (derivatization agent) as acceptor phase and submersed in the alkalinized plasma sample. The system was submitted to stirring at 800rpm for 50min at 40°C. Different experimental parameters were systematically evaluated by response surface methodology. Under the optimized conditions, the calibration curve was linear in the range of 1-100ng/mL (r=0.9991) with a limit of detection of 0.1ng/mL (S/N=3). The precision estimated as the relative standard deviation (RSD) was less than 4.5% and the accuracy was 94.3-100.7%. The present method was successfully applied to determine MT in human plasma samples.

  8. Effects of a Nonthermal Atmospheric Pressure Plasma Jet on Human Gingival Fibroblasts for Biomedical Application.

    PubMed

    Lee, Jung-Hwan; Kim, Kyoung-Nam

    2016-01-01

    Nonthermal atmospheric pressure plasma jets (APPJ) have been developed and applied in biomedical research as a cancer treatment or bacterial sterilization. However, the drawback of APPJ on normal oral cells during plasma treatment and underlying cell death mechanisms have not been studied and clearly explained, although there is known to be an influence from reactive oxygen species (ROS). Hence, this study investigates whether and how a nonthermal atmospheric pressure air plasma jet kills human normal gingival cells using immortalized human gingival fibroblasts (hTERT-hNOF cells). In this study, a set of physicochemical or biological methods were used to illuminate the killing mechanisms. It was found that ROS were induced intracellularly without a breakdown of the cell wall and apoptosis was involved in cell death when an air APPJ treatment was performed on the cells directly without media; the air treatment only supported a detachment of the cells without increase of ROS. It was also revealed that a correlation between intracellular ROS concentration and cells viability existed. These results indicated that the direct air APPJ treatment possibly raises safety issue to normal tissue and thereby APPJ application in biomedical field needs more in vitro and in vivo study to optimize it.

  9. Effects of a Nonthermal Atmospheric Pressure Plasma Jet on Human Gingival Fibroblasts for Biomedical Application

    PubMed Central

    2016-01-01

    Nonthermal atmospheric pressure plasma jets (APPJ) have been developed and applied in biomedical research as a cancer treatment or bacterial sterilization. However, the drawback of APPJ on normal oral cells during plasma treatment and underlying cell death mechanisms have not been studied and clearly explained, although there is known to be an influence from reactive oxygen species (ROS). Hence, this study investigates whether and how a nonthermal atmospheric pressure air plasma jet kills human normal gingival cells using immortalized human gingival fibroblasts (hTERT-hNOF cells). In this study, a set of physicochemical or biological methods were used to illuminate the killing mechanisms. It was found that ROS were induced intracellularly without a breakdown of the cell wall and apoptosis was involved in cell death when an air APPJ treatment was performed on the cells directly without media; the air treatment only supported a detachment of the cells without increase of ROS. It was also revealed that a correlation between intracellular ROS concentration and cells viability existed. These results indicated that the direct air APPJ treatment possibly raises safety issue to normal tissue and thereby APPJ application in biomedical field needs more in vitro and in vivo study to optimize it. PMID:27597959

  10. Effects of a Nonthermal Atmospheric Pressure Plasma Jet on Human Gingival Fibroblasts for Biomedical Application.

    PubMed

    Lee, Jung-Hwan; Kim, Kyoung-Nam

    2016-01-01

    Nonthermal atmospheric pressure plasma jets (APPJ) have been developed and applied in biomedical research as a cancer treatment or bacterial sterilization. However, the drawback of APPJ on normal oral cells during plasma treatment and underlying cell death mechanisms have not been studied and clearly explained, although there is known to be an influence from reactive oxygen species (ROS). Hence, this study investigates whether and how a nonthermal atmospheric pressure air plasma jet kills human normal gingival cells using immortalized human gingival fibroblasts (hTERT-hNOF cells). In this study, a set of physicochemical or biological methods were used to illuminate the killing mechanisms. It was found that ROS were induced intracellularly without a breakdown of the cell wall and apoptosis was involved in cell death when an air APPJ treatment was performed on the cells directly without media; the air treatment only supported a detachment of the cells without increase of ROS. It was also revealed that a correlation between intracellular ROS concentration and cells viability existed. These results indicated that the direct air APPJ treatment possibly raises safety issue to normal tissue and thereby APPJ application in biomedical field needs more in vitro and in vivo study to optimize it. PMID:27597959

  11. MCP-1 binds to oxidized LDL and is carried by lipoprotein(a) in human plasma

    PubMed Central

    Wiesner, Philipp; Tafelmeier, Maria; Chittka, Dominik; Choi, Soo-Ho; Zhang, Li; Byun, Young Sup; Almazan, Felicidad; Yang, Xiaohong; Iqbal, Navaid; Chowdhury, Punam; Maisel, Alan; Witztum, Joseph L.; Handel, Tracy M.; Tsimikas, Sotirios; Miller, Yury I.

    2013-01-01

    Lipoprotein oxidation plays an important role in pathogenesis of atherosclerosis. Oxidized low density lipoprotein (OxLDL) induces profound inflammatory responses in vascular cells, such as production of monocyte chemoattractant protein-1 (MCP-1) [chemokine (C-C motif) ligand 2], a key chemokine in the initiation and progression of vascular inflammation. Here we demonstrate that OxLDL also binds MCP-1 and that the OxLDL-bound MCP-1 retains its ability to recruit monocytes. A human MCP-1 mutant in which basic amino acids Arg-18 and Lys-19 were replaced with Ala did not bind to OxLDL. The MCP-1 binding to OxLDL was inhibited by the monoclonal antibody E06, which binds oxidized phospholipids (OxPLs) in OxLDL. Because OxPLs are carried by lipoprotein(a) [Lp(a)] in human plasma, we tested to determine whether Lp(a) binds MCP-1. Recombinant wild-type but not mutant MCP-1 added to human plasma bound to Lp(a), and its binding was inhibited by E06. Lp(a) captured from human plasma contained MCP-1 and the Lp(a)-associated endogenous MCP-1 induced monocyte migration. These results demonstrate that OxLDL and Lp(a) bind MCP-1 in vitro and in vivo and that OxPLs are major determinants of the MCP-1 binding. The association of MCP-1 with OxLDL and Lp(a) may play a role in modulating monocyte trafficking during atherogenesis. PMID:23667177

  12. Considerations in the development of a sensitive HPLC assay for human epidermal growth factors in human plasma.

    PubMed

    Kagel, J R; Rossi, D T; Nordblom, G D; Dudeck, R C; Barksdale, C M; Kuo, B S; Wright, D S

    1995-09-01

    A sensitive assay was developed for human epidermal growth factors (hEGF) 1-48 (dosed), hEGF 1-53 (endogenous), without interference from potential metabolites hEGFs 1-47 or 1-46. Spiked human plasma samples were injected directly, utilizing on-line immunoaffinity HPLC (anti-hEGF) clean-up. No change in capacity was noted after 81 cycles. After release from the immunoaffinity column, the fragments were further resolved by strong cation-exchange (SCX) via a column switching valve. Method development also required interfacing immunoaffinity, ion-exchange, and detection components. Immunoassays on collected fractions yielded a detection limit of 1 microgram ml-1, although a detection limit of 75 pg ml-1 appears feasible.

  13. Determination of intact oxaliplatin in human plasma using high performance liquid chromatography-tandem mass spectrometry.

    PubMed

    Zhang, Wenjiang; Seymour, Lesley; Chen, Eric X

    2008-12-15

    A HPLC-tandem mass spectrometry method was developed and validated for the quantitation of intact oxaliplatin in human plasma. Plasma ultrafiltrates were precipitated with acetonitrile and separation was performed on a 250 mm Beckman ODS reverse phase column using a gradient mobile phase. The mass spectrometer was operated in positive ionization mode using TurboionSpray and precursor-product ion combinations of m/z 391.1-->305.1 and 371.1-->247.0 were monitored for oxaliplatin and carboplatin, the internal standard, respectively. The lower limit of quantitation for oxaliplatin was 20 ng/ml. The linear range of the method was 20-1000 ng/ml. The between- and within-day relative standard deviations ranged from 3.1 to 7.7%, and accuracy was within 5%. This method was successfully applied in a clinical study of oxaliplatin.

  14. Influence of low and high pressure baroreceptors on plasma renin activity in humans

    NASA Technical Reports Server (NTRS)

    Mark, A. L.; Abboud, F. M.; Fitz, A. E.

    1977-01-01

    The effects of low and high pressure baroreceptors on plasma renin activity (immunoassay) were evaluated using graded lower body suction (LBS) in six healthy men. LBS at -10 and -20 mmHg for 10 min decreased central venous pressure without changing arterial pressure and thereby presumably reduced low but not high pressure baroreceptor inhibition of renin release. LBS at these levels produced forearm vasoconstriction, but did not increase renin. LBS at -40 mmHG decreased central venous and arterial pulse pressure and thus reduced both low and high pressure baroreceptor inhibition. LBS at this level produced forearm vasoconstriction and tachycardia and increased renin. In summary, reduction in low pressure baroreceptor inhibition in humans did not increase renin in the presence of physiological tonic inhibition from high pressure baroreceptors. Increases in renin did not occur until there was combined reduction of high and low pressure baroreceptor inhibition on plasma renin activity.

  15. Structural difference at the active site of dibucaine resistant variant of human plasma cholinesterase.

    PubMed Central

    Muensch, H; Yoshida, A; Altland, K; Jensen, W; Goedde, H W

    1978-01-01

    Human plasma cholinesterase from five different genotypes -- E1U E1U, E1U E1A, E1A E1A, E1U E1S, E1A E1S, and E1U E1U C5+ -- was purified 8,000 fold from serum by a two-step procedure involving chromatography on DEAE-cellulose and preparative disc electrophoresis. The esterases were labeled with diisopropyl-1, 3-C14-fluorophosphate (DFP) aminoethylated, and digested by trypsin. The trytic digests were subjected to high voltage electrophoresis, and the radioactive peptides were detected by radioautography. Comparison of the peptides revealed different electrophoretic mobilities of the usual and atypical (dibucaine resistant) plasma cholinesterase peptides. The results are consistent with a structural abnormality of the active center in the variant enzyme. No difference was observed an the esteratic site of the enzyme with C5 component. Images Fig. 1 PMID:677127

  16. Separation of proteins from human plasma by sample displacement chromatography in hydrophobic interaction mode

    PubMed Central

    Josic, Djuro; Breen, Lucas; Clifton, James; Gajdosik, Martina Srajer; Gaso-Sokac, Dajana; Rucevic, Marijana; Müller, Egbert

    2013-01-01

    Sample displacement chromatography (SDC) in reversed-phase and ion-exchange modes was introduced approximately twenty years ago. This method was first used for the preparative purification of peptides and proteins. Recently, SDC in ion-exchange mode was also successfully used for enrichment of low abundance proteins from human plasma. In this paper, the use of SDC for the separation of plasma proteins in hydrophobic interaction mode is demonstrated. By use of two or more columns coupled in series during sample application, and subsequent elution of detached columns in parallel, additional separation of bound proteins was achieved. Further low-abundance, physiologically active proteins could be highly enriched and detected by ESI-MS/MS. PMID:22740472

  17. Separation of proteins from human plasma by sample displacement chromatography in hydrophobic interaction mode.

    PubMed

    Josic, Djuro; Breen, Lucas; Clifton, James; Gajdosik, Martina Srajer; Gaso-Sokac, Dajana; Rucevic, Marijana; Müller, Egbert

    2012-07-01

    Sample displacement chromatography (SDC) in reversed-phase and ion-exchange modes was introduced approximately 20 years ago. This method was first used for the preparative purification of peptides and proteins. Recently, SDC in ion-exchange mode was also successfully used for enrichment of low-abundance proteins from human plasma. In this paper, the use of SDC for the separation of plasma proteins in hydrophobic interaction mode is demonstrated. By use of two or more columns coupled in series during sample application, and subsequent elution of detached columns in parallel, additional separation of bound proteins was achieved. Further low-abundance, physiologically active proteins could be highly enriched and detected by ESI-MS/MS.

  18. In vitro human chondrocyte culture on plasma-treated poly(glycerol sebacate) scaffolds.

    PubMed

    Theerathanagorn, Tharinee; Klangjorhor, Jeerawan; Sakulsombat, Morakot; Pothacharoen, Peraphan; Pruksakorn, Dumnoensun; Kongtawelert, Prachya; Janvikul, Wanida

    2015-01-01

    Porous poly(glycerol sebacate) (PGS) scaffolds were prepared using a salt leaching technique and subsequently surface modified by a low oxygen plasma treatment prior to the use in the in vitro culture of human chondrocytes. Condensation polymerization of glycerol and sebacic acid used at various mole ratios, i.e. 1:1, 1:1.25, and 1:1.5, was initially conducted to prepare PGS prepolymers. Porous elastomeric PGS scaffolds were directly fabricated from the mixtures of each prepolymer and 90% (w/w) NaCl particles and then subjected to the plasma treatment to enhance the surface hydrophilicity of the materials. The properties of both untreated and plasma-treated PGS scaffolds were comparatively evaluated, in terms of surface morphology, surface chemical composition, porosity, and storage modulus using scanning electron microscopy (SEM), X-ray photoelectron spectroscopy, micro-computed tomography, and dynamic mechanical analysis, respectively. The responses of chondrocytes cultured on individual PGS scaffolds were assessed, in terms of cell proliferation and ECM production. The results revealed that average pore sizes and porosity of the scaffolds were increased with an increasing sebacic acid concentration used. The storage moduli of the scaffolds were raised after the plasma treatment, possibly due to the further crosslinking of PGS upon treatment. Moreover, the scaffold prepared with a higher sebacic acid content demonstrated a greater capability of promoting cell infiltration, proliferation, and ECM production, especially when it was plasma-treated; the greatest HA, sGAG, uronic acid, and collagen contents were detected in matrix of this scaffold. The H & E and safranin O staining results also strongly supported this finding. The storage modulus of the scaffold was intensified after incubation with the chondrocytes for 21 days, indicating the accretion and retention of matrix ECM on the cell-cultured scaffold.

  19. In vitro human chondrocyte culture on plasma-treated poly(glycerol sebacate) scaffolds.

    PubMed

    Theerathanagorn, Tharinee; Klangjorhor, Jeerawan; Sakulsombat, Morakot; Pothacharoen, Peraphan; Pruksakorn, Dumnoensun; Kongtawelert, Prachya; Janvikul, Wanida

    2015-01-01

    Porous poly(glycerol sebacate) (PGS) scaffolds were prepared using a salt leaching technique and subsequently surface modified by a low oxygen plasma treatment prior to the use in the in vitro culture of human chondrocytes. Condensation polymerization of glycerol and sebacic acid used at various mole ratios, i.e. 1:1, 1:1.25, and 1:1.5, was initially conducted to prepare PGS prepolymers. Porous elastomeric PGS scaffolds were directly fabricated from the mixtures of each prepolymer and 90% (w/w) NaCl particles and then subjected to the plasma treatment to enhance the surface hydrophilicity of the materials. The properties of both untreated and plasma-treated PGS scaffolds were comparatively evaluated, in terms of surface morphology, surface chemical composition, porosity, and storage modulus using scanning electron microscopy (SEM), X-ray photoelectron spectroscopy, micro-computed tomography, and dynamic mechanical analysis, respectively. The responses of chondrocytes cultured on individual PGS scaffolds were assessed, in terms of cell proliferation and ECM production. The results revealed that average pore sizes and porosity of the scaffolds were increased with an increasing sebacic acid concentration used. The storage moduli of the scaffolds were raised after the plasma treatment, possibly due to the further crosslinking of PGS upon treatment. Moreover, the scaffold prepared with a higher sebacic acid content demonstrated a greater capability of promoting cell infiltration, proliferation, and ECM production, especially when it was plasma-treated; the greatest HA, sGAG, uronic acid, and collagen contents were detected in matrix of this scaffold. The H & E and safranin O staining results also strongly supported this finding. The storage modulus of the scaffold was intensified after incubation with the chondrocytes for 21 days, indicating the accretion and retention of matrix ECM on the cell-cultured scaffold. PMID:26387514

  20. Quantification of human immunodeficiency virus type 1 RNA from dried plasma spots collected on filter paper.

    PubMed Central

    Cassol, S; Gill, M J; Pilon, R; Cormier, M; Voigt, R F; Willoughby, B; Forbes, J

    1997-01-01

    To assess dried plasma spots (DPSs) as a source of material for virus quantification, human immunodeficiency virus type 1 (HIV-1) RNA levels were quantified in matched DPS and liquid plasma samples from 73 infected patients, including 5 neonates and 4 adult patients with acute HIV-1 infection. Quantifications were performed by commercially available assays (NASBA [nucleic acid sequence-based amplification] or Amplicor, or both). There was a strong correlation between HIV-1 RNA levels in plasma and DPSs. More importantly, there was no decline in HIV-1 RNA levels in DPSs stored for as long as 2 weeks at 20 degrees C. Similarly, storage of DPSs for 3 days at 37 degrees C resulted in no decrease in viral RNA levels. For patients with primary infection, the DPS method allowed for the measurement of RNA levels in plasma during the initial spike in the level of viremia and in the subsequent period of suppressed viral replication. DPS quantification was equally informative in the neonatal setting, with all five newborns showing HIV-1 RNA loads of greater than 4.991 log10 copies/ml. We conclude that the viral RNA levels in DPSs are equivalent to those measured in fresh-frozen plasma. The ease and economy of DPS sampling, the minute volumes required, and the unexpected stability of dried RNA suggest that the use of DPSs will be particularly valuable for small-volume neonatal samples and large, population-based studies in which cold storage and transportation present special problems, as is often the case in developing countries. The ability to measure viral changes during primary infection suggests that the method will be useful for assessing vaccine efficacy in large field trials. PMID:9350736

  1. Direct measurement by single photon counting of lipid hydroperoxides in human plasma and lipoproteins.

    PubMed

    Zamburlini, A; Maiorino, M; Barbera, P; Roveri, A; Ursini, F

    1995-11-20

    A single photon counting procedure for measuring lipid hydroperoxides in human plasma or LDL-VLDL, escaping from extraction and chromatography, is described. This appears to be a relevant procedure because the recovery of phospholipid hydroperoxides from plasma is a critical point which, in our hands, was limited and poorly reproducible. The sample is added to a reaction mixture containing luminol, hemin, and Triton X-100 in an alkaline buffer, the photon emission is recorded, and the data are processed using the monoexponential decay of the photon emission rate. The measurement is applied to (a) plasma passed through a "desalting" cartridge to eliminate the small water-soluble antioxidants which inhibit the chemiluminescent process or (b) apo-B-containing lipoproteins (LDL-VLDL) isolated by heparin-Sepharose affinity chromatography. The content of lipid hydroperoxides is calculated using an internal calibration with palmitoyllinoleoylphosphatidylcholine hydroperoxide. This procedure, based on a single photon counting technology, was adopted to produce reliable results using samples from which inhibitors of the photon emission process have not been completely eliminated. The specificity of the signal for lipid hydroperoxides was validated by its complete disappearance following incubation of the sample with glutathione and phospholipid-hydroperoxide glutathione peroxidase (EC 1.11.1.12), the sole enzyme specific for all classes of lipid hydroperoxides in lipoproteins. The interassay variability was < 10%. The results indicated that the concentration of lipid hydroperoxides in the plasma of 20 healthy subjects was 353 +/- 78 nM. In different subjects, LDL-VLDL accounted for 40-80% of the lipid hydroperoxides in plasma. PMID:8600817

  2. Association between plasma selenium level and NRF2 target genes expression in humans.

    PubMed

    Reszka, Edyta; Wieczorek, Edyta; Jablonska, Ewa; Janasik, Beata; Fendler, Wojciech; Wasowicz, Wojciech

    2015-04-01

    Animal studies in rodent and in vitro studies indicate compensatory role of nuclear factor (erythroid-derived 2)-like (Nrf2) and Nrf2-regulated antioxidant and phase II biotransformation enzymes for the dietary selenium (Se) deficiency or for the loss of selenoproteins. To explore associations between plasma Se level and NRF2-regulated cytoprotective genes expression, an observational study was conducted in a population of 96 healthy non-smoking men living in Central Poland aged 18-83 years with relatively low plasma Se level. NRF2, KEAP2, CAT, EPHX1, GCLC, GCLM, GPX2, GSR, GSTA1, GSTM1, GSTP1, GSTT1, HMOX1, NQO1, PRDX1, SOD1, SOD2, TXNRD1 transcript levels in peripheral blood leukocytes and polymorphism of NRF2-617C/A (rs6721961) in blood genomic DNA were determined by means of quantitative real-time PCR. Mean plasma Se level was found to be 51.10±15.25μg/L (range 23.86-96.18μg/L). NRF2 mRNA level was positively correlated with expression of investigated NRF2-target genes. The multivariate linear regression adjusting for selenium status showed that plasma Se level was significantly inversely associated only with expression of GSTP1 (β-coef.=-0.270, p=0.009), PRDXR1 (β-coef.=-0.245, p=0.017) and SOD2 with an inverse trend toward significance (β-coef.=-0.186, p=0.074), but without an effect of NRF2 gene variants. NRF2 expression was inversely associated with age (r=-0.23, p=0.03) and body mass index (r=-0.29, p<0.001). The findings may suggest a possible link between plasma Se level and cytoprotective response at gene level in humans. PMID:25524402

  3. Quantification of Transthyretin Kinetic Stability in Human Plasma Using Subunit Exchange

    PubMed Central

    2015-01-01

    The transthyretin (TTR) amyloidoses are a group of degenerative diseases caused by TTR aggregation, requiring rate-limiting tetramer dissociation. Kinetic stabilization of TTR, by preferential binding of a drug to the native tetramer over the dissociative transition state, dramatically slows the progression of familial amyloid polyneuropathy. An established method for quantifying the kinetic stability of recombinant TTR tetramers in buffer is subunit exchange, in which tagged TTR homotetramers are added to untagged homotetramers at equal concentrations to measure the rate at which the subunits exchange. Herein, we report a subunit exchange method for quantifying the kinetic stability of endogenous TTR in human plasma. The subunit exchange reaction is initiated by the addition of a substoichiometric quantity of FLAG-tagged TTR homotetramers to endogenous TTR in plasma. Aliquots of the subunit exchange reaction, taken as a function of time, are then added to an excess of a fluorogenic small molecule, which immediately arrests further subunit exchange. After binding, the small molecule reacts with the TTR tetramers, rendering them fluorescent and detectable in human plasma after subsequent ion exchange chromatography. The ability to report on the extent of TTR kinetic stabilization resulting from treatment with oral tafamidis is important, especially for selection of the appropriate dose for patients carrying rare mutations. This method could also serve as a surrogate biomarker for the prediction of the clinical outcome. Subunit exchange was used to quantify the stabilization of WT TTR from senile systemic amyloidosis patients currently being treated with tafamidis (20 mg orally, once daily). TTR kinetic stability correlated with the tafamidis plasma concentration. PMID:24661308

  4. Protection by polyphenols of postprandial human plasma and low-density lipoprotein modification: the stomach as a bioreactor.

    PubMed

    Kanner, Joseph; Gorelik, Shlomit; Roman, Sirota; Kohen, Ron

    2012-09-12

    Recent studies dramatically showed that the removal of circulating modified low-density lipoprotein (LDL) results in complete prevention of atherosclerosis. The gastrointestinal tract is constantly exposed to food, some of it containing oxidized compounds. Lipid oxidation in the stomach was demonstrated by ingesting heated red meat in rats. Red wine polyphenols added to the rats' meat diet prevented lipid peroxidation in the stomach and absorption of malondialdehyde (MDA) in rat plasma. In humans, postprandial plasma MDA levels rose by 3-fold after a meal of red meat cutlets. MDA derived from meat consumption caused postprandial plasma LDL modification in human. The levels of plasma MDA showed a 75% reduction by consumption of red wine polyphenols during the meat meal. Locating the main biological site of action of polyphenols in the stomach led to a revision in the understanding of how antioxidants work in vivo and may help to elucidate the mechanism involved in the protective effects of polyphenols in human health.

  5. The Dipeptidyl Peptidase Family, Prolyl Oligopeptidase, and Prolyl Carboxypeptidase in the Immune System and Inflammatory Disease, Including Atherosclerosis

    PubMed Central

    Waumans, Yannick; Baerts, Lesley; Kehoe, Kaat; Lambeir, Anne-Marie; De Meester, Ingrid

    2015-01-01

    Research from over the past 20 years has implicated dipeptidyl peptidase (DPP) IV and its family members in many processes and different pathologies of the immune system. Most research has been focused on either DPPIV or just a few of its family members. It is, however, essential to consider the entire DPP family when discussing any one of its members. There is a substantial overlap between family members in their substrate specificity, inhibitors, and functions. In this review, we provide a comprehensive discussion on the role of prolyl-specific peptidases DPPIV, FAP, DPP8, DPP9, dipeptidyl peptidase II, prolyl carboxypeptidase, and prolyl oligopeptidase in the immune system and its diseases. We highlight possible therapeutic targets for the prevention and treatment of atherosclerosis, a condition that lies at the frontier between inflammation and cardiovascular disease. PMID:26300881

  6. Vacuolar carboxypeptidase Y of Saccharomyces cerevisiae is glycosylated, sorted and matured in the fission yeast Schizosaccharomyces pombe.

    PubMed

    Simeon, A; Egner, R; Gascon, S; Suarez-Rendueles, P

    1995-03-01

    Vacuolar carboxypeptidase Y of Saccharomyces cerevisiae (CPYsc) has been expressed in a Schizosaccharomyces pombe strain devoid of the endogenous equivalent peptidase, employing a 2 mu derived plasmid. Immunoblot analysis revealed that CPYsc produced in the fission yeast has a higher molecular mass than mature CPYsc produced by the budding yeast. CPYsc is glycosylated when expressed in S. pombe and uses four N-linked glycosylation sites as shown by endoglycosidase H digestion. Carbohydrate removal leads to a protein moiety which is indistinguishable in size from deglycosylated CPYsc produced by S. cerevisiae. CPYsc isolated from S. pombe soluble extracts is enzymatically active and thus is presumed to undergo correct proteolytic maturation. Subcellular fractionation experiments showed a cofractionation of CPYsc with the S. pombe endoproteinases PrA and PrB, suggesting that the protein is correctly sorted to the vacuole and that these peptidases might be responsible for zymogen activation.

  7. Synthesis of Mevalonate- and Fluorinated Mevalonate Prodrugs and Their in vitro Human Plasma Stability

    PubMed Central

    Kang, Soosung; Watanabe, Mizuki; Jacobs, JC; Yamaguchi, Masaya; Dahesh, Samira; Nizet, Victor; Leyh, Thomas S.; Silverman, Richard B.

    2014-01-01

    The mevalonate pathway is essential for the production of many important molecules in lipid biosynthesis. Inhibition of this pathway is the mechanism of statin cholesterol-lowering drugs, as well as the target of drugs to treat osteoporosis, to combat parasites, and to inhibit tumor cell growth. Unlike the human mevalonate pathway, the bacterial pathway appears to be regulated by diphosphomevalonate (DPM). Enzymes in the mevalonate pathway act to produce isopentenyl diphosphate, the product of the DPM decarboxylase reaction, utilize phosphorylated (charged) intermediates, which are poorly bioavailable. It has been shown that fluorinated DPMs (6-fluoro- and 6,6,6-trifluoro-5-diphosphomevalonate) are excellent inhibitors of the bacterial pathway; however, highly charged DPM and analogues are not bioavailable. To increase cellular permeability of mevalonate analogues, we have synthesized various prodrugs of mevalonate and 6-fluoro- and 6,6,6-trifluoromevalonate that can be enzymatically transformed to the corresponding DPM or fluorinated DPM analogues by esterases or amidases. To probe the required stabilities as potentially bioavailable prodrugs, we measured the half-lives of esters, amides, carbonates, acetals, and ketal promoieties of mevalonate and the fluorinated mevalonate analogues in human blood plasma. Stability studies showed that the prodrugs are converted to the mevalonates in human plasma with a wide range of half-lives. These studies provide stability data for a variety of prodrug options having varying stabilities and should be very useful in the design of appropriate prodrugs of mevalonate and fluorinated mevalonates. PMID:25461893

  8. A contaminant-free assessment of Endogenous Retroviral RNA in human plasma

    PubMed Central

    Karamitros, Timokratis; Paraskevis, Dimitrios; Hatzakis, Angelos; Psichogiou, Mina; Elefsiniotis, Ioannis; Hurst, Tara; Geretti, Anna-Maria; Beloukas, Apostolos; Frater, John; Klenerman, Paul; Katzourakis, Aris; Magiorkinis, Gkikas

    2016-01-01

    Endogenous retroviruses (ERVs) comprise 6–8% of the human genome. HERVs are silenced in most normal tissues, up-regulated in stem cells and in placenta but also in cancer and HIV-1 infection. Crucially, there are conflicting reports on detecting HERV RNA in non-cellular clinical samples such as plasma that suggest the study of HERV RNA can be daunting. Indeed, we find that the use of real-time PCR in a quality assured clinical laboratory setting can be sensitive to low-level proviral contamination. We developed a mathematical model for low-level contamination that allowed us to design a laboratory protocol and standard operating procedures for robust measurement of HERV RNA. We focus on one family, HERV-K HML-2 (HK2) that has been most recently active even though they invaded our ancestral genomes almost 30 millions ago. We extensively validated our experimental design on a model cell culture system showing high sensitivity and specificity, totally eliminating the proviral contamination. We then tested 236 plasma samples from patients infected with HIV-1, HCV or HBV and found them to be negative. The study of HERV RNA for human translational studies should be performed with extensively validated protocols and standard operating procedures to control the widespread low-level human DNA contamination. PMID:27640347

  9. Radioimmunoassay for bupropion in human plasma: comparison of tritiated and iodinated radioligands

    SciTech Connect

    Butz, R.F.; Smith, P.G.; Schroeder, D.H.; Findlay, J.W.

    1983-03-01

    We evaluated the potential usefulness of /sup 125/I-labeled p-hydroxybupropion in a direct radioimmunoassay for bupropion in human plasma as compared with a currently used (/sup 3/H)bupropion dextran-coated charcoal method. In both radioimmunoassay methods succinoylpropylbupropion antiserum was used that was highly specific for unchanged drug, cross reactivities with known bupropion metabolites being less than 0.3%. However, the use of /sup 125/I-labeled p-hydroxybupropion afforded greater sensitivity (0.3 microgram/L vs 0.6 microgram/L with (/sup 3/H)bupropion) and was readily adaptable to the more convenient polyethylene glycol separation method. Between-assay CVs were 3.8 to 12.2% (mean 7.6%) with the /sup 125/I-based radioimmunoassay and 5.1 to 11.5% (mean 7.5%) with the /sup 3/H-based assay. Agreement between the two radioimmunoassay determinations of buproprion in human plasma samples collected over a 60-h period after oral drug administration was excellent (slope . 1.086, r . 0.989). We find the /sup 125/I-based assay a convenient and suitable alternative to the (/sup 3/H)bupropion assay in pharmacokinetic studies in humans.

  10. A simple and sensitive HPLC method for quantification of the metabolin of meclofenoxate in human plasma.

    PubMed

    Ni, Bin; Zhang, Junren; Zou, Jianjun; Zhao, Wei; Li, JianHua

    2010-01-01

    A simple and sensitive high-performance liquid chromatographic method was developed for quantification of the metabolin of meclofenoxate, chlorophenoxyacetic acid, in human plasma. Ibuprofen was used as an internal standard. The present method used protein precipitation for extraction of chlorophenoxyacetic acid from human plasma. Separation was carried out on a reversed-phase C(18) column. The column effluent was monitored by UV detection at 254 nm. The mobile phase was a mixture of methanol and water containing 1.0% glacial acetic acid (70:30 v/v) at a flow rate of 1.0 mL/min. The column temperature was 20 degrees C. This method was linear over the range of 0.047-28.20 microg/mL with a regression coefficient greater than 0.99. The mean recovery of chlorophenoxyacetic acid and IS were (79.54 +/- 6.33)% and (78.48 +/- 2.14)%, respectively, and the method was found to be precise, accurate, and specific during the study. The method was successfully applied for pharmacokinetic study of chlorophenoxyacetic acid in human.

  11. Synthesis of mevalonate- and fluorinated mevalonate prodrugs and their in vitro human plasma stability.

    PubMed

    Kang, Soosung; Watanabe, Mizuki; Jacobs, J C; Yamaguchi, Masaya; Dahesh, Samira; Nizet, Victor; Leyh, Thomas S; Silverman, Richard B

    2015-01-27

    The mevalonate pathway is essential for the production of many important molecules in lipid biosynthesis. Inhibition of this pathway is the mechanism of statin cholesterol-lowering drugs, as well as the target of drugs to treat osteoporosis, to combat parasites, and to inhibit tumor cell growth. Unlike the human mevalonate pathway, the bacterial pathway appears to be regulated by diphosphomevalonate (DPM). Enzymes in the mevalonate pathway act to produce isopentenyl diphosphate, the product of the DPM decarboxylase reaction, utilize phosphorylated (charged) intermediates, which are poorly bioavailable. It has been shown that fluorinated DPMs (6-fluoro- and 6,6,6-trifluoro-5-diphosphomevalonate) are excellent inhibitors of the bacterial pathway; however, highly charged DPM and analogs are not bioavailable. To increase cellular permeability of mevalonate analogs, we have synthesized various prodrugs of mevalonate and 6-fluoro- and 6,6,6-trifluoromevalonate that can be enzymatically transformed to the corresponding DPM or fluorinated DPM analogs by esterases or amidases. To probe the required stabilities as potentially bioavailable prodrugs, we measured the half-lives of esters, amides, carbonates, acetals, and ketal promoieties of mevalonate and the fluorinated mevalonate analogs in human blood plasma. Stability studies showed that the prodrugs are converted to the mevalonates in human plasma with a wide range of half-lives. These studies provide stability data for a variety of prodrug options having varying stabilities and should be very useful in the design of appropriate prodrugs of mevalonate and fluorinated mevalonates.

  12. A contaminant-free assessment of Endogenous Retroviral RNA in human plasma.

    PubMed

    Karamitros, Timokratis; Paraskevis, Dimitrios; Hatzakis, Angelos; Psichogiou, Mina; Elefsiniotis, Ioannis; Hurst, Tara; Geretti, Anna-Maria; Beloukas, Apostolos; Frater, John; Klenerman, Paul; Katzourakis, Aris; Magiorkinis, Gkikas

    2016-01-01

    Endogenous retroviruses (ERVs) comprise 6-8% of the human genome. HERVs are silenced in most normal tissues, up-regulated in stem cells and in placenta but also in cancer and HIV-1 infection. Crucially, there are conflicting reports on detecting HERV RNA in non-cellular clinical samples such as plasma that suggest the study of HERV RNA can be daunting. Indeed, we find that the use of real-time PCR in a quality assured clinical laboratory setting can be sensitive to low-level proviral contamination. We developed a mathematical model for low-level contamination that allowed us to design a laboratory protocol and standard operating procedures for robust measurement of HERV RNA. We focus on one family, HERV-K HML-2 (HK2) that has been most recently active even though they invaded our ancestral genomes almost 30 millions ago. We extensively validated our experimental design on a model cell culture system showing high sensitivity and specificity, totally eliminating the proviral contamination. We then tested 236 plasma samples from patients infected with HIV-1, HCV or HBV and found them to be negative. The study of HERV RNA for human translational studies should be performed with extensively validated protocols and standard operating procedures to control the widespread low-level human DNA contamination. PMID:27640347

  13. A tandem Kunitz protease inhibitor (KPI106)-serine carboxypeptidase (SCP1) controls mycorrhiza establishment and arbuscule development in Medicago truncatula.

    PubMed

    Rech, Stefanie S; Heidt, Sven; Requena, Natalia

    2013-09-01

    Plant proteases and protease inhibitors are involved in plant developmental processes including those involving interactions with microbes. Here we show that a tandem between a Kunitz protease inhibitor (KPI106) and a serine carboxypeptidase (SCP1) controls arbuscular mycorrhiza development in the root cortex of Medicago truncatula. Both proteins are only induced during mycorrhiza formation and belong to large families whose members are also mycorrhiza-specific. Furthermore, the interaction between KPI106 and SCP1 analysed using the yeast two-hybrid system is specific, indicating that each family member might have a defined counterpart. In silico docking analysis predicted a putative P1 residue in KPI106 (Lys173) that fits into the catalytic pocket of SCP1, suggesting that KPI106 might inhibit the enzyme activity by mimicking the protease substrate. In vitro mutagenesis of the Lys173 showed that this residue is important in determining the strength and specificity of the interaction. The RNA interference (RNAi) inactivation of the serine carboxypeptidase SCP1 produces aberrant mycorrhizal development with an increased number of septated hyphae and degenerate arbuscules, a phenotype also observed when overexpressing KPI106. Protease and inhibitor are both secreted as observed when expressed in Nicotiana benthamiana epidermal cells. Taken together we envisage a model in which the protease SCP1 is secreted in the apoplast where it produces a peptide signal critical for proper fungal development within the root. KPI106 also at the apoplast would modulate the spatial and/or temporal activity of SCP1 by competing with the protease substrate.

  14. Crystal structure of the E. coli dipeptidyl carboxypeptidase Dcp: further indication of a ligand-dependent hinge movement mechanism.

    PubMed

    Comellas-Bigler, M; Lang, R; Bode, W; Maskos, K

    2005-05-27

    Dcp from Escherichia coli is a 680 residue cytoplasmic peptidase, which shows a strict dipeptidyl carboxypeptidase activity. Although Dcp had been assigned to the angiotensin I-converting enzymes (ACE) due to blockage by typical ACE inhibitors, it is currently grouped into the M3 family of mono zinc peptidases, which also contains the endopeptidases neurolysin and thimet oligopeptidase (TOP). We have cloned, expressed, purified, and crystallized Dcp in the presence of an octapeptide "inhibitor", and have determined its 2.0A crystal structure using MAD methods. The analysis revealed that Dcp consists of two half shell-like subdomains, which enclose an almost closed two-chamber cavity. In this cavity, two dipeptide products presumably generated by Dcp cleavage of the octapeptide bind to the thermolysin-like active site fixed to side-chains, which are provided by both subdomains. In particular, an Arg side-chain backed by a Glu residue, together with two Tyr phenolic groups provide a charged anchor for fixing the C-terminal carboxylate group of the P2' residue of a bound substrate, explaining the strict dipeptidyl carboxypeptidase specificity of Dcp. Tetrapeptidic substrates are fixed only via their main-chain functions from P2 to P2', suggesting a broad residue specificity for Dcp. Both subdomains exhibit very similar chain folds as the equivalent but abducted subdomains of neurolysin and TOP. Therefore, this "product-bound" Dcp structure seems to represent the inhibitor/substrate-bound "closed" form of the M3 peptidases, generated from the free "open" substrate-accessible form by a hinge-bending mechanism. A similar mechanism has recently been demonstrated experimentally for ACE2.

  15. Elucidation of N-glycosites within human plasma glycoproteins for cancer biomarker discovery.

    PubMed

    Drake, Penelope; Schilling, Birgit; Gibson, Brad; Fisher, Susan

    2013-01-01

    Glycans are an important class of post-translational modifications that decorate a wide array of protein substrates. These cell-type specific molecules, which are modulated during developmental and disease processes, are attractive biomarker candidates as biology regarding altered glycosylation can be used to guide the experimental design. The mass spectrometry (MS)-based workflow described here incorporates chromatography on affinity matrices formed from lectins, proteins that bind specific glycan motifs. The goal was to design a relatively simple method for the rapid analysis of small plasma volumes (e.g., clinical specimens). As increases in sialylation and fucosylation are prominent among cancer-associated modifications, we focused on Sambucus nigra agglutinin and AAL, which bind sialic acid- and fucose-containing structures, respectively. Positive controls (fucosylated and sialylated human lactoferrin glycopeptides), and negative controls (high-mannose glycopeptides from Saccharomyces cerevisiae invertase) were used to monitor the specificity of lectin capture and optimize the workflow. Multiple Affinity Removal System 14-depleted, trypsin-digested human plasma from healthy donors served as the target analyte. Samples were loaded onto the lectin columns and separated by high performance liquid chromatography (HPLC) into flow through and bound fractions, which were treated with PNGase F, an amidase that removes N-linked glycans and marks the underlying asparagine glycosite by a +1 Da mass shift. The deglycosylated peptide fractions were interrogated by HPLC ESI-MS/MS on a quadrupole time-of-flight mass spectrometer. The method allowed identification of 122 human plasma glycoproteins containing 247 unique glycosites. Notably, glycoproteins that circulate at ng/mL levels (e.g., cadherin-5 at 0.3-4.9 ng/mL, and neutrophil gelatinase-associated lipocalin which is present at ∼2.5 ng/mL) were routinely observed, suggesting that this method enables the detection of

  16. Inhibitory effects of kiwifruit extract on human platelet aggregation and plasma angiotensin-converting enzyme activity.

    PubMed

    Dizdarevic, Lili L; Biswas, Dipankar; Uddin, M D Main; Jørgenesen, Aud; Falch, Eva; Bastani, Nasser E; Duttaroy, Asim K

    2014-01-01

    Previous human studies suggest that supplementation with kiwifruits lowers several cardiovascular risk factors such as platelet hyperactivity, blood pressure and plasma lipids. The cardiovascular health benefit of fruit and vegetables is usually attributed to the complex mixture of phytochemicals therein; however, kiwifruit's cardioprotective factors are not well studied. In this study, we investigated the effects of kiwifruit extract on human blood platelet aggregation and plasma angiotensin-converting enzyme (ACE) activity. A sugar-free, heat-stable aqueous extract with molecular mass less than 1000 Da was prepared from kiwifruits. Typically, 100 g kiwifruits produced 66.3 ± 5.8 mg (1.2 ± 0.1 mg CE) of sugar-free kiwifruit extract (KFE). KFE inhibited both human platelet aggregation and plasma ACE activity in a dose-dependent manner. KFE inhibited platelet aggregation in response to ADP, collagen and arachidonic acid, and inhibitory action was mediated in part by reducing TxA2 synthesis. The IC50 for ADP-induced platelet aggregation was 1.6 ± 0.2 mg/ml (29.0 ± 3.0 μg CE/ml), whereas IC50 for serum ACE was 0.6 ± 0.1 mg/ml (11.0 ± 1.2 μg CE/ml). Consuming 500 mg of KFE (9.0 mg CE) in 10 g margarine inhibited ex vivo platelet aggregation by 12.7%, 2 h after consumption by healthy volunteers (n = 9). All these data indicate that kiwifruit contains very potent antiplatelet and anti-ACE components. Consuming kiwifruits might be beneficial as both preventive and therapeutic regime in cardiovascular disease. PMID:24219176

  17. Enzymatic degradation of in vitro Staphylococcus aureus biofilms supplemented with human plasma

    PubMed Central

    Watters, Chase M; Burton, Tarea; Kirui, Dickson K; Millenbaugh, Nancy J

    2016-01-01

    Enzymatic debridement is a therapeutic strategy used clinically to remove necrotic tissue from wounds. Some of the enzymes utilized for debridement have been tested against bacterial pathogens, but the effectiveness of these agents in dispersing clinically relevant biofilms has not been fully characterized. Here, we developed an in vitro Staphylococcus aureus biofilm model that mimics wound-like conditions and employed this model to investigate the antibiofilm activity of four enzymatic compounds. Human plasma at concentrations of 0%–50% was supplemented into growth media and used to evaluate biofilm biomass accumulation over 24 hours and 48 hours in one methicillin-sensitive and five methicillin-resistant strains of S. aureus. Supplementation of media with 10% human plasma resulted in the most robust biofilms in all six strains. The enzymes α-amylase, bromelain, lysostaphin, and papain were then tested against S. aureus biofilms cultured in 10% human plasma. Quantification of biofilms after 2 hours and 24 hours of treatment using the crystal violet assay revealed that lysostaphin decreased biomass by up to 76%, whereas α-amylase, bromelain, and papain reduced biomass by up to 97%, 98%, and 98%, respectively. Scanning electron microscopy confirmed that the dispersal agents detached the biofilm exopolysaccharide matrix and bacteria from the growth surface. Lysostaphin caused less visible dispersal of the biofilms, but unlike the other enzymes, induced morphological changes indicative of bacterial cell damage. Overall, our results indicate that use of enzymes may be an effective means of eradicating biofilms and a promising strategy to improve treatment of multidrug-resistant bacterial infections. PMID:27175088

  18. Enzymatic degradation of in vitro Staphylococcus aureus biofilms supplemented with human plasma.

    PubMed

    Watters, Chase M; Burton, Tarea; Kirui, Dickson K; Millenbaugh, Nancy J

    2016-01-01

    Enzymatic debridement is a therapeutic strategy used clinically to remove necrotic tissue from wounds. Some of the enzymes utilized for debridement have been tested against bacterial pathogens, but the effectiveness of these agents in dispersing clinically relevant biofilms has not been fully characterized. Here, we developed an in vitro Staphylococcus aureus biofilm model that mimics wound-like conditions and employed this model to investigate the antibiofilm activity of four enzymatic compounds. Human plasma at concentrations of 0%-50% was supplemented into growth media and used to evaluate biofilm biomass accumulation over 24 hours and 48 hours in one methicillin-sensitive and five methicillin-resistant strains of S. aureus. Supplementation of media with 10% human plasma resulted in the most robust biofilms in all six strains. The enzymes α-amylase, bromelain, lysostaphin, and papain were then tested against S. aureus biofilms cultured in 10% human plasma. Quantification of biofilms after 2 hours and 24 hours of treatment using the crystal violet assay revealed that lysostaphin decreased biomass by up to 76%, whereas α-amylase, bromelain, and papain reduced biomass by up to 97%, 98%, and 98%, respectively. Scanning electron microscopy confirmed that the dispersal agents detached the biofilm exopolysaccharide matrix and bacteria from the growth surface. Lysostaphin caused less visible dispersal of the biofilms, but unlike the other enzymes, induced morphological changes indicative of bacterial cell damage. Overall, our results indicate that use of enzymes may be an effective means of eradicating biofilms and a promising strategy to improve treatment of multidrug-resistant bacterial infections. PMID:27175088

  19. Inhibitory effects of kiwifruit extract on human platelet aggregation and plasma angiotensin-converting enzyme activity.

    PubMed

    Dizdarevic, Lili L; Biswas, Dipankar; Uddin, M D Main; Jørgenesen, Aud; Falch, Eva; Bastani, Nasser E; Duttaroy, Asim K

    2014-01-01

    Previous human studies suggest that supplementation with kiwifruits lowers several cardiovascular risk factors such as platelet hyperactivity, blood pressure and plasma lipids. The cardiovascular health benefit of fruit and vegetables is usually attributed to the complex mixture of phytochemicals therein; however, kiwifruit's cardioprotective factors are not well studied. In this study, we investigated the effects of kiwifruit extract on human blood platelet aggregation and plasma angiotensin-converting enzyme (ACE) activity. A sugar-free, heat-stable aqueous extract with molecular mass less than 1000 Da was prepared from kiwifruits. Typically, 100 g kiwifruits produced 66.3 ± 5.8 mg (1.2 ± 0.1 mg CE) of sugar-free kiwifruit extract (KFE). KFE inhibited both human platelet aggregation and plasma ACE activity in a dose-dependent manner. KFE inhibited platelet aggregation in response to ADP, collagen and arachidonic acid, and inhibitory action was mediated in part by reducing TxA2 synthesis. The IC50 for ADP-induced platelet aggregation was 1.6 ± 0.2 mg/ml (29.0 ± 3.0 μg CE/ml), whereas IC50 for serum ACE was 0.6 ± 0.1 mg/ml (11.0 ± 1.2 μg CE/ml). Consuming 500 mg of KFE (9.0 mg CE) in 10 g margarine inhibited ex vivo platelet aggregation by 12.7%, 2 h after consumption by healthy volunteers (n = 9). All these data indicate that kiwifruit contains very potent antiplatelet and anti-ACE components. Consuming kiwifruits might be beneficial as both preventive and therapeutic regime in cardiovascular disease.

  20. Enzymatic degradation of in vitro Staphylococcus aureus biofilms supplemented with human plasma.

    PubMed

    Watters, Chase M; Burton, Tarea; Kirui, Dickson K; Millenbaugh, Nancy J

    2016-01-01

    Enzymatic debridement is a therapeutic strategy used clinically to remove necrotic tissue from wounds. Some of the enzymes utilized for debridement have been tested against bacterial pathogens, but the effectiveness of these agents in dispersing clinically relevant biofilms has not been fully characterized. Here, we developed an in vitro Staphylococcus aureus biofilm model that mimics wound-like conditions and employed this model to investigate the antibiofilm activity of four enzymatic compounds. Human plasma at concentrations of 0%-50% was supplemented into growth media and used to evaluate biofilm biomass accumulation over 24 hours and 48 hours in one methicillin-sensitive and five methicillin-resistant strains of S. aureus. Supplementation of media with 10% human plasma resulted in the most robust biofilms in all six strains. The enzymes α-amylase, bromelain, lysostaphin, and papain were then tested against S. aureus biofilms cultured in 10% human plasma. Quantification of biofilms after 2 hours and 24 hours of treatment using the crystal violet assay revealed that lysostaphin decreased biomass by up to 76%, whereas α-amylase, bromelain, and papain reduced biomass by up to 97%, 98%, and 98%, respectively. Scanning electron microscopy confirmed that the dispersal agents detached the biofilm exopolysaccharide matrix and bacteria from the growth surface. Lysostaphin caused less visible dispersal of the biofilms, but unlike the other enzymes, induced morphological changes indicative of bacterial cell damage. Overall, our results indicate that use of enzymes may be an effective means of eradicating biofilms and a promising strategy to improve treatment of multidrug-resistant bacterial infections.

  1. Use of Human Plasma Samples to Identify Circulating Drug Metabolites that Inhibit Cytochrome P450 Enzymes.

    PubMed

    Eng, Heather; Obach, R Scott

    2016-08-01

    Drug interactions elicited through inhibition of cytochrome P450 (P450) enzymes are important in pharmacotherapy. Recently, greater attention has been focused on not only parent drugs inhibiting P450 enzymes but also on possible inhibition of these enzymes by circulating metabolites. In this report, an ex vivo method whereby the potential for circulating metabolites to be inhibitors of P450 enzymes is described. To test this method, seven drugs and their known plasma metabolites were added to control human plasma at concentrations previously reported to occur in humans after administration of the parent drug. A volume of plasma for each drug based on the known inhibitory potency and time-averaged concentration of the parent drug was extracted and fractionated by high-pressure liquid chromatography-mass spectrometry, and the fractions were tested for inhibition of six human P450 enzyme activities (CYP1A2, CYP2C8, CYP2C9, CYP2C19, CYP2D6, and CYP3A4). Observation of inhibition in fractions that correspond to the retention times of metabolites indicates that the metabolite has the potential to contribute to P450 inhibition in vivo. Using this approach, norfluoxetine, hydroxyitraconazole, desmethyldiltiazem, desacetyldiltiazem, desethylamiodarone, hydroxybupropion, erythro-dihydrobupropion, and threo-dihydrobupropion were identified as circulating metabolites that inhibit P450 activities at a similar or greater extent as the parent drug. A decision tree is presented outlining how this method can be used to determine when a deeper investigation of the P450 inhibition properties of a drug metabolite is warranted. PMID:27271369

  2. Human plasma contains cross-reactive Abeta conformer-specific IgG antibodies.

    PubMed

    O'Nuallain, Brian; Acero, Luis; Williams, Angela D; Koeppen, Helen P McWilliams; Weber, Alfred; Schwarz, Hans P; Wall, Jonathan S; Weiss, Deborah T; Solomon, Alan

    2008-11-25

    Two conformers of aggregated Abeta, i.e., fibrils and oligomers, have been deemed important in the pathogenesis of Alzheimer's disease. We now report that intravenous immune globulin (IVIG) derived from pools of human plasma contains IgGs that recognize conformational epitopes present on fibrils and oligomers, but not their soluble monomeric precursor. We have used affinity chromatography to isolate these antibodies and have shown that they cross-reacted with comparable nanomolar avidity with both types of Abeta aggregates; notably, binding was not inhibited by soluble Abeta monomers. Our studies provide further support for investigating the therapeutic use of IVIG in Alzheimer's disease.

  3. Comparison of functional aspects of the coagulation cascade in human and sea turtle plasmas.

    PubMed

    Soslau, Gerald; Wallace, Bryan; Vicente, Catherine; Goldenberg, Seth J; Tupis, Todd; Spotila, James; George, Robert; Paladino, Frank; Whitaker, Brent; Violetta, Gary; Piedra, Rotney

    2004-08-01

    Functional hemostatic pathways are critical for the survival of all vertebrates and have been evolving for more than 400 million years. The overwhelming majority of studies of hemostasis in vertebrates have focused on mammals with very sparse attention paid to reptiles. There have been virtually no studies of the coagulation pathway in sea turtles whose ancestors date back to the Jurassic period. Sea turtles are often exposed to rapidly altered environmental conditions during diving periods. This may reduce their blood pH during prolonged hypoxic dives. This report demonstrates that five species of turtles possess only one branch of the mammalian coagulation pathway, the extrinsic pathway. Mixing studies of turtle plasmas with human factor-deficient plasmas indicate that the intrinsic pathway factors VIII and IX are present in turtle plasma. These two factors may play a significant role in supporting the extrinsic pathway by feedback loops. The intrinsic factors, XI and XII are not detected which would account for the inability of reagents to induce coagulation via the intrinsic pathway in vitro. The analysis of two turtle factors, factor II (prothrombin) and factor X, demonstrates that they are antigenically/functionally similar to the corresponding human factors. The turtle coagulation pathway responds differentially to both pH and temperature relative to each turtle species and relative to human samples. The coagulation time (prothrombin time) increases as the temperature decreases between 37 and 15 degrees C. The increased time follows a linear relationship, with similar slopes for loggerhead, Kemps ridley and hawksbill turtles as well as for human samples. Leatherback turtle samples show a dramatic nonlinear increased time below 23 degrees C, and green turtle sample responses were similar but less dramatic. All samples also showed increased prothrombin times as the pH decreased from 7.8 to 6.4, except for three turtle species. The prothrombin times decreased

  4. Comparison of functional aspects of the coagulation cascade in human and sea turtle plasmas.

    PubMed

    Soslau, Gerald; Wallace, Bryan; Vicente, Catherine; Goldenberg, Seth J; Tupis, Todd; Spotila, James; George, Robert; Paladino, Frank; Whitaker, Brent; Violetta, Gary; Piedra, Rotney

    2004-08-01

    Functional hemostatic pathways are critical for the survival of all vertebrates and have been evolving for more than 400 million years. The overwhelming majority of studies of hemostasis in vertebrates have focused on mammals with very sparse attention paid to reptiles. There have been virtually no studies of the coagulation pathway in sea turtles whose ancestors date back to the Jurassic period. Sea turtles are often exposed to rapidly altered environmental conditions during diving periods. This may reduce their blood pH during prolonged hypoxic dives. This report demonstrates that five species of turtles possess only one branch of the mammalian coagulation pathway, the extrinsic pathway. Mixing studies of turtle plasmas with human factor-deficient plasmas indicate that the intrinsic pathway factors VIII and IX are present in turtle plasma. These two factors may play a significant role in supporting the extrinsic pathway by feedback loops. The intrinsic factors, XI and XII are not detected which would account for the inability of reagents to induce coagulation via the intrinsic pathway in vitro. The analysis of two turtle factors, factor II (prothrombin) and factor X, demonstrates that they are antigenically/functionally similar to the corresponding human factors. The turtle coagulation pathway responds differentially to both pH and temperature relative to each turtle species and relative to human samples. The coagulation time (prothrombin time) increases as the temperature decreases between 37 and 15 degrees C. The increased time follows a linear relationship, with similar slopes for loggerhead, Kemps ridley and hawksbill turtles as well as for human samples. Leatherback turtle samples show a dramatic nonlinear increased time below 23 degrees C, and green turtle sample responses were similar but less dramatic. All samples also showed increased prothrombin times as the pH decreased from 7.8 to 6.4, except for three turtle species. The prothrombin times decreased

  5. Saliva versus plasma bioequivalence of rusovastatin in humans: validation of class III drugs of the salivary excretion classification system.

    PubMed

    Idkaidek, Nasir; Arafat, Tawfiq

    2015-03-01

    Bioequivalence of rusovastatin in healthy human volunteers was done using saliva and plasma matrices in order to investigate the robustness of using saliva instead of plasma as a surrogate for bioequivalence of class III drugs according to the salivary excretion classification system (SECS). Saliva and plasma samples were collected for 72 h after oral administration of rusovastatin 40 mg to 12 healthy humans. Saliva and plasma pharmacokinetic parameters were calculated by non-compartmental analysis. Analysis of variance, 90 % confidence intervals, and intra-subject and inter-subject variability values of pharmacokinetic parameters were calculated using Kinetica program V5. Human effective intestinal permeability was also calculated by SimCYP program V13. Rusovastatin falls into class III (high permeability/low fraction unbound to plasma proteins) and hence was subjected to salivary excretion. A correlation coefficient of 0.99 between saliva and plasma concentrations, and a saliva/plasma concentration ratio of 0.175 were observed. The 90 % confidence limits of area under the curve (AUClast) and maximum concentration (C max) showed similar trends in both saliva and plasma. On the other hand, inter- and intra-subject variability values in saliva were higher than in plasma, leading to the need for a slightly higher number of subjects to be used in saliva studies. Non-invasive saliva sampling instead of the invasive plasma sampling method can be used as a surrogate for bioequivalence of SECS class III drugs when an adequate sample size is used.

  6. Human plasma concentrations of cytochrome P450 probes extrapolated from pharmacokinetics in cynomolgus monkeys using physiologically based pharmacokinetic modeling.

    PubMed

    Shida, Satomi; Utoh, Masahiro; Murayama, Norie; Shimizu, Makiko; Uno, Yasuhiro; Yamazaki, Hiroshi

    2015-01-01

    1. Cynomolgus monkeys are widely used in preclinical studies as non-human primate species. Pharmacokinetics of human cytochrome P450 probes determined in cynomolgus monkeys after single oral or intravenous administrations were extrapolated to give human plasma concentrations. 2. Plasma concentrations of slowly eliminated caffeine and R-/S-warfarin and rapidly eliminated omeprazole and midazolam previously observed in cynomolgus monkeys were scaled to human oral biomonitoring equivalents using known species allometric scaling factors and in vitro metabolic clearance data with a simple physiologically based pharmacokinetic (PBPK) model. Results of the simplified human PBPK models were consistent with reported experimental PK data in humans or with values simulated by a fully constructed population-based simulator (Simcyp). 3. Oral administrations of metoprolol and dextromethorphan (human P450 2D probes) in monkeys reportedly yielded plasma concentrations similar to their quantitative detection limits. Consequently, ratios of in vitro hepatic intrinsic clearances of metoprolol and dextromethorphan determined in monkeys and humans were used with simplified PBPK models to extrapolate intravenous PK in monkeys to oral PK in humans. 4. These results suggest that cynomolgus monkeys, despite their rapid clearance of some human P450 substrates, could be a suitable model for humans, especially when used in conjunction with simple PBPK models.

  7. [Simultaneous quantitative determination of amprenavir and indinavir in human plasma by high-performance liquid chromatography].

    PubMed

    Laussine, S; Roux, A; Delhotal-Landes, B; Flouvat, B

    2001-01-01

    A reversed-phase high-performance liquid chromatographic assay for the determination of the HIV protease inhibitors amprenavir (Agenerase) and indinavir (Crixivan) in human plasma is described, using a mobile phase consisting of 0.50 M phosphate buffer (adjusted to pH 5,5) - Milli-Q water - acetonitrile (120: 1,080: 800, v/v/v). A solid-phase extraction using C18 extraction columns (Discovery columns 100 mg, 1 ml Supelco) and a liquid-liquid extraction with 0.5 ml hydrogenocarbonate/carbonate buffer (adjusted to pH 10.6) and 6 ml methyl ter-butyl ether have been compared. The liquid-liquid extraction has been chosen to be easier and cheaper. The method has been validated over the range of 60 to 3,000 ng/ml for amprenavir and 20 to 3,000 ng/ml for indinavir using a 0.5 ml sample volume. The specificity, linearity, accuracy and precision have been studied. The limit of detection was respectively for amprenavir and indinavir 15 and 4 ng, and the limit of quantification was 60 and 20 ng/ml. Stability tests under various conditions were performed. This assay can readily be used in a hospital laboratory for the routine monitoring of plasma concentrations of amprenavir in HIV-infected patients. The trough plasma concentrations average has been determined in patients treated by amprenavir and indinavir for seven months. PMID:11282520

  8. Computer simulation of speciation of trivalent aluminum, gadolinium and yttrium ions in human blood plasma.

    PubMed

    Jakovljević, Ivan; Petrović, Đorđe; Joksović, Ljubinka; Lazarević, Ivan; Đurđević, Predrag

    2013-01-01

    The speciation of Al3+, Gd3+ and Y3+ ions in human plasma has been studied by computer simulation using the program HySS2009. A literature computer model of blood plasma was updated and comprised 9 metals, 43 ligands and over 6100 complexes. To this model critically evaluated data of Al3+, Gd3+ and Y3+ constants with blood plasma ligands have been added. Low molecular mass (LMM) speciation of Al3+ ion strongly depends upon the chosen equilibrium model of the metal - phosphate and metal - citrate systems. The obtained computer simulation of LMM speciation data of Al3+ ion were: AlPO4Cit (40.7%), AlPO4CitOH (22.9%), AlCitOH (19.2%) and AlPO4(OH) (12.7%) (% of total LMM Al species pool); for Gd3+ ion: GdAspCit (30%) and GdCit(OH)2 (20%) (% of total [Gd]) and for Y3+ ion: YCit (48%), Y(CO3)2 (32%) and Y(CO3) (11%) (% of total [Y]). Citrate appears as the important binding and mobilizing ligand for all examined ions, while the dominating species are the ternary ones.

  9. A rapid bioluminescence assay for measuring myeloperoxidase activity in human plasma.

    PubMed

    Goiffon, Reece J; Martinez, Sara C; Piwnica-Worms, David

    2015-02-10

    Myeloperoxidase (MPO) is a circulating cardiovascular disease (CVD) biomarker used to estimate clinical risk and patient prognosis. Current enzyme-linked immunosorbent assays (ELISA) for MPO concentration are costly and time-intensive. Here we report a novel bioluminescence assay, designated MPO activity on a polymer surface (MAPS), for measuring MPO activity in human plasma samples using the bioluminescent substrate L-012. The method delivers a result in under an hour and is resistant to confounding effects from endogenous MPO inhibitors. In a pilot clinical study, we compared MAPS and two clinical ELISAs using 72 plasma samples from cardiac catheterization patients. Results from parallel MAPS and ELISAs were concordant within 2±11 μg l(-1) MPO with similar uncertainty and reproducibility. Results between parallel MAPS and ELISA were in better agreement than those between independent ELISAs. MAPS may provide an inexpensive and rapid assay for determining MPO activity in plasma samples from patients with CVD or potentially other immune and inflammatory disorders.

  10. Electrically neutral microheterogeneity of human plasma transthyretin (prealbumin) detected by isoelectric focusing in urea gradients.

    PubMed

    Altland, K; Winter, P; Sauerborn, M K

    1999-06-01

    Mutants of the human plasma transthyretin (TTR, prealbumin) have attracted interest due to their rather frequent association with the autosomal dominant disease familial amyloidotic polyneuropathy (FAP). Some three quarters of known TTR mutations produce electrically neutral amino acid substitutions undetectable via separation by charge. We have developed an electrophoretic procedure sensitive to differences in the stability of tetramers and monomers under partially denaturing conditions. The differential folding states were found to be fully reversible. Applying the procedure we found 14 electrically silent mutants of TTR among 2000 plasma samples from German donors. We demonstrate that the normal TTR monomer exists in different forms of variable stability and/or charge due to binding of sulfhydryls from plasma to the unique cysteine at position 10 of the primary structure as well as due to modification by treatment with an oxidant. We found that reduction of Cys10 increases the stability of the folded monomeric and tetrameric conformations. The conformational changes of TTR induced by isoelectric focusing in a urea gradient were found to be associated by a gain of three positive charge units. Using published crystallographic data we present structural sites in the TTR molecule which could explain the observed effects.

  11. A rapid bioluminescence assay for measuring myeloperoxidase activity in human plasma

    PubMed Central

    Goiffon, Reece J.; Martinez, Sara C.; Piwnica-Worms, David

    2015-01-01

    Myeloperoxidase (MPO) is a circulating cardiovascular disease (CVD) biomarker used to estimate clinical risk and patient prognosis. Current enzyme-linked immunosorbent assays (ELISA) for MPO concentration are costly and time-intensive. Here we report a novel bioluminescence assay, designated MPO activity on a polymer surface (MAPS), for measuring MPO activity in human plasma samples using the bioluminescent substrate L-012. The method delivers a result in under an hour and is resistant to confounding effects from endogenous MPO inhibitors. In a pilot clinical study, we compared MAPS and two clinical ELISAs using 72 plasma samples from cardiac catheterization patients. Results from parallel MAPS and ELISAs were concordant within 2±11 μg l−1 MPO with similar uncertainty and reproducibility. Results between parallel MAPS and ELISA were in better agreement than those between independent ELISAs. MAPS may provide an inexpensive and rapid assay for determining MPO activity in plasma samples from patients with CVD or potentially other immune and inflammatory disorders. PMID:25666092

  12. Quantification of artemisinin in human plasma using liquid chromatography coupled to tandem mass spectrometry

    PubMed Central

    Lindegardh, N.; Tarning, J.; Toi, P.V.; Hien, T.T.; Farrar, J.; Singhasivanon, P.; White, N.J.; Ashton, M.; Day, N.P.J.

    2009-01-01

    A liquid chromatographic tandem mass spectroscopy method for the quantification of artemisinin in human heparinised plasma has been developed and validated. The method uses Oasis HLB™ μ-elution solid phase extraction 96-well plates to facilitate a high throughput of 192 samples a day. Artesunate (internal standard) in a plasma–water solution was added to plasma (50 μL) before solid phase extraction. Artemisinin and its internal standard artesunate were analysed by liquid chromatography and MS/MS detection on a Hypersil Gold C18 (100 mm × 2.1 mm, 5 μm) column using a mobile phase containing acetonitrile–ammonium acetate 10 mM pH 3.5 (50:50, v/v) at a flow rate of 0.5 mL/min. The method has been validated according to published FDA guidelines and showed excellent performance. The within-day, between-day and total precisions expressed as R.S.D., were lower than 8% at all tested quality control levels including the upper and lower limit of quantification. The limit of detection was 0.257 ng/mL for artemisinin and the calibration range was 1.03–762 ng/mL using 50 μL plasma. The method was free from matrix effects as demonstrated both graphically and quantitatively. PMID:19162422

  13. Analytical performance of nano-LC-SRM using nondepleted human plasma over an 18-month period.

    PubMed

    Song, Xiaomin; Amirkhani, Ardeshir; Wu, Jemma X; Pascovici, Dana; Zaw, Thiri; Xavier, Dylan; Clarke, Stephen J; Molloy, Mark P

    2016-08-01

    A standardized procedure for label-free nano-LC-SRM analysis of 32 high-medium abundance proteins from nondepleted human plasma was established and SRM data were acquired on 45 separate days for a control sample that was independently prepared on 39 distinct dates over an 18-month period (542 days). This case study enabled us to assess quantitative variance associated with nano-LC-SRM plasma analysis, mimicking experimental conditions that would be experienced with clinical trial biomarker studies. We assessed sample preparation variability attributed to different technicians and sample storage stability. Instrument performance varied over the 18-month period requiring ion path cleaning, so we assessed the impact of declining performance on specific peptide ion sensitivity and evaluated how various data normalization strategies could compensate for these changes. Our analysis demonstrated that while sample preparation was the main contributor for data variances when MS data were acquired within days, variability in SRM sensitivity was a far greater source of variance when data were acquired over a long period. The overall median multiplexed assay CV was 13% over the 18-month period. This case study is illustrative of large-scale plasma biomarker studies using nano-LC-SRM over extended periods and highlights aspects of bioanalysis that require careful attention to ensure reliable quantitation.

  14. Determination of linsidomine in human plasma by tandem LC-MS with ESI.

    PubMed

    Sutherland, F C; de Jager, A D; Swart, K J; Hundt, H K; Scanes, T; Hundt, A F

    2000-04-01

    A sensitive method for the determination of linsidomine in plasma was developed, using high-performance liquid chromatographic (HPLC) separation with tandem mass spectrometric detection. Linsidomine was derivatised with propyl chloroformate and extracted with tert-butyl methyl ether/1,2-dichloroethane (55:45, v/v), back-extracted into HCl (0.01 M) followed by alkalinisation and back-extraction into ether; the final ether extract evaporated, reconstituted in mobile phase and then separated on a Phenomenex Luna C18 (2) 5 micron 2.1 x 150 mm column with a mobile phase consisting of methanol water formic acid (98/100%) (400:600:0.05, v/v/v) at a flow-rate of 0.4 ml min(-1). Detection was achieved by a Finnigan MAT mass spectrometer (LCQ) at unit resolution in the selected reaction monitoring (SRM) mode monitoring the transition of the protonated molecular ion m/z 257.0 to the product ion m/z 86.0. The mean recovery for linsidomine was 51% with a lower limit of quantification of 0.70 ng/ml using 1 ml plasma for extraction. This LC-MS/MS method for the determination of linsidomine in human plasma allows for better specificity and a higher sample throughput than the traditional LC-UV methods. It also demonstrates the profound effect that the composition of acidic modifiers and matrix constituents can have on the electrospray ionisation (ESI) of the analyte.

  15. Extracellular vesicles are rapidly purified from human plasma by PRotein Organic Solvent PRecipitation (PROSPR)

    PubMed Central

    Gallart-Palau, Xavier; Serra, Aida; Wong, Andrew See Weng; Sandin, Sara; Lai, Mitchell K. P.; Chen, Christopher P.; Kon, Oi Lian; Sze, Siu Kwan

    2015-01-01

    Extracellular vesicles (EVs) such as exosomes and microvesicles mediate intercellular communication and regulate a diverse range of crucial biological processes. Host cells that are damaged, infected or transformed release biomarker-containing EVs into the peripheral circulation, where they can be readily accessed for use in diagnostic or prognostic testing. However, current methods of EV isolation from blood plasma are complex and often require relatively large sample volumes, hence are inefficient for widespread use in clinical settings. Here, we report a novel and inexpensive method of rapidly isolating EVs from small volumes of human blood plasma by PRotein Organic Solvent PRecipitation (PROSPR). PROSPR encompasses a rapid three-step protocol to remove soluble proteins from plasma via precipitation in cold acetone, leaving the lipid-encapsulated EVs behind in suspension. This generates higher purity EVs that can then be obtained from filtration or classical ultracentrifugation methods. We foresee that PROSPR-based purification of EVs will significantly accelerate the discovery of new disease biomarkers and the characterization of EVs with potential for clinical applications. PMID:26419333

  16. Simulation of Human Plasma Concentrations of Thalidomide and Primary 5-Hydroxylated Metabolites Explored with Pharmacokinetic Data in Humanized TK-NOG Mice.

    PubMed

    Nishiyama, Sayako; Suemizu, Hiroshi; Shibata, Norio; Guengerich, F Peter; Yamazaki, Hiroshi

    2015-11-16

    Plasma concentrations of thalidomide and primary 5-hydroxylated metabolites including 5,6-dihydroxythalidomide and glutathione (GSH) conjugate(s) were investigated in chimeric mice with highly "humanized" liver cells harboring cytochrome P450 3A5*1. Following oral administration of thalidomide (100 mg/kg), plasma concentrations of GSH conjugate(s) of 5-hydroxythalidomide were higher in humanized mice than in controls. Simulation of human plasma concentrations of thalidomide were achieved with a simplified physiologically based pharmacokinetic model in accordance with reported thalidomide concentrations. The results indicate that the pharmacokinetics in humans of GSH conjugate and/or catechol primary 5-hydroxylated thalidomide contributing in vivo activation can be estimated for the first time. PMID:26492539

  17. Simulation of Human Plasma Concentrations of Thalidomide and Primary 5-Hydroxylated Metabolites Explored with Pharmacokinetic Data in Humanized TK-NOG Mice.

    PubMed

    Nishiyama, Sayako; Suemizu, Hiroshi; Shibata, Norio; Guengerich, F Peter; Yamazaki, Hiroshi

    2015-11-16

    Plasma concentrations of thalidomide and primary 5-hydroxylated metabolites including 5,6-dihydroxythalidomide and glutathione (GSH) conjugate(s) were investigated in chimeric mice with highly "humanized" liver cells harboring cytochrome P450 3A5*1. Following oral administration of thalidomide (100 mg/kg), plasma concentrations of GSH conjugate(s) of 5-hydroxythalidomide were higher in humanized mice than in controls. Simulation of human plasma concentrations of thalidomide were achieved with a simplified physiologically based pharmacokinetic model in accordance with reported thalidomide concentrations. The results indicate that the pharmacokinetics in humans of GSH conjugate and/or catechol primary 5-hydroxylated thalidomide contributing in vivo activation can be estimated for the first time.

  18. Proteomic analysis identifies interleukin 11 regulated plasma membrane proteins in human endometrial epithelial cells in vitro

    PubMed Central

    2011-01-01

    Background During the peri-implantation period, the embryo adheres to an adequately prepared or receptive endometrial surface epithelium. Abnormal embryo adhesion to the endometrium results in embryo implantation failure and infertility. Endometrial epithelial cell plasma membrane proteins critical in regulating adhesion may potentially be infertility biomarkers or targets for treating infertility. Interleukin (IL) 11 regulates human endometrial epithelial cells (hEEC) adhesion. Its production is abnormal in women with infertility. The objective of the study was to identify IL11 regulated plasma membrane proteins in hEEC in vitro using a proteomic approach. Methods Using a 2D-differential in-gel electrophoresis (DIGE) electrophoresis combined with LCMS/MS mass spectrometry approach, we identified 20 unique plasma membrane proteins differentially regulated by IL11 in ECC-1 cells, a hEEC derived cell line. Two IL11 regulated proteins with known roles in cell adhesion, annexin A2 (ANXA2) and flotillin-1 (FLOT1), were validated by Western blot and immunocytochemistry in hEEC lines (ECC-1 and an additional cell line, Ishikawa) and primary hEEC. Flotilin-1 was further validated by immunohistochemistry in human endometrium throughout the menstrual cycle (n = 6-8/cycle). Results 2D-DIGE analysis identified 4 spots that were significantly different between control and IL11 treated group. Of these 4 spots, there were 20 proteins that were identified with LCMS/MS. Two proteins; ANXA2 and FLOT1 were chosen for further analyses and have found to be significantly up-regulated following IL11 treatment. Western blot analysis showed a 2-fold and a 2.5-fold increase of ANXA2 in hEEC membrane fraction of ECC-1 and Ishikawa cells respectively. Similarly, a 1.8-fold and a 2.3/2.4-fold increase was also observed for FLOT1 in hEEC membrane fraction of ECC-1 and Ishikawa cells respectively. In vitro, IL11 induced stronger ANXA2 expression on cell surface of primary hEEC and ECC-1 whilst

  19. Hemocompatible control of sulfobetaine-grafted polypropylene fibrous membranes in human whole blood via plasma-induced surface zwitterionization.

    PubMed

    Chen, Sheng-Han; Chang, Yung; Lee, Kueir-Rarn; Wei, Ta-Chin; Higuchi, Akon; Ho, Feng-Ming; Tsou, Chia-Chun; Ho, Hsin-Tsung; Lai, Juin-Yih

    2012-12-21

    In this work, the hemocompatibility of zwitterionic polypropylene (PP) fibrous membranes with varying grafting coverage of poly(sulfobetaine methacrylate) (PSBMA) via plasma-induced surface polymerization was studied. Charge neutrality of PSBMA-grafted layers on PP membrane surfaces was controlled by the low-pressure and atmospheric plasma treatment in this study. The effects of grafting composition, surface hydrophilicity, and hydration capability on blood compatibility of the membranes were determined. Protein adsorption onto the different PSBMA-grafted PP membranes from human fibrinogen solutions was measured by enzyme-linked immunosorbent assay (ELISA) with monoclonal antibodies. Blood platelet adhesion and plasma clotting time measurements from a recalcified platelet-rich plasma solution were used to determine if platelet activation depends on the charge bias of the grafted PSBMA layer. The charge bias of PSBMA layer deviated from the electrical balance of positively and negatively charged moieties can be well-controlled via atmospheric plasma-induced interfacial zwitterionization and was further tested with human whole blood. The optimized PSBMA surface graft layer in overall charge neutrality has a high hydration capability and keeps its original blood-inert property of antifouling, anticoagulant, and antithrmbogenic activities when it comes into contact with human blood. This work suggests that the hemocompatible nature of grafted PSBMA polymers by controlling grafting quality via atmospheric plasma treatment gives a great potential in the surface zwitterionization of hydrophobic membranes for use in human whole blood. PMID:23181727

  20. The comparison of DNA damage induced by micro DBD plasma and low energy electron for curing human diseases

    NASA Astrophysics Data System (ADS)

    Park, Yeunsoo

    2015-09-01

    It is well known that low energy electrons (LEE, especially below 10 eV) can generate DNA damage via indirect action named dissociative electron attachment (DEA). We can now explain some parts of the exact mechanism on DNA damage by LEE collision with direct ionization effect when cancer patients get the radiotherapy. It is kind of remarkable information in the field of radiation therapy. However, it is practically very difficult to directly apply this finding to human disease cure due to difficulty of LEE therapy actualization and request of further clinical studies. Recently, there is a novel challenge in plasma application, that is, how we can apply plasma technology to diagnosis and treatment of many serious diseases like cancer. Cold atmospheric pressure plasma (CAPP) is a very good source to apply to plasma medicine and bio-applications because of low temperature, low cost, and easy handling. Some scientists have already reported good results related to clinical plasma application. The purposes of this study are to further find out exact mechanisms of DNA damage by LEE at the molecular level, to verify new DNA damage like structural alteration on DNA subunits and to compare DNA damage by LEE and plasma source. We will keep expanding our study to DNA damage by plasma source to develop plasma-based new medical and biological applications. We will show some recent results, DNA damage by LEE and non-thermal plasma.

  1. Individual variation in macronutrient regulation measured by proton magnetic resonance spectroscopy of human plasma.

    PubMed

    Park, Youngja; Kim, Seoung Bum; Wang, Bing; Blanco, Roberto A; Le, Ngoc-Anh; Wu, Shaoxiong; Accardi, Carolyn J; Alexander, R Wayne; Ziegler, Thomas R; Jones, Dean P

    2009-07-01

    Proton nuclear magnetic resonance ((1)H-NMR) spectroscopy of plasma provides a global metabolic profiling method that shows promise for clinical diagnostics. However, cross-sectional studies are complicated by a lack of understanding of intraindividual variation, and this limits experimental design and interpretation of data. The present study determined the diurnal variation detected by (1)H NMR spectroscopy of human plasma. Data reduction methods revealed three time-of-day metabolic patterns, which were associated with morning, afternoon, and night. Major discriminatory regions for these time-of-day patterns included the various kinds of lipid signals (-CH(2)- and -CH(2)OCOR), and the region between 3 and 4 ppm heavily overlapped with amino acids that had alpha-CH and alpha-CH(2). The phasing and duration of time-of-day patterns were variable among individuals, apparently because of individual difference in food processing/digestion and absorption and clearance of macronutrient energy sources (fat, protein, carbohydrate). The times of day that were most consistent among individuals, and therefore most useful for cross-sectional studies, were fasting morning (0830-0930), postprandial afternoon (1430-1630), and nighttime samples (0430-0530). Importantly, the integrated picture of metabolism provided by (1)H-NMR spectroscopy of plasma suggests that this approach is suitable to study complex regulatory processes, including eating patterns/eating disorders, upper gastrointestinal functions (gastric emptying, pancreatic, biliary functions), and absorption/clearance of macronutrients. Hence, (1)H-NMR spectroscopy of plasma could provide a global metabolic tolerance test to assess complex processes involved in disease, including eating disorders and the range of physiological processes causing dysregulation of energy homeostasis.

  2. Choline intake and genetic polymorphisms influence choline metabolite concentrations in human breast milk and plasma123

    PubMed Central

    Fischer, Leslie M; da Costa, Kerry Ann; Galanko, Joseph; Sha, Wei; Stephenson, Brigitte; Vick, Julie; Zeisel, Steven H

    2010-01-01

    Background: Choline is essential for infant nutrition, and breast milk is a rich source of this nutrient. Common single nucleotide polymorphisms (SNPs) change dietary requirements for choline intake. Objective: The aim of this study was to determine whether total choline intake and/or SNPs influence concentrations of choline and its metabolites in human breast milk and plasma. Design: We gave a total of 103 pregnant women supplemental choline or a placebo from 18 wk gestation to 45 d postpartum and genotyped the women for 370 common SNPs. At 45 d postpartum, we measured choline metabolite concentrations in breast milk and plasma and assessed the dietary intake of choline by using a 3-d food record. Results: On average, lactating women in our study ate two-thirds of the recommended intake for choline (Adequate Intake = 550 mg choline/d). Dietary choline intake (no supplement) correlated with breast-milk phosphatidylcholine and plasma choline concentrations. A supplement further increased breast-milk choline, betaine, and phosphocholine concentrations and increased plasma choline and betaine concentrations. We identified 5 SNPs in MTHFR that altered the slope of the intake–metabolite concentration relations, and we identified 2 SNPs in PEMT that shifted these curves upward. Individuals who shared sets of common SNPs were outliers in plots of intake–metabolite concentration curves; we suggest that these SNPs should be further investigated to determine how they alter choline metabolism. Conclusion: Total intake of choline and genotype can influence the concentrations of choline and its metabolites in the breast milk and blood of lactating women and thereby affect the amount of choline available to the developing infant. This study was registered at clinicaltrials.gov as NCT00678925. PMID:20534746

  3. Physiologically relevant plasma d,l-homocysteine concentrations mobilize Cd from human serum albumin.

    PubMed

    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. PMID:27294530

  4. Determination of triapine, a ribonucleotide reductase inhibitor, in human plasma by liquid chromatography tandem mass spectrometry.

    PubMed

    Feng, Ye; Kunos, Charles A; Xu, Yan

    2015-09-01

    Triapine is an inhibitor of ribonucleotide reductase (RNR). Studies have shown that triapine significantly decreases the activity of RNR and enhanced the radiation-mediated cytotoxicity in cervical and colon cancer. In this work, we have developed and validated a selective and sensitive LC-MS/MS method for the determination of triapine in human plasma. In this method, 2-[(3-fluoro-2-pyridinyl)methylene] hydrazinecarbothioamide (NSC 266749) was used as the internal standard (IS); plasma samples were prepared by deproteinization with acetonitrile; tripaine and the IS were separated on a Waters Xbridge Shield RP18 column (3.5 µm; 2.1 × 50 mm) using a mobile phase containing 25.0% methanol and 75.0% ammonium bicarbonate buffer (10.0 mM, pH 8.50; v/v); column eluate was monitored by positive turbo-ionspray tandem mass spectrometry; and quantitation of triapine was carried out in multiple-reaction-monitoring mode. The method developed had a linear calibration range of 0.250-50.0 ng/mL with correlation coefficient of 0.999 for triapine in human plasma. The IS-normalized recovery and the IS-normalized matrix factor of triapine were 101-104% and 0.89-1.05, respectively. The accuracy expressed as percentage error and precision expressed as coefficient of variation were ≤±6 and ≤8%, respectively. The validated LC-MS/MS method was applied to the measurement of triapine in patient samples from a phase I clinical trial. PMID:25677991

  5. Physiologically relevant plasma d,l-homocysteine concentrations mobilize Cd from human serum albumin.

    PubMed

    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.

  6. Direct measurement of the precursors of adrenocorticotropin in human plasma by two-site immunoradiometric assay

    SciTech Connect

    Crosby, S.R.; Stewart, M.F.; Ratcliffe, J.G.; White, A.

    1988-12-01

    An immunoradiometric assay (IRMA) for the direct measurement of the precursors of ACTH in unextracted human plasma has been developed and evaluated clinically in normal subjects and patients with disorders of the hypothalamic-pituitary-adrenal axis. The IRMA is based on an iodinated monoclonal antibody to ACTH and a monoclonal antibody to gamma MSH coupled to Sephacryl S300. The assay detects only peptides containing both epitopes, i.e. POMC (31K) and pro-ACTH (22K). The reference standard was partially purified POMC from culture medium of human corticotroph adenoma cells. The detection limit (greater than +2.5SD of the 0 standard) was 2.0 pmol/L and the within-assay coefficient of variation was less than 10% between 29 and 2600 pmol/L. Plasma concentrations of ACTH precursor peptides in 11 normal subjects sampled at 0930 h ranged from 5-34 pmol/L. The concentrations in the patient groups studied were: 260-2300 pmol/L in 5 patients with the ectopic ACTH syndrome associated with small cell lung cancer, less than 2.0-104 pmol/L in 10 patients with pituitary-dependent Cushing's disease, 23 pmol/L in a patient with Nelson's syndrome, and 3.0-230 pmol/L in 5 patients with Addison's disease. We conclude that this IRMA offers a simple and reliable method for measuring ACTH precursors in unextracted plasma. The proportionately greater elevation of ACTH precursors compared to ACTH in patients with the ectopic ACTH syndrome associated with small cell lung cancer but not in pituitary-dependent Cushing's syndrome, suggests that this assay may be clinically useful.

  7. Determination of febuxostat in human plasma using ultra-performance liquid chromatography tandem mass spectrometry.

    PubMed

    Lukram, Ojikumar; Parmar, Shivaji; Hande, Amit

    2013-06-01

    A rapid and sensitive liquid chromatography-tandem mass spectrometry method has been developed and validated for the determination of febuxostat in human plasma. The liquid-liquid extraction technique was used for the extraction of febuxostat from human plasma using trandolapril as the internal standard (IS). Chromatography was performed on a ultra-performance liquid chromatography (UPLC) BEH C18, 50 mm X 2.1 mm, 1.7 µm particle size column, with the mobile phase consisting of 0.1% formic acid and acetonitrile (in a 25:75 ratio), followed by detection using mass spectrometry. The method involves a simple reversed isocratic chromatography condition and mass spectrometry detection, which enables detection at sub-microgram levels. The method was validated and the lower limit of quantification for febuxostat was found to be 0.075 µg/ml. The mean recovery for febuxostat ranged from 100.9 to 106.5%. This method increased the sensitivity and selectivity; resulting in high-throughput analysis of febuxostat using commercially available IS for pharmacokinetic, bioavailability, and bioequivalence studies, with a chromatographic run time of 1.5 min only.

  8. Flow induced dispersion analysis rapidly quantifies proteins in human plasma samples.

    PubMed

    Poulsen, Nicklas N; Andersen, Nina Z; Østergaard, Jesper; Zhuang, Guisheng; Petersen, Nickolaj J; Jensen, Henrik

    2015-07-01

    Rapid and sensitive quantification of protein based biomarkers and drugs is a substantial challenge in diagnostics and biopharmaceutical drug development. Current technologies, such as ELISA, are characterized by being slow (hours), requiring relatively large amounts of sample and being subject to cumbersome and expensive assay development. In this work a new approach for quantification based on changes in diffusivity is presented. The apparent diffusivity of an indicator molecule interacting with the protein of interest is determined by Taylor Dispersion Analysis (TDA) in a hydrodynamic flow system. In the presence of the analyte the apparent diffusivity of the indicator changes due to complexation. This change in diffusivity is used to quantify the analyte. This approach, termed Flow Induced Dispersion Analysis (FIDA), is characterized by being fast (minutes), selective (quantification is possible in a blood plasma matrix), fully automated, and being subject to a simple assay development. FIDA is demonstrated for quantification of the protein Human Serum Albumin (HSA) in human plasma as well as for quantification of an antibody against HSA. The sensitivity of the FIDA assay depends on the indicator-analyte dissociation constant which in favourable cases is in the sub-nanomolar to picomolar range for antibody-antigen interactions. PMID:26031223

  9. Ultra-performance liquid chromatographic determination of tocopherols and retinol in human plasma.

    PubMed

    Bell, Edward C; John, Mathew; Hughes, Rodney J; Pham, Thu

    2014-10-01

    A rapid, selective and sensitive ultra-performance liquid chromatography method has been developed for the detection and quantification of tocopherols and retinol in human plasma. Alpha-tocopherol, gamma-tocopherol and retinol are assayed using fluorescence detection. Excitation/emission wavelengths are 295/330 nm and 325/470 nm for the analysis of both tocopherols and retinol, respectively. Retinol acetate is employed as the internal standard. The reversed-phase method incorporates gradient elution with a mobile phase consisting of methanol and acetonitrile. Separation of vitamin compounds is achieved using a bridged ethyl hybrid C18 column. The retention times for retinol, retinol acetate, gamma-tocopherol and alpha-tocopherol are 1.6, 1.8, 3.9 and 4.3 min, respectively. The limits of quantification for retinol, gamma-tocopherol and alpha-tocopherol were 0.02, 0.02 and 0.1 µg/mL, respectively. The assay method is suitable for the analysis of tocopherols and retinol in human plasma. The method may be applied following the ingestion of foods fortified with these fat-soluble vitamins.

  10. High-Throughput Quantitative Lipidomics Analysis of Nonesterified Fatty Acids in Human Plasma.

    PubMed

    Christinat, Nicolas; Morin-Rivron, Delphine; Masoodi, Mojgan

    2016-07-01

    We present a high-throughput, nontargeted lipidomics approach using liquid chromatography coupled to high-resolution mass spectrometry for quantitative analysis of nonesterified fatty acids. We applied this method to screen a wide range of fatty acids from medium-chain to very long-chain (8 to 24 carbon atoms) in human plasma samples. The method enables us to chromatographically separate branched-chain species from their straight-chain isomers as well as separate biologically important ω-3 and ω-6 polyunsaturated fatty acids. We used 51 fatty acid species to demonstrate the quantitative capability of this method with quantification limits in the nanomolar range; however, this method is not limited only to these fatty acid species. High-throughput sample preparation was developed and carried out on a robotic platform that allows extraction of 96 samples simultaneously within 3 h. This high-throughput platform was used to assess the influence of different types of human plasma collection and preparation on the nonesterified fatty acid profile of healthy donors. Use of the anticoagulants EDTA and heparin has been compared with simple clotting, and only limited changes have been detected in most nonesterified fatty acid concentrations. PMID:27185515

  11. Analysis of Amisulpride in Human Plasma by SPE and LC with Fluorescence Detection

    PubMed Central

    Kudris, Illia V.; Skakun, Nona N.; Orlova, Irina N.; Libina, Victoria V.

    2011-01-01

    A rapid, precise, accurate, and selective high-performance liquid chromatographic method with fluorescence detection has been validated and used for analysis of amisulpride in human plasma after a simple solid-phase extraction procedure. Compounds were separated on a CN column with 0.03 M potassium dihydrogen phosphate (pH 6.5)–acetonitrile 65:35 (v/v) as mobile phase. Fluorescence detection was performed at excitation and emission wavelengths of 274 and 370 nm, respectively. Calibration plots were linear over the concentration range 10–1,000 ng mL−1 in human plasma, and the lower limit of quantification was 10 ng mL−1. Accuracy was between 0.4 and 6.4% and precision was between 3.1 and 7.5%. Amisulpride was sufficiently stable through three freeze–thaw cycles, during storage for 6 h at room temperature, and for 2 months at −22 °C. The method is suitable for the analysis of clinical samples from pharmacokinetic studies. PMID:21423321

  12. Plasma Membrane Abundance of Human Aquaporin 5 Is Dynamically Regulated by Multiple Pathways.

    PubMed

    Kitchen, Philip; Öberg, Fredrik; Sjöhamn, Jennie; Hedfalk, Kristina; Bill, Roslyn M; Conner, Alex C; Conner, Matthew T; Törnroth-Horsefield, Susanna

    2015-01-01

    Aquaporin membrane protein channels mediate cellular water flow. Human aquaporin 5 (AQP5) is highly expressed in the respiratory system and secretory glands where it facilitates the osmotically-driven generation of pulmonary secretions, saliva, sweat and tears. Dysfunctional trafficking of AQP5 has been implicated in several human disease states, including Sjögren's syndrome, bronchitis and cystic fibrosis. In order to investigate how the plasma membrane expression levels of AQP5 are regulated, we studied real-time translocation of GFP-tagged AQP5 in HEK293 cells. We show that AQP5 plasma membrane abundance in transfected HEK293 cells is rapidly and reversibly regulated by at least three independent mechanisms involving phosphorylation at Ser156, protein kinase A activity and extracellular tonicity. The crystal structure of a Ser156 phosphomimetic mutant indicates that its involvement in regulating AQP5 membrane abundance is not mediated by a conformational change of the carboxy-terminus. We suggest that together these pathways regulate cellular water flow. PMID:26569106

  13. A New Method to Simultaneously Quantify the Antioxidants: Carotenes, Xanthophylls, and Vitamin A in Human Plasma

    PubMed Central

    Colmán-Martínez, Mariel; Martínez-Huélamo, Miriam; Miralles, Esther; Estruch, Ramón; Lamuela-Raventós, Rosa M.

    2016-01-01

    A simple and accurate reversed phase high-performance liquid chromatography coupled with diode array detector (HPLC-DAD) method for simultaneously determining and quantifying the antioxidants carotenes, xanthophylls, and retinol in human plasma is presented in this paper. Compounds were extracted with hexane, a C30 column, and a mobile phase of methanol, methyl tert-butyl ether, and water were used for the separation of the compounds. A total of 8 carotenoids, 3 Z-β-carotene isomers, and 1 fat-soluble vitamin (retinol) were resolved within 72 min at a flow rate of 0.6 mL/min. Detection was achieved at 450 nm for carotenoids and 330 nm for retinol. To evaluate the effectiveness of themethod, it has been applied to an intervention study conducted on eight volunteers. Results. Limits of detection were between 0.1 μg/mL for lycopene and astaxanthin and 1.3 μg/mL for 15-Z-β-carotene. Recoveries were ranged between 89% and 113% for α-carotene and astaxanthin, respectively. Accuracy was between 90.7% and 112.2% and precision was between 1% and 15% RSD. In human plasma samples compounds studied were identified besides three lycopene isomers, demonstrated to be suitable for application in dietary intervention studies. Conclusions. Due to its accuracy, precision, selectivity, and reproducibility, this method is suitable to dietary habits and/or antioxidants status studies. PMID:26697143

  14. Plasma from human mothers of fetuses with severe arthrogryposis multiplex congenita causes deformities in mice

    PubMed Central

    Jacobson, Leslie; Polizzi, Agata; Morriss-Kay, Gillian; Vincent, Angela

    1999-01-01

    Arthrogryposis multiplex congenita (AMC) is characterized by fixed joint contractures and other deformities, sometimes resulting in fetal death. The cause is unknown in most cases, but some women with fetuses affected by severe AMC have serum antibodies that inhibit fetal acetylcholine receptor (AChR) function, and antibodies to fetal antigens might play a pathogenic role in other congenital disorders. To investigate this possibility, we have established a model by injecting pregnant mice with plasma from four anti-AChR antibody–positive women whose fetuses had severe AMC. We found that human antibodies can be transferred efficiently to the mouse fetus during the last few days of fetal life. Many of the fetuses of dams injected with AMC maternal plasmas or Ig were stillborn and showed fixed joints and other deformities. Moreover, similar changes were found in mice after injection of a serum from one anti-AChR antibody–negative mother who had had four AMC fetuses. Thus, we have confirmed the role of maternal antibodies in cases of AMC associated with maternal anti-AChR, and we have demonstrated the existence of pathogenic maternal factors in one other case. Importantly, this approach can be used to look at the effects of other maternal human antibodies on development of the fetus. PMID:10194476

  15. Plasma Membrane Abundance of Human Aquaporin 5 Is Dynamically Regulated by Multiple Pathways

    PubMed Central

    Kitchen, Philip; Öberg, Fredrik; Sjöhamn, Jennie; Hedfalk, Kristina; Bill, Roslyn M.; Conner, Alex C.; Conner, Matthew T.; Törnroth-Horsefield, Susanna

    2015-01-01

    Aquaporin membrane protein channels mediate cellular water flow. Human aquaporin 5 (AQP5) is highly expressed in the respiratory system and secretory glands where it facilitates the osmotically-driven generation of pulmonary secretions, saliva, sweat and tears. Dysfunctional trafficking of AQP5 has been implicated in several human disease states, including Sjögren’s syndrome, bronchitis and cystic fibrosis. In order to investigate how the plasma membrane expression levels of AQP5 are regulated, we studied real-time translocation of GFP-tagged AQP5 in HEK293 cells. We show that AQP5 plasma membrane abundance in transfected HEK293 cells is rapidly and reversibly regulated by at least three independent mechanisms involving phosphorylation at Ser156, protein kinase A activity and extracellular tonicity. The crystal structure of a Ser156 phosphomimetic mutant indicates that its involvement in regulating AQP5 membrane abundance is not mediated by a conformational change of the carboxy-terminus. We suggest that together these pathways regulate cellular water flow. PMID:26569106

  16. Rapid and sensitive determination of nalmefene in human plasma by gas chromatography-mass spectrometry.

    PubMed

    Xie, Shan; Suckow, Raymond F; Mason, Barbara J; Allen, David; Cooper, Thomas B

    2002-06-25

    A rapid gas chromatography-mass spectrometric method for the determination of nalmefene in human plasma is described. The procedure involves protein precipitation, extraction with ethanol-chloroform mixture and derivatization with pentafluropropionic anhydride. The deuterated analog of nalmefene, 6beta-naltrexol-d(7), was used as the internal standard. Quantitation was achieved on a HP-1 column (12 mx0.2 mm I.D.) with negative chemical ionization (NCI) using methane:ammonia (95:5) as the reagent gas. The standard curves were fitted using a quadratic equation with the curve encompassing a range of 0.5 to 200 ng/ml, and the intra- and inter-assay variations for three different nalmefene levels were less than 10% throughout. The limit of quantitation was found to be 0.5 ng/ml. The method described is highly specific and reproducible, and could also be applied for the determination of naltrexone and 6beta-naltrexol. Application of the method to actual human plasma samples is demonstrated.

  17. Efficient Methods To Isolate Human Monoclonal Antibodies from Memory B Cells and Plasma Cells.

    PubMed

    Corti, Davide; Lanzavecchia, Antonio

    2014-10-01

    In this article, we highlight the advantages of isolating human monoclonal antibodies from the human memory B cells and plasma cell repertoires by using high-throughput cellular screens. Memory B cells are immortalized with high efficiency using Epstein-Barr virus (EBV) in the presence of a toll-like receptor (TLR) agonist, while plasma cells are maintained in single-cell cultures by using interleukin 6 (IL-6) or stromal cells. In both cases, multiple parallel assays, including functional assays, can be used to identify rare cells that produce antibodies with unique properties. Using these methods, we have isolated potent and broadly neutralizing antibodies against a variety of viruses, in particular, a pan-influenza-A-neutralizing antibody and an antibody that neutralizes four different paramyxoviruses. Given the high throughput and the possibility of directly screening for function (rather than just binding), these methods are instrumental to implement a target-agnostic approach to identify the most effective antibodies and, consequently, the most promising targets for vaccine design. This approach is exemplified by the identification of unusually potent cytomegalovirus-neutralizing antibodies that led to the identification of the target, a pentameric complex that we are developing as a candidate vaccine. PMID:26104354

  18. [Determination of ambroxol and clenbuterol in human plasma by LC-MS/MS method].

    PubMed

    Lin, Nan; Chen, Xiao-Yan; Song, Bo; Zhong, Da-Fang

    2007-03-01

    Ambroxol and clenbuterol were extracted from human plasma samples by liquid-liquid extraction, ambroxol was separated on a Zorbax XDB-C18 column and detected by tandem mass spectrometry with an atmospheric pressure chemical ionization interface after oral administration of a compound preparation. Clenbuterol was separated on a Zorbax XDB-C8 column and detected by tandem mass spectrometry with an electrospray ionization interface. Diphenhydramine is used as the internal standard. The linear concentration ranges of the calibration curves for ambroxol and clenbuterol were 0.080 - 400 microg x L(-1) and 5.0 - 5 000 ng x L(-1), respectively. The lower limits of quantification were 0.080 microg x L(-1) for ambroxol and 5.0 ng x L(-1) for clenbuterol, individually. The inter-day and intra-day precision (RSD) across three validation run over the entire concentration range was below 7.5%, and the accuracy (RE) was within +/- 2.5% for both ambroxol and clenbuterol. The methods were used to determine the pharmacokinetic parameters of ambroxol and clenbuterol in human plasma after oral administration of a compound preparation containing 60 mg ambroxol hydrochloride and 40 microg clenbuterol hydrochloride. The method was proved to be highly sensitive, selective and suitable for the pharmacokinetic study of different compound preparations containing ambroxol and clenbuterol.

  19. A New Method to Simultaneously Quantify the Antioxidants: Carotenes, Xanthophylls, and Vitamin A in Human Plasma.

    PubMed

    Colmán-Martínez, Mariel; Martínez-Huélamo, Miriam; Miralles, Esther; Estruch, Ramón; Lamuela-Raventós, Rosa M

    2015-01-01

    A simple and accurate reversed phase high-performance liquid chromatography coupled with diode array detector (HPLC-DAD) method for simultaneously determining and quantifying the antioxidants carotenes, xanthophylls, and retinol in human plasma is presented in this paper. Compounds were extracted with hexane, a C30 column, and a mobile phase of methanol, methyl tert-butyl ether, and water were used for the separation of the compounds. A total of 8 carotenoids, 3 Z-β-carotene isomers, and 1 fat-soluble vitamin (retinol) were resolved within 72 min at a flow rate of 0.6 mL/min. Detection was achieved at 450 nm for carotenoids and 330 nm for retinol. To evaluate the effectiveness of themethod, it has been applied to an intervention study conducted on eight volunteers. Results. Limits of detection were between 0.1 μg/mL for lycopene and astaxanthin and 1.3 μg/mL for 15-Z-β-carotene. Recoveries were ranged between 89% and 113% for α-carotene and astaxanthin, respectively. Accuracy was between 90.7% and 112.2% and precision was between 1% and 15% RSD. In human plasma samples compounds studied were identified besides three lycopene isomers, demonstrated to be suitable for application in dietary intervention studies. Conclusions. Due to its accuracy, precision, selectivity, and reproducibility, this method is suitable to dietary habits and/or antioxidants status studies. PMID:26697143

  20. High precision quantification of human plasma proteins using the automated SISCAPA Immuno-MS workflow.

    PubMed

    Razavi, Morteza; Leigh Anderson, N; Pope, Matthew E; Yip, Richard; Pearson, Terry W

    2016-09-25

    Efficient robotic workflows for trypsin digestion of human plasma and subsequent antibody-mediated peptide enrichment (the SISCAPA method) were developed with the goal of improving assay precision and throughput for multiplexed protein biomarker quantification. First, an 'addition only' tryptic digestion protocol was simplified from classical methods, eliminating the need for sample cleanup, while improving reproducibility, scalability and cost. Second, methods were developed to allow multiplexed enrichment and quantification of peptide surrogates of protein biomarkers representing a very broad range of concentrations and widely different molecular masses in human plasma. The total workflow coefficients of variation (including the 3 sequential steps of digestion, SISCAPA peptide enrichment and mass spectrometric analysis) for 5 proteotypic peptides measured in 6 replicates of each of 6 different samples repeated over 6 days averaged 3.4% within-run and 4.3% across all runs. An experiment to identify sources of variation in the workflow demonstrated that MRM measurement and tryptic digestion steps each had average CVs of ∼2.7%. Because of the high purity of the peptide analytes enriched by antibody capture, the liquid chromatography step is minimized and in some cases eliminated altogether, enabling throughput levels consistent with requirements of large biomarker and clinical studies.

  1. An antigenic study of human plasma cells in normal tissue and in myeloma: identification of a novel plasma cell associated antigen.

    PubMed Central

    Nathan, P D; Walker, L; Hardie, D; Richardson, P; Khan, M; Johnson, G D; Ling, N R

    1986-01-01

    A mouse monoclonal antibody named BU11 which detects an antigen strongly expressed on human plasma cells is described. The antibody stains plasma cells in tonsil sections, fresh and cultured plasmacytoid cells from the bone marrow of patients with multiple myeloma and cells of the plasmacytoid cell line RPMI 8226 used as the immunogen. In vitro studies of pokeweed mitogen (PWM) stimulated peripheral blood B cells and Epstein-Barr virus (EBV) stimulated tonsil B cells show that the antigen is present mainly on cells coexpressing the OKT10 antigen and containing cytoplasmic immunoglobulin (cIg). The BU11 antigen is expressed weakly on some normal B cells and is not present on T cells, monocytes or granulocytes. The antigen is of molecular weight 58kD under reducing conditions and is biochemically distinct from previously described plasma cell antigens. Images Fig. 4 PMID:3024883

  2. Effect of antigravity suit inflation on cardiovascular, PRA, and PVP responses in humans. [Plasma Renin Activity and Plasma VasoPressin

    NASA Technical Reports Server (NTRS)

    Kravik, S. E.; Keil, L. C.; Geelen, G.; Wade, C. E.; Barnes, P. R.

    1986-01-01

    The effects of lower body and abdominal pressure, produced by antigravity suit inflation, on blood pressure, pulse rate, fluid and electrolyte shift, plasma vasopressin and plasma renin activity in humans in upright postures were studied. Five men and two women stood upright for 3 hr with the suit being either inflated or uninflated. In the control tests, the suit was inflated only during the latter part of the trials. Monitoring was carried out with a sphygnomanometer, with sensors for pulse rates, and using a photometer and osmometer to measure blood serum characteristics. The tests confirmed earlier findings that the anti-g suit eliminates increases in plasma renin activity. Also, the headward redistribution of blood obtained in the tests commends the anti-g suit as an alternative to water immersion or bed rest for initial weightlessness studies.

  3. Dextrose-mediated aggregation of therapeutic monoclonal antibodies in human plasma: Implication of isoelectric precipitation of complement proteins.

    PubMed

    Luo, Shen; Zhang, Baolin

    2015-01-01

    Many therapeutic monoclonal antibodies (mAbs) are clinically administered through intravenous infusion after mixing with a diluent, e.g., saline, 5% dextrose. Such a clinical setting increases the likelihood of interactions among mAb molecules, diluent, and plasma components, which may adversely affect product safety and efficacy. Avastin® (bevacizumab) and Herceptin® (trastuzumab), but not Remicade® (infliximab), were shown to undergo rapid aggregation upon dilution into 5% dextrose when mixed with human plasma in vitro; however, the biochemical pathways leading to the aggregation were not clearly defined. Here, we show that dextrose-mediated aggregation of Avastin or Herceptin in plasma involves isoelectric precipitation of complement proteins. Using mass spectrometry, we found that dextrose-induced insoluble aggregates were composed of mAb itself and multiple abundant plasma proteins, namely complement proteins C3, C4, factor H, fibronectin, and apolipoprotein. These plasma proteins, which are characterized by an isoelectronic point of 5.5-6.7, lost solubility at the resulting pH in the mixture with formulated Avastin (pH 6.2) and Herceptin (pH 6.0). Notably, switching formulation buffers for Avastin (pH 6.2) and Remicade (pH 7.2) reversed their aggregation profiles. Avastin formed little, if any, insoluble aggregates in dextrose-plasma upon raising the buffer pH to 7.2 or above. Furthermore, dextrose induced pH-dependent precipitation of plasma proteins, with massive insoluble aggregates being detected at pH 6.5-6.8. These data show that isoelectric precipitation of complement proteins is a prerequisite of dextrose-induced aggregation of mAb in human plasma. This finding highlights the importance of assessing the compatibility of a therapeutic mAb with diluent and human plasma during product development. PMID:26338058

  4. Dextrose-mediated aggregation of therapeutic monoclonal antibodies in human plasma: Implication of isoelectric precipitation of complement proteins.

    PubMed

    Luo, Shen; Zhang, Baolin

    2015-01-01

    Many therapeutic monoclonal antibodies (mAbs) are clinically administered through intravenous infusion after mixing with a diluent, e.g., saline, 5% dextrose. Such a clinical setting increases the likelihood of interactions among mAb molecules, diluent, and plasma components, which may adversely affect product safety and efficacy. Avastin® (bevacizumab) and Herceptin® (trastuzumab), but not Remicade® (infliximab), were shown to undergo rapid aggregation upon dilution into 5% dextrose when mixed with human plasma in vitro; however, the biochemical pathways leading to the aggregation were not clearly defined. Here, we show that dextrose-mediated aggregation of Avastin or Herceptin in plasma involves isoelectric precipitation of complement proteins. Using mass spectrometry, we found that dextrose-induced insoluble aggregates were composed of mAb itself and multiple abundant plasma proteins, namely complement proteins C3, C4, factor H, fibronectin, and apolipoprotein. These plasma proteins, which are characterized by an isoelectronic point of 5.5-6.7, lost solubility at the resulting pH in the mixture with formulated Avastin (pH 6.2) and Herceptin (pH 6.0). Notably, switching formulation buffers for Avastin (pH 6.2) and Remicade (pH 7.2) reversed their aggregation profiles. Avastin formed little, if any, insoluble aggregates in dextrose-plasma upon raising the buffer pH to 7.2 or above. Furthermore, dextrose induced pH-dependent precipitation of plasma proteins, with massive insoluble aggregates being detected at pH 6.5-6.8. These data show that isoelectric precipitation of complement proteins is a prerequisite of dextrose-induced aggregation of mAb in human plasma. This finding highlights the importance of assessing the compatibility of a therapeutic mAb with diluent and human plasma during product development.

  5. Differential partitioning of antioxidants, including hydroxytyrosol, in human plasma and LDL: implications for their antioxidant activity in vivo.

    PubMed

    Visioli, Francesco; Colombo, Claudio; Galli, Claudio

    2012-05-01

    In vivo studies of LDL oxidation following consumption of natural phenolic compounds have yielded mixed results. It is reported that the amphiphilic hydroxytyrosol, after addition to human plasma, does not accumulate in LDL but protects plasma lipids, which are extracted together with hydroxytyrosol, from chemically-induced oxidation. Thus, a novel methodology was proposed, which does not rely on LDL separation and subsequent oxidation but is based on the oxidation of total lipids - simultaneously extracted from plasma with antioxidants - to evaluate the effects of micronutrients that do not partition into LDL, after in vivo supplementation.

  6. Protein binding of fentanyl and its metabolite nor-fentanyl in human plasma, albumin and α-1 acid glycoprotein.

    PubMed

    Bista, Sudeep Raj; Haywood, Alison; Hardy, Janet; Lobb, Michael; Tapuni, Angela; Norris, Ross

    2015-03-01

    1.Fentanyl is a highly lipophilic opioid commonly used to treat cancer pain. Plasma protein binding (PPB) of fentanyl in human plasma is reported as 80-85%, however it is unclear whether fentanyl binds primarily to albumin (ALB) or α-1 acid glycoprotein (AAG) and no studies have been conducted on the metabolite, nor-fentanyl. Fentanyl is also known to bind to plasticware and ultrafiltration (UF) devices which impacts adversely on binding experiments. 2.PPB of fentanyl and nor-fentanyl to ALB and AAG in isotonic phosphate buffer solution and seeded human plasma was quantified. PPB was also performed in plasma samples obtained from cancer patients receiving transdermal fentanyl. The adsorption of fentanyl and nor-fentanyl to UF devices and plasticware commonly used in PPB studies was also assessed. 3.Fentanyl was shown to bind primarily to ALB as opposed to AAG, with nor-fentanyl exhibiting negligible binding to plasma proteins. Total PPB of fentanyl was 86-89% in seeded human plasma. PPB in 56 cancer patient samples was 95.1 ± 3.52% for fentanyl and 32.4 ± 21.9% for nor-fentanyl. 4.UF was shown to be a reliable and convenient method for PPB studies, thereby removing the need for complex testing for adsorption of the drug to plasticware during UF.

  7. 3D Structure of Sulfolobus solfataricus Carboxypeptidase Developed by Molecular Modeling is Confirmed by Site-Directed Mutagenesis and Small Angle X-Ray Scattering

    PubMed Central

    Occhipinti, Emanuela; Martelli, Pier Luigi; Spinozzi, Francesco; Corsi, Federica; Formantici, Cristina; Molteni, Laura; Amenitsch, Heintz; Mariani, Paolo; Tortora, Paolo; Casadio, Rita

    2003-01-01

    Sulfolobus solfataricus carboxypeptidase (CPSso) is a thermostable zinc-metalloenzyme with a Mr of 43,000. Taking into account the experimentally determined zinc content of one ion per subunit, we developed two alternative 3D models, starting from the available structures of Thermoactinomyces vulgaris carboxypeptidase (Model A) and Pseudomonas carboxypeptidase G2 (Model B). The former enzyme is monomeric and has one metal ion in the active site, while the latter is dimeric and has two bound zinc ions. The two models were computed by exploiting the structural alignment of the one zinc- with the two zinc-containing active sites of the two templates, and with a threading procedure. Both computed structures resembled the respective template, with only one bound zinc with tetrahedric coordination in the active site. With these models, two different quaternary structures can be modeled: one using Model A with a hexameric symmetry, the other from Model B with a tetrameric symmetry. Mutagenesis experiments directed toward the residues putatively involved in metal chelation in either of the models disproved Model A and supported Model B, in which the metal-binding site comprises His108, Asp109, and His168. We also identified Glu142 as the acidic residue interacting with the water molecule occupying the fourth chelation site. Furthermore, the overall fold and the oligomeric structure of the molecule was validated by small angle x-ray scattering (SAXS). An ab initio original approach was used to reconstruct the shape of the CPSso in solution from the experimental curves. The results clearly support a tetrameric structure. The Monte Carlo method was then used to compare the crystallographic coordinates of the possible quaternary structures for CPSso with the SAXS profiles. The fitting procedure showed that only the model built using the Pseudomonas carboxypeptidase G2 structure as a template fitted the experimental data. PMID:12885660

  8. Relationship between plasma IGF-I levels, in vitro correlates of immunity, and human senescence.

    PubMed

    Krishnaraj, R; Zaks, A; Unterman, T

    1998-09-01

    Insulin-like growth factor-I (IGF-I) is a polypeptide mitogen which is regulated by growth hormone (GH). IGF-I mediates many of the biological functions of GH, including the maintenance of lymphoid mass and functions. Since GH secretion declines with age, we asked whether changes in the availability of IGF-I might contribute to age-associated alterations in immune functions. As a first step, we examined relationships between plasma levels of IGF-I and in vitro correlates of immunity in young and elderly subjects. Heparinized plasma and lymphocytes were collected from the peripheral blood of 34 healthy young (aged 27 +/- 0.9 years, mean +/- SEM) and 41 elderly (79 +/- 1.3 years) volunteers (31 males and 44 females in total). Plasma levels of IGF-I, measured by radioimmunoassay after the removal of IGF-I-binding proteins, were reduced among elders compared to young controls (138 +/- 8.7 ng/mL vs 80.2 +/- 4.7 ng/mL, P < 0.001). The number of circulating lymphocytes did not change with age. The proliferative response ([3H]thymidine uptake into DNA) of T-cells to concanavalin A and B-cells to pokeweed mitogen were reduced among elders (P < 0.05). An increased spontaneous antitumor natural killer (NK) activity (P < 0.001) was accompanied by a higher percentage of CD16(+) NK cells among lymphocytes in older subjects (P < 0.001). The NK cell number was positively related to IGF-I levels in young volunteers but not among elders. Correlation analysis demonstrated a highly significant relationship between plasma IGF-I levels and T-cell (but not B-cell) proliferative response during aging (r = 0.492, P < 0.001). Our results imply that reduced immunocompetence may be one of the consequences of reduced IGF-I levels in human aging. Among the three types of immune cells tested, the T-cells were most sensitive to fluctuations in IGF-I levels. Reduced IGF-I availability may be one of the determinants of the decline in T-cell-mediated immune function in the elderly. To our knowledge

  9. LC-MS/MS for the simultaneous analysis of arachidonic acid and 32 related metabolites in human plasma: Basal plasma concentrations and aspirin-induced changes of eicosanoids.

    PubMed

    Shinde, Dhananjay D; Kim, Kwon-Bok; Oh, Kyung-Suk; Abdalla, Nagi; Liu, Kwang-Hyeon; Bae, Soo Kyung; Shon, Ji-Hong; Kim, Ho-Sook; Kim, Dong-Hyun; Shin, Jae Gook

    2012-12-12

    Eicosanoids play an important role in various biological responses and can be used as biomarkers for specific diseases. Therefore, we developed a highly selective, sensitive, and robust liquid chromatography-tandem mass spectrometric method to measure arachidonic acid and its 32 metabolites in human plasma. Sample preparation involved solid phase extraction, which efficiently removed sources of interference present in human plasma. Chromatographic separation was performed using a Luna C(8)-column with 0.5mM ammonium formate buffer and acetonitrile as the mobile phase under gradient conditions. Detection was performed using tandem mass spectrometry equipped with an electrospray ionization interface in negative ion mode. The matrix did not affect the reproducibility and reliability of the assay. All analytes showed good linearity over the investigated concentration range (r>0.997). The validated lower limit of quantitation for the analytes ranged from 10 to 400pg/mL. Intra- and inter-day precision (RDS%) over the concentration ranges for all eicosanoids were within 16.8%, and accuracy ranged between 88.1 and 108.2%. This assay was suitable for the determination of basal plasma levels of eicosanoids and the evaluation of effect of aspirin on eicosanoid plasma levels in healthy subjects. PMID:23217314

  10. Lipid-protein interactions in plasma membranes of fiber cells isolated from the human eye lens.

    PubMed

    Raguz, Marija; Mainali, Laxman; O'Brien, William J; Subczynski, Witold K

    2014-03-01

    The protein content in human lens membranes is extremely high, increases with age, and is higher in the nucleus as compared with the cortex, which should strongly affect the organization and properties of the lipid bilayer portion of intact membranes. To assess these effects, the intact cortical and nuclear fiber cell plasma membranes isolated from human lenses from 41- to 60-year-old donors were studied using electron paramagnetic resonance spin-labeling methods. Results were compared with those obtained for lens lipid membranes prepared from total lipid extracts from human eyes of the same age group [Mainali, L., Raguz, M., O'Brien, W. J., and Subczynski, W. K. (2013) Biochim. Biophys. Acta]. Differences were considered to be mainly due to the effect of membrane proteins. The lipid-bilayer portions of intact membranes were significantly less fluid than lipid bilayers of lens lipid membranes, prepared without proteins. The intact membranes were found to contain three distinct lipid environments termed the bulk lipid domain, boundary lipid domain, and trapped lipid domain. However, the cholesterol bilayer domain, which was detected in cortical and nuclear lens lipid membranes, was not detected in intact membranes. The relative amounts of bulk and trapped lipids were evaluated. The amount of lipids in domains uniquely formed due to the presence of membrane proteins was greater in nuclear membranes than in cortical membranes. Thus, it is evident that the rigidity of nuclear membranes is greater than that of cortical membranes. Also the permeability coefficients for oxygen measured in domains of nuclear membranes were significantly lower than appropriate coefficients measured in cortical membranes. Relationships between the organization of lipids into lipid domains in fiber cells plasma membranes and the organization of membrane proteins are discussed.

  11. Lipid-Protein Interactions in Plasma Membranes of Fiber Cells Isolated from the Human Eye Lens

    PubMed Central

    Raguz, Marija; Mainali, Laxman; O’Brien, William J.; Subczynski, Witold K.

    2014-01-01

    The protein content in human lens membranes is extremely high, increases with age, and is higher in the nucleus as compared with the cortex, which should strongly affect the organization and properties of the lipid bilayer portion of intact membranes. To assess these effects, the intact cortical and nuclear fiber cell plasma membranes isolated from human lenses from 41- to 60-year-old donors were studied using electron paramagnetic resonance spin-labeling methods. Results were compared with those obtained for lens lipid membranes prepared from total lipid extracts from human eyes of the same age group [Mainali,L., Raguz, M., O’Brien, W. J., and Subczynski, W. K. (2013) Biochim. Biophys. Acta]. Differences were considered to be mainly due to the effect of membrane proteins. The lipid-bilayer portions of intact membranes were significantly less fluid than lipid bilayers of lens lipid membranes, prepared without proteins. The intact membranes were found to contain three distinct lipid environments termed the bulk lipid domain, boundary lipid domain, and trapped lipid domain. However, the cholesterol bilayer domain, which was detected in cortical and nuclear lens lipid membranes, was not detected in intact membranes. The relative amounts of bulk and trapped lipids were evaluated. The amount of lipids in domains uniquely formed due to the presence of membrane proteins was greater in nuclear membranes than in cortical membranes. Thus, it is evident that the rigidity of nuclear membranes is greater than that of cortical membranes. Also the permeability coefficients for oxygen measured in domains of nuclear membranes were significantly lower than appropriate coefficients measured in cortical membranes. Relationships between the organization of lipids into lipid domains in fiber cells plasma membranes and the organization of membrane proteins are discussed. PMID:24486794

  12. Development and validation of an UPLC-PDA method to quantify daptomycin in human plasma and in dried plasma spots.

    PubMed

    Baietto, Lorena; D'Avolio, Antonio; Pace, Simone; Simiele, Marco; Marra, Cristina; Ariaudo, Alessandra; Di Perri, Giovanni; De Rosa, Francesco Giuseppe

    2014-01-01

    Quantification of daptomycin in plasma samples may be useful in optimizing therapy especially in special patients' population. Nevertheless, therapeutic drug monitoring of daptomycin is still limited probably for the low number of laboratories which perform this analysis and for high shipment costs. We developed and validated a new UPLC-PDA method to quantify daptomycin in plasma and in dried plasma spots (DPS) collected on dried sample spots devices (DSSD). Daptomycin and quinoxaline, used as internal standard, were monitored at 262nm and 253nm, respectively. Daptomycin was extracted from plasma using acetonitrile and from DPS using an extraction solution (ethyl acetate-acetic acid-acetone-water; 50:20:20:10, v/v/v/v). Both assays were linear over the calibration range of 0.781 to 200μg/ml. Considering the method of extraction from plasma, mean intra and inter-day accuracy was -1.18% and -2.79%, respectively. Mean intra and inter-day precision was 7.91% and 9.22%, respectively. Regarding the extraction method from DPS, mean intra and inter-day accuracy was 2.21% and 2.41%, respectively. Mean intra and inter-day precision was 8.01% and 9.19%, respectively. Daptomycin in DPS was found to be stable for 7 days at room temperature (20-25°C) and for at least 30 days at 4°C. A statistically significant (p<0.001) linear correlation was found between daptomycin extracted from plasma and from DPS (r(2)=0.919). DPS represents a safe and cheap strategy to store and ship plasma samples. Thus, it is suited for pharmacokinetic studies and therapeutic drug monitoring of daptomycin in hospitals without a therapeutic drug monitoring laboratory.

  13. NtSCP1 from Tobacco Is an Extracellular Serine Carboxypeptidase III That Has an Impact on Cell Elongation1[C][W][OA

    PubMed Central

    Bienert, Manuela Désirée; Delannoy, Mélanie; Navarre, Catherine; Boutry, Marc

    2012-01-01

    The leaf extracellular space contains several peptidases, most of which are of unknown function. We isolated cDNAs for two extracellular serine carboxypeptidase III genes from tobacco (Nicotiana tabacum), NtSCP1 and NtSCP2, belonging to a phylogenetic clade not yet functionally characterized in plants. NtSCP1 and NtSCP2 are orthologs derived from the two ancestors of tobacco. Reverse transcription-polymerase chain reaction analysis showed that NtSCP1 and NtSCP2 are expressed in root, stem, leaf, and flower tissues. Expression analysis of the β-glucuronidase reporter gene fused to the NtSCP1 transcription promoter region confirmed this expression profile. Western blotting of NtSCP1 and expression of an NtSCP1-green fluorescent protein fusion protein showed that the protein is located in the extracellular space of tobacco leaves and culture cells. Purified His-tagged NtSCP1 had carboxypeptidase activity in vitro. Transgenic tobacco plants overexpressing NtSCP1 showed a reduced flower length due to a decrease in cell size. Etiolated seedlings of these transgenic plants had shorter hypocotyls. These data provide support for a role of an extracellular type III carboxypeptidase in the control of cell elongation. PMID:22214816

  14. Plasma clearance of human low-density lipoprotein in human apolipoprotein B transgenic mice is related to particle diameter.

    PubMed

    Berneis, Kaspar; Shames, David M; Blanche, Patricia J; La Belle, Michael; Rizzo, Manfredi; Krauss, Ronald M

    2004-04-01

    To test for intrinsic differences in metabolic properties of low-density lipoprotein (LDL) as a function of particle size, we examined the kinetic behavior of 6 human LDL fractions ranging in size from 251 to 265 A injected intravenously into human apolipoprotein (apo) B transgenic mice. A multicompartmental model was formulated and fitted to the data by standard nonlinear regression using the Simulation, Analysis and Modeling (SAAM II) program. Smaller sized LDL particles (251 to 257 A) demonstrated a significantly slower fractional catabolic rate (FCR) (0.050 +/- 0.045 h(-1)) compared with particles of larger size (262 to 265 A) (0.134 +/- -0.015 h(-1), P <.03), and there was a significant correlation between FCR and the peak LDL diameter of the injected fractions (R(2) =.71, P <.034). The sum of the equilibration parameters, k(2,1) and k(1,2), for smaller LDL (0.255 h(-1) and 0.105 h(-1), respectively) was significantly smaller than that for larger LDL (0.277 h(-1) and 0.248 h(-1), respectively; P <.01), indicative of slower intravascular-extravascular exchange for smaller LDL. Therefore in this mouse model, smaller LDL particles are cleared more slowly from plasma than larger LDL and are exchanged more slowly with the extravascular space. This might be due to compositional or structural features of smaller LDL that lead to retarded clearance.

  15. Sphingosine 1-phosphate, a bioactive sphingolipid abundantly stored in platelets, is a normal constituent of human plasma and serum.

    PubMed

    Yatomi, Y; Igarashi, Y; Yang, L; Hisano, N; Qi, R; Asazuma, N; Satoh, K; Ozaki, Y; Kume, S

    1997-05-01

    Although sphingosine 1-phosphate (Sph-1-P) is reportedly involved in diverse cellular processes and the physiological roles of this bioactive sphingolipid have been strongly suggested, few studies have revealed the presence of Sph-1-P in human samples, including body fluids and cells, under physiological conditions. In this study, we identified Sph-1-P as a normal constituent of human plasma and serum. The Sph-1-P levels in plasma and serum were 191+/-79 and 484+/-82 pmol/ml (mean+/-SD, n=8), respectively. Furthermore, when Sph-1-P was measured in paired plasma and serum samples obtained from 6 healthy adults, the serum Sph-1-P/plasma Sph-1-P ratio was found to be 2.65+/-1.26 (mean+/-SD). It is most likely that the source of discharged Sph-1-P during blood clotting is platelets, because platelets abundantly store Sph-1-P compared with other blood cells, and release part of their stored Sph-1-P extracellularly upon stimulation. We also studied Sph-1-P-related metabolism in plasma. [3H]Sph was stable and not metabolized at all in plasma, but was rapidly incorporated into platelets and metabolized mainly to Sph-1-P in platelet-rich plasma. [3H]Sph-1-P was found to be unchanged in plasma, revealing that plasma does not contain the enzymes needed for Sph-1-P degradation. In summary, platelets can convert Sph into Sph-1-P, and are storage sites for the latter in the blood. In view of the diverse biological effects of Sph-1-P, the release of Sph-1-P from activated platelets may be involved in a variety of physiological and pathophysiological processes, including thrombosis, hemostasis, atherosclerosis and wound healing.

  16. Studies on the Coumarin Anticoagulant Drugs: Interaction of Human Plasma Albumin and Warfarin Sodium*

    PubMed Central

    O'Reilly, Robert A.

    1967-01-01

    In studies by continuous flow electrophoresis the coumarin anticoagulant drug warfarin sodium was found to be bound solely to the albumin fraction of the plasma proteins. The interaction was studied in detail by equilibrium dialysis of solutions of crystalline human plasma albumin and warfarin sodium. Analysis of the data showed that albumin possesses a single strong binding site for warfarin with an association constant of 154,000 at 3° C and secondary classes of several sites with a much lower affinity. The free energy of binding for the first anion determined at 3° and 37° C was -6.54 and -7.01 kcal per mole, respectively. The standard enthalpy change for the interaction was -3.48 kcal per mole, and the entropy change was +11.2 U. The negative enthalpy change was surprisingly large and the positive entropy change small for an anion-albumin interaction, suggesting significant nonionic binding. The inability to saturate the albumin binding sites, even when high concentrations of warfarin were used, is consistent with a reversible configurational alteration of the albumin molecule during the binding process. The thermodynamic data indicate that the albumin binding sites for warfarin sodium are formed during the process of binding, rather than being performed as in antigen-antibody reactions. The strength of the binding process suggests that many of the pharmacodynamic characteristics of warfarin sodium in man are determined by its strong interaction with plasma albumin. Such correlations of the physicochemical interactions and biologic effects of the coumarin anticoagulant drugs should lead to a better understanding of their mechanisms of action. Images PMID:6025484

  17. Rapid quantitative method for the detection of phenylalanine and tyrosine in human plasma using pillar array columns and gradient elution.

    PubMed

    Song, Yanting; Takatsuki, Katsuya; Sekiguchi, Tetsushi; Funatsu, Takashi; Shoji, Shuichi; Tsunoda, Makoto

    2016-07-01

    This study reports a fast and quantitative determination method for phenylalanine (Phe) and tyrosine (Tyr) in human plasma using on-chip pressure-driven liquid chromatography. A pillar array column with low-dispersion turns and a gradient elution system was used. The separation of fluorescent derivatives of Phe, Tyr, and other hydrophobic amino acids was successfully performed within 140 s. Under the optimized conditions, Phe and Tyr in human plasma were quantified. The developed method is promising for rapid diagnosis in the clinical field.

  18. The forkhead transcription factor FOXP1 represses human plasma cell differentiation.

    PubMed

    van Keimpema, Martine; Grüneberg, Leonie J; Mokry, Michal; van Boxtel, Ruben; van Zelm, Menno C; Coffer, Paul; Pals, Steven T; Spaargaren, Marcel

    2015-10-29

    Expression of the forkhead transcription factor FOXP1 is essential for early B-cell development, whereas downregulation of FOXP1 at the germinal center (GC) stage is required for GC B-cell function. Aberrantly high FOXP1 expression is frequently observed in diffuse large B-cell lymphoma and mucosa-associated lymphoid tissue lymphoma, being associated with poor prognosis. Here, by gene expression analysis upon ectopic overexpression of FOXP1 in primary human memory B cells (MBCs) and B-cell lines, combined with chromatin immunoprecipitation and sequencing, we established that FOXP1 directly represses expression of PRDM1, IRF4, and XBP1, transcriptional master regulators of plasma cell (PC) differentiation. In accordance, FOXP1 is prominently expressed in primary human naive and MBCs, but expression strongly decreases during PC differentiation. Moreover, as compared with immunoglobulin (Ig) M(+) MBCs, IgG(+) MBCs combine lower expression of FOXP1 with an enhanced intrinsic PC differentiation propensity, and constitutive (over)expression of FOXP1 in B-cell lines and primary human MBCs represses their ability to differentiate into PCs. Taken together, our data indicate that proper control of FOXP1 expression plays a critical role in PC differentiation, whereas aberrant expression of FOXP1 might contribute to lymphomagenesis by blocking this terminal B-cell differentiation. PMID:26289642

  19. The forkhead transcription factor FOXP1 represses human plasma cell differentiation

    PubMed Central

    van Keimpema, Martine; Grüneberg, Leonie J.; Mokry, Michal; van Boxtel, Ruben; van Zelm, Menno C.; Coffer, Paul; Pals, Steven T.

    2015-01-01

    Expression of the forkhead transcription factor FOXP1 is essential for early B-cell development, whereas downregulation of FOXP1 at the germinal center (GC) stage is required for GC B-cell function. Aberrantly high FOXP1 expression is frequently observed in diffuse large B-cell lymphoma and mucosa-associated lymphoid tissue lymphoma, being associated with poor prognosis. Here, by gene expression analysis upon ectopic overexpression of FOXP1 in primary human memory B cells (MBCs) and B-cell lines, combined with chromatin immunoprecipitation and sequencing, we established that FOXP1 directly represses expression of PRDM1, IRF4, and XBP1, transcriptional master regulators of plasma cell (PC) differentiation. In accordance, FOXP1 is prominently expressed in primary human naive and MBCs, but expression strongly decreases during PC differentiation. Moreover, as compared with immunoglobulin (Ig) M+ MBCs, IgG+ MBCs combine lower expression of FOXP1 with an enhanced intrinsic PC differentiation propensity, and constitutive (over)expression of FOXP1 in B-cell lines and primary human MBCs represses their ability to differentiate into PCs. Taken together, our data indicate that proper control of FOXP1 expression plays a critical role in PC differentiation, whereas aberrant expression of FOXP1 might contribute to lymphomagenesis by blocking this terminal B-cell differentiation. PMID:26289642

  20. Large-scale and high-confidence proteomic analysis of human seminal plasma

    PubMed Central

    Pilch, Bartosz; Mann, Matthias

    2006-01-01

    Background The development of mass spectrometric (MS) techniques now allows the investigation of very complex protein mixtures ranging from subcellular structures to tissues. Body fluids are also popular targets of proteomic analysis because of their potential for biomarker discovery. Seminal plasma has not yet received much attention from the proteomics community but its characterization could provide a future reference for virtually all studies involving human sperm. The fluid is essential for the survival of spermatozoa and their successful journey through the female reproductive tract. Results Here we report the high-confidence identification of 923 proteins in seminal fluid from a single individual. Fourier transform MS enabled parts per million mass accuracy, and two consecutive stages of MS fragmentation allowed confident identification of proteins even by single peptides. Analysis with GoMiner annotated two-thirds of the seminal fluid proteome and revealed a large number of extracellular proteins including many proteases. Other proteins originated from male accessory glands and have important roles in spermatozoan survival. Conclusion This high-confidence characterization of seminal plasma content provides an inventory of proteins with potential roles in fertilization. When combined with quantitative proteomics methodologies, it should be useful for studies of fertilization, male infertility, and prostatic and testicular cancers. PMID:16709260

  1. Aqueous two phase system based on ionic liquid for isolation of quinine from human plasma sample.

    PubMed

    Flieger, J; Czajkowska-Żelazko, A

    2015-01-01

    Aqueous two phase system was applied for selective extraction of quinine from human plasma. Bi-phase was constructed from ionic liquid: butyl-methyl-imidazolium chloride after addition kosmotropic salts K₃PO₄ or KH₂PO₄. Quinine was determined in plasma samples after drinking of tonic containing quinine. Determination was performed by HPLC on 5-μm Zorbax SB-CN column and eluent containing 40% acetonitrile (v/v), 20 mM phosphate buffer at pH 3 and 40 mM NaPF₆ using external standard method. The spectrophotometric detection was set λ=214 nm. Selective fluorescence detection was performed at excitation of 325 nm and emission of 375 nm. Proposed strategy provides suitable sample purification and gives extraction yields in the range of 89-106%. The determination coefficient (R(2)) has a value ≥0.997 in the range of 50-800 ng/ml quinine concentration. The limit of quantification was set at 27.9 ng/ml and the detection limit was found to be 8.4 ng/ml under fluorescence detection. PMID:25053040

  2. LC/MS Method for the Determination of Stable Isotope Labeled Promethazine in Human Plasma

    NASA Technical Reports Server (NTRS)

    Zuwei, Wang; Boyd, Jason; Berens, Kurt L.; Putcha, Lakshmi

    2004-01-01

    Promethazine (PMZ) is taken by astronauts orally (PO), intramuscularly (IM) or rectally (PR) for space motion sickness. LC/MS method was developed with off-line solid phase extraction to measure plasma concentrations of PMZ given as stable isotope-labeled (SIL) formulations by the three different routes of administration simultaneously. Samples (0.5ml) were loaded on to Waters Oasis HLB co-polymer cartridges and eluted with 1.0 mL methanol. HPLC separation of the eluted sample was performed using an Agilent Zorbax SB-CN column (50 x 2.1 mm) at a flow rate of 0.2 mL/min for 6 min. Acetonitrile/ ammonium acetate (30 mM) in water (3:2, v/v), pH 5.6 plus or minus 0.1, was used as the mobile phase for separation. Concentrations of PMZ, PMZ-d4 and PMZ-d7 and chlorpromazine (internal standard) were determined using a Micromass ZMD single quadrupole mass spectrometer with Electrospray Ionization (ESI). ESI mass spectra were acquired in positive ion mode with selected ion monitoring of [M+ H]dot plus. The method is rapid, reproducible and the assay specific parameters are listed in a table. A novel, sensitive and specific method for the measurement of PMZ and SIL PMZ in human plasma is reported.

  3. Aqueous two phase system based on ionic liquid for isolation of quinine from human plasma sample.

    PubMed

    Flieger, J; Czajkowska-Żelazko, A

    2015-01-01

    Aqueous two phase system was applied for selective extraction of quinine from human plasma. Bi-phase was constructed from ionic liquid: butyl-methyl-imidazolium chloride after addition kosmotropic salts K₃PO₄ or KH₂PO₄. Quinine was determined in plasma samples after drinking of tonic containing quinine. Determination was performed by HPLC on 5-μm Zorbax SB-CN column and eluent containing 40% acetonitrile (v/v), 20 mM phosphate buffer at pH 3 and 40 mM NaPF₆ using external standard method. The spectrophotometric detection was set λ=214 nm. Selective fluorescence detection was performed at excitation of 325 nm and emission of 375 nm. Proposed strategy provides suitable sample purification and gives extraction yields in the range of 89-106%. The determination coefficient (R(2)) has a value ≥0.997 in the range of 50-800 ng/ml quinine concentration. The limit of quantification was set at 27.9 ng/ml and the detection limit was found to be 8.4 ng/ml under fluorescence detection.

  4. Ultra performance liquid chromatography PDA method for determination of tigecycline in human plasma.

    PubMed

    D'Avolio, Antonio; Peila, Emanuela; Simiele, Marco; Pensi, Debora; Baietto, Lorena; Cusato, Jessica; Cinnirella, Giacoma; De Rosa, Francesco; Di Perri, Giovanni

    2013-12-01

    : A simple ultra performance liquid chromatography with photodiode array method for the quantification of human plasma concentrations of tigecycline was developed and validated. Quinaxoline, used as an internal standard, was added to 500 μL of plasma before adding 1 mL of protein precipitation solution. The extracts were dried in a vacuum centrifuge system at 60°C and reconstituted with 60 μL of water and acetonitrile (95:5, vol/vol), and 5 μL was injected onto an ACQUITY UPLC H-Class system. Chromatographic separation was performed on a C18 ACQUITY UPLC HSS T3 column using a gradient of potassium phosphate buffer (pH 3.2) and acetonitrile. Detection was performed using a photodiode array detector at 350 nm. Relative error at 3 quality control concentrations ranged from -2.49% to -8.74%. Intraday and interday (percent relative standard error) precision ranged from 3.93% to 12.27% and from 9.53% to 13.32%, respectively. Limit of quantification and limit of detection were 0.024 and 0.006 μg/mL, respectively. Mean recovery was 95%. The calibration curve was linear up to 6 μg/mL. This concentration range proved to be adequate to measure tigecycline concentrations in patients treated with the drug, therefore this method would be suitable for therapeutic drug monitoring.

  5. Quantification of faropenem in human plasma by high-performance liquid chromatography.

    PubMed

    Nirogi, Ramakrishna V S; Kandikere, Vishwottam N; Shrivastava, Wishu; Mudigonda, Koteshwara

    2005-01-01

    A simple, sensitive and specific high-performance liquid chromatography (HPLC) method with ultraviolet detection (315 nm) was developed and validated for quantitation of faropenem (CAS 106560-14-9), the newest addition to the group of beta-lactam antimicrobials, in human plasma. Following solid-phase extraction using Waters Oasis SPE cartridges, the analyte and internal standard (hydrochlorothiazide, CAS 58-93-5) were separated using an isocratic mobile phase of 10 mmol/L acetate buffer (pH adjusted to 7.0 with dilute acetic acid) / methanol / triethyl amine (70/30/0.03, v/v/v) on reverse phase Waters symmetry C18 column. The lower limit of quantitation was 200 ng/mL, with a relative standard deviation of less than 2 %. A linear range of 200 to 25000 ng/mL was established. This HPLC method was validated with between-batch and within-batch precision of 1.6 to 2.3 % and 0.4 to 1.6 %, respectively. The between-batch and within-batch bias was -3.1 to 5.3 % and -6.0 to 1.5 %, respectively. Frequently coadministered drugs did not interfere with the described methodology. The stability of faropenem in plasma was excellent, with no evidence of degradation during sample processing (autosampler) and 30 days storage in a freezer. This validated method is sensitive, simple and repeatable enough to be used in pharmacokinetic studies. PMID:16430031

  6. Determination of Rivaroxaban in Human Plasma by Solid-Phase Extraction-High Performance Liquid Chromatography.

    PubMed

    Çelebier, Mustafa; Reçber, Tuba; Koçak, Engin; Altınöz, Sacide; Kır, Sedef

    2016-02-01

    In this study, a solid-phase extraction (SPE)-high performance liquid chromatography (HPLC)-ultra violet (UV) method was developed for the determination of rivaroxaban (RIV), an oral anticoagulant drug, in human plasma samples. The concentration of RIV in plasma samples was increased 7.5 times and the interference coming from matrix components was avoided by using SPE. The extracted samples of RIV were analyzed by using an HPLC-UV method. RIV was approved in 2008 and many studies have been published in recent years in order to investigate its pharmacokinetic profile in various groups. In light of this information, it is clear that the RIV pharmacokinetic profile should be investigated in further studies; the HPLC-UV method presented in this study might be an easy method to apply, as it is a cheap and rapid alternative to HPLC-MS-MS for this purpose. A Phenomenex Luna 5-µm C18 100 Å LC column (250 × 4.6 mm) was used for the separation of RIV and prednisolone (internal standard). The total analysis time was <6 min. The method was validated according to the FDA guidelines and can be proposed for pharmacokinetic studies of RIV. PMID:26351327

  7. Simultaneous determination of chlorpheniramine and pseudoephedrine in human plasma by liquid chromatography-tandem mass spectrometry.

    PubMed

    Chen, Xiaoyan; Zhang, Yong; Zhong, Dafang

    2004-05-01

    A sensitive and specific procedure for simultaneous quantitation of chlorpheniramine and pseudoephedrine in human plasma has been developed and validated. Analytes were extracted from plasma samples by liquid-liquid extraction, separated on a Diamonsil C18 column (250 x 4.6 mm i.d.) and detected by tandem mass spectrometry with an atmospheric pressure chemical ionization interface. Diphenhydramine was used as the internal standard. The method has a lower limit of quantitation of 0.2 and 2.0 ng/mL for chlorpheniramine and pseudoephedrine, respectively. The intra- and inter-day relative standard deviation, calculated from quality control (QC) samples were below 4.3% for chlorpheniramine and below 9.5% for pseudoephedrine. The inter-day relative error as determined from QC samples was within 4.7% for each analyte. The overall extraction recoveries of chlorpheniramine and pseudoephedrine were 77 and 61% on average, respectively. The method was successfully applied to pharmaockinetic study of chlorpheniramine and pseudoephedrine in volunteers receiving formulations containing 4 mg of chlorpheniramine maleate and 60 mg of pseudoephedrine hydrochloride.

  8. Determination of Rivaroxaban in Human Plasma by Solid-Phase Extraction-High Performance Liquid Chromatography.

    PubMed

    Çelebier, Mustafa; Reçber, Tuba; Koçak, Engin; Altınöz, Sacide; Kır, Sedef

    2016-02-01

    In this study, a solid-phase extraction (SPE)-high performance liquid chromatography (HPLC)-ultra violet (UV) method was developed for the determination of rivaroxaban (RIV), an oral anticoagulant drug, in human plasma samples. The concentration of RIV in plasma samples was increased 7.5 times and the interference coming from matrix components was avoided by using SPE. The extracted samples of RIV were analyzed by using an HPLC-UV method. RIV was approved in 2008 and many studies have been published in recent years in order to investigate its pharmacokinetic profile in various groups. In light of this information, it is clear that the RIV pharmacokinetic profile should be investigated in further studies; the HPLC-UV method presented in this study might be an easy method to apply, as it is a cheap and rapid alternative to HPLC-MS-MS for this purpose. A Phenomenex Luna 5-µm C18 100 Å LC column (250 × 4.6 mm) was used for the separation of RIV and prednisolone (internal standard). The total analysis time was <6 min. The method was validated according to the FDA guidelines and can be proposed for pharmacokinetic studies of RIV.

  9. Parallel artificial liquid membrane extraction as an efficient tool for removal of phospholipids from human plasma.

    PubMed

    Ask, Kristine Skoglund; Bardakci, Turgay; Parmer, Marthe Petrine; Halvorsen, Trine Grønhaug; Øiestad, Elisabeth Leere; Pedersen-Bjergaard, Stig; Gjelstad, Astrid

    2016-09-10

    Generic Parallel Artificial Liquid Membrane Extraction (PALME) methods for non-polar basic and non-polar acidic drugs from human plasma were investigated with respect to phospholipid removal. In both cases, extractions in 96-well format were performed from plasma (125μL), through 4μL organic solvent used as supported liquid membranes (SLMs), and into 50μL aqueous acceptor solutions. The acceptor solutions were subsequently analysed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) using in-source fragmentation and monitoring the m/z 184→184 transition for investigation of phosphatidylcholines (PC), sphingomyelins (SM), and lysophosphatidylcholines (Lyso-PC). In both generic methods, no phospholipids were detected in the acceptor solutions. Thus, PALME appeared to be highly efficient for phospholipid removal. To further support this, qualitative (post-column infusion) and quantitative matrix effects were investigated with fluoxetine, fluvoxamine, and quetiapine as model analytes. No signs of matrix effects were observed. Finally, PALME was evaluated for the aforementioned drug substances, and data were in accordance with European Medicines Agency (EMA) guidelines. PMID:27433988

  10. Metabolism of low-density lipoprotein free cholesterol by human plasma lecithin-cholesterol acyltransferase

    SciTech Connect

    Fielding, P.E.; Miida, Takashi; Fielding, C.J. )

    1991-09-03

    The metabolism of cholesterol derived from ({sup 3}H) cholesterol-labeled low-density lipoprotein (LDL) was determined in human blood plasma. LDL-derived free cholesterol first appeared in large {alpha}-migrating HDL (HDL{sub 2}) and was then transferred to small {alpha}-HDL (HDL{sub 3}) for esterification. The major part of such esters was retained within HDL of increasing size in the course of lecithin-cholesterol acyltransferase (LCAT) activity; the balance was recovered in LDL. Transfer of preformed cholesteryl esters within HDL contributed little to the labeled cholesteryl ester accumulating HDL{sub 2}. When cholesterol for esterification was derived instead from cell membranes, a significantly smaller proportion of this cholesteryl ester was subsequently recovered in LDL. These data suggest compartmentation of cholesteryl esters within plasma that have been formed from cell membrane or LDL free cholesterol, and the role for HDL{sub 2} as a relatively unreactive sink for LCAT-derived cholesteryl esters.

  11. Cross-tolerance of human placental plasma membranes of smokers to fluidizing effects of alcohol

    SciTech Connect

    Sastry, B.V.R.; Horst, M.A.; Naukam, R.J. )

    1991-03-11

    There is cross-tolerance between ethanol and several centrally acting drugs at the membrane level. In order to evaluate cross-tolerance between maternal smoking during pregnancy and alcohol, the authors have prepared plasma membranes of human term placentas from nonsmokers (NS, n=5) and smokers (S, 24 {plus minus} 8 cigarettes/day, n=5) and studied their microviscosities by steady state fluorescence polarization using trans-1,6-diphenyl-1,3,5-hexatriene as a fluorescent probe. These experiments gave the following results: (a) microviscosity was increased by maternal smoking; (b) alcohol decreased microviscosity of the membranes of smokers; (c) exogenous nicotine did not exert any significant effect on the membranes of smokers and nonsmokers. Therefore, the increase in the rigidity of placental plasma membranes is due to chronic smoking, and these membranes are tolerant to the fluidizing effects of alcohol. Cross-tolerance between smoking and ethanol suggests a common hydrophobic locus of the apparent adaptation at the membrane level.

  12. The interaction of the carbon nanoparticles with human cell plasma membrane

    NASA Astrophysics Data System (ADS)

    Overchuk, M.; Prylutska, S.; Bilyy, Rostyslav; Prylutsky, Yu.; Ritter, U.

    2013-09-01

    The study of carbon nanostructures is a highly topical branch of bionanotechnology because of their potential application in biomedicine. Carbon nanotubes (CNTs) are known for their ability to kill tumor cells causing hyperthermia shock and can be used in photothermal therapy respectively. Also chemically modified CNTs can be used for drug delivery. The needle-like shape of CNTs allows them to penetrate into the cell plasma membrane without killing the cell. C60 fullerenes are regarded as valuable nanocarriers for different hydrophobic molecules as well as potential antiviral agents or photosensitizers. In our previous studies we have demonstrated that all types of carbon nanoparticles cause externalization of phosphatidylserine (PS) from the inner to the outer layer of the cell membrane in the small local patches (points of contact), leaving the other parts of plasma membrane PS-negative. In the current work there were studied the interactions of pristine C60 fullerenes and different types of CNTs with human blood cells (erythrocytes and Jurkat T-cells). We have shown, that carbon nanoparticles do not have any hemolytic effects, if judged by the dynamics of acidic hemolysis, although they are capable of permeabilizating the cells and facilitating the internalization of propidium iodide into the nuclei.

  13. A mathematical model of diurnal variations in human plasma melatonin levels

    NASA Technical Reports Server (NTRS)

    Brown, E. N.; Choe, Y.; Shanahan, T. L.; Czeisler, C. A.

    1997-01-01

    Studies in animals and humans suggest that the diurnal pattern in plasma melatonin levels is due to the hormone's rates of synthesis, circulatory infusion and clearance, circadian control of synthesis onset and offset, environmental lighting conditions, and error in the melatonin immunoassay. A two-dimensional linear differential equation model of the hormone is formulated and is used to analyze plasma melatonin levels in 18 normal healthy male subjects during a constant routine. Recently developed Bayesian statistical procedures are used to incorporate correctly the magnitude of the immunoassay error into the analysis. The estimated parameters [median (range)] were clearance half-life of 23.67 (14.79-59.93) min, synthesis onset time of 2206 (1940-0029), synthesis offset time of 0621 (0246-0817), and maximum N-acetyltransferase activity of 7.17(2.34-17.93) pmol x l(-1) x min(-1). All were in good agreement with values from previous reports. The difference between synthesis offset time and the phase of the core temperature minimum was 1 h 15 min (-4 h 38 min-2 h 43 min). The correlation between synthesis onset and the dim light melatonin onset was 0.93. Our model provides a more physiologically plausible estimate of the melatonin synthesis onset time than that given by the dim light melatonin onset and the first reliable means of estimating the phase of synthesis offset. Our analysis shows that the circadian and pharmacokinetics parameters of melatonin can be reliably estimated from a single model.

  14. Isolation of human complex-forming glycoprotein, heterogeneous in charge (protein HC), and its IgA complex from plasma. Physiochemical and immunochemical properties, normal plasma concentration.

    PubMed

    Grubb, A O; López, C; Tejler, L; Mendez, E

    1983-12-10

    Human complex-forming glycoprotein, heterogeneous in charge (protein HC) has previously been isolated from urine and immunochemically shown to be present in low and high molecular weight forms in blood plasma (Tejler, L., and Grubb, A. O. (1976) Biochim. Biophys. Acta 439, 82-94). In the present work, the major low and high molecular weight forms of the protein were isolated from plasma by immunosorption followed by gel chromatography. The plasma low molecular weight protein HC and the urinary protein had similar, if not identical, molecular weight, amino acid composition, NH2-terminal and carboxyl-terminal amino acid sequences and electrophoretic mobility. The low molecular weight plasma protein HC carried a yellow chromophore like the urinary protein, but its molar extinction coefficient at 280 nm was lower and its charge heterogeneity less pronounced than that of urinary protein HC. The plasma high molecular weight protein HC had a hydrodynamic volume which was greater than that of monomeric IgA but smaller than that of dimeric IgA. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the isolated high molecular weight protein followed by electrophoretic blotting and immunochemical analysis demonstrated that the protein contained four polypeptide chains: two light immunoglobulin chains (Mr = 23,000), one IgA alpha-chain (Mr = 54,000), and one chain with Mr approximately 90,000 which carried both alpha-chain and protein HC antigenic determinants. Whether the protein HC X IgA complex is a functionally significant part of the humoral immune system cannot be decided without further experimentation, but the complex was found to be completely absent from the blood plasma of patients with a selective deficiency of IgA-secreting immunocytes. The isolated low and high molecular weight plasma protein HC components were used as standard proteins in the construction of a quantitative crossed immunoelectrophoretic assay for the simultaneous quantitation of the two

  15. Radioimmunoassay of human growth hormone: technique and application to plasma, cerebrospinal fluid, and pituitary extracts

    PubMed Central

    Thomas, Frances J.; Lloyd, H. M.; Thomas, M. J.

    1972-01-01

    A radioimmunoassay for human growth hormone using activated charcoal is described and its precision, accuracy, and sensitivity are defined. Results are presented for growth hormone measurements in plasma obtained during hypoglycaemia induced with insulin in patients of short stature and during glucose tolerance tests in patients with acromegaly. The method was used to measure growth hormone concentrations in cerebrospinal fluid and in extracts of pituitary tumours. No growth hormone was detected in the cerebrospinal fluid of patients without acromegaly. In patients with acromegaly, the concentration of growth hormone in cerebrospinal fluid was measurable and was considerably elevated in one patient with extrasellar extension of a pituitary tumour. Extracts of chromophobe pituitary tumours contained very small concentrations of growth hormone. In extracts of pituitary tumours removed from acromegalic patients, concentrations fell either below or within the normal range. PMID:5086220

  16. Monoclonal antibody against the N-terminal end of human plasma fibronectin.

    PubMed Central

    Vartio, T; Salonen, E M; De Petro, G; Barlati, S; Miggiano, V; Stähli, C; Virgallita, G; Takács, B; Vaheri, A

    1983-01-01

    Purified human plasma fibronectin was digested with cathepsin G and the degradation products were tested for reactivity towards a monoclonal antibody. In an immunoblotting assay, after sodium dodecyl sulphate/polyacrylamide-gel electrophoresis of the digestion products, the 85 000-Mr and 72 000-Mr gelatin- and heparin-binding fragments as well as the N-terminal 30 000-Mr heparin-binding fragment reacted with the antibody, whereas the 64 000-Mr gelatin- and heparin-binding fragment did not. In enzyme immunoassay the antibody reacted with intact fibronectin and the 30 000-Mr fragment but not with a 40 000-Mr gelatin-binding fragment. The alignment of the binding domains in these fragments and in the intact molecule [Vartio (1982) Eur. J. Biochem. 123, 223-233] localizes the antigenic determinant to the 21 000 Da N-terminal Staphylococcus aureus-binding region of fibronectin. Images Fig. 1. Fig. 2. PMID:6194791

  17. Crystallization and Preliminary X-Ray Crystallographic Analysis of Human Plasma Platelet Activating Factor Acetylhydrolase

    SciTech Connect

    Samanta, U.; Wilder, C; Bahnson, B

    2009-01-01

    The plasma form of the human enzyme platelet activating factor acetylhydrolase (PAF-AH) has been crystallized, and X-ray diffraction data were collected at a synchrotron source to a resolution of 1.47 {angstrom}. The crystals belong to space group C2, with unit cell parameters of a = 116.18, b = 83.06, c = 96.71 {angstrom}, and {beta} = 115.09 and two molecules in the asymmetric unit. PAF-AH functions as a general anti-inflammatory scavenger by reducing the levels of the signaling molecule PAF. Additionally, the LDL bound enzyme has been linked to atherosclerosis due to its hydrolytic activities of pro-inflammatory agents, such as sn-2 oxidatively fragmented phospholipids.

  18. Determination of steroid hormones in human plasma by GC-triple quadrupole MS.

    PubMed

    Matysik, Silke; Schmitz, Gerd

    2015-07-01

    A fast and sensitive GC-MS/MS method is proposed to determine pregnenolone, dehydroepiandrosterone (DHEA), testosterone and dihydrotestosterone from human plasma. Steroids were extracted by liquid/liquid extraction, and derivatized with N-methyl-N-trimethylsilyl-trifluoracetamide. Electron ionization at 30eV was applied to generate high abundant precursor ions. Specific precursor/product transitions were generated a priori for each compound of interest and these assays were then used to selectively detect and quantify these steroids. Using the selected reaction monitoring mode, detection limits in the pg/ml (subnmol/l) range could be achieved for all four steroid compounds. The method was validated for imprecision, and recovery and applied in a pilot study in patients with hypercholesterolemia. Associations of increased cholesterol biosynthesis and low testosterone and dihydrotestosterone concentrations were found.

  19. Electrochemistry of raloxifene on glassy carbon electrode and its determination in pharmaceutical formulations and human plasma.

    PubMed

    Bagheri, Akbar; Hosseini, Hadi

    2012-12-01

    The electrochemical behavior of raloxifene (RLX) on the surface of a glassy carbon electrode (GCE) has been studied by cyclic voltammetry (CV). The CV studies were performed in various supporting electrolytes, wide range of potential scan rates, and pHs. The results showed an adsorption-controlled and quasi-reversible process for the electrochemical reaction of RLX, and a probable redox mechanism was suggested. Under the optimum conditions, differential pulse voltammetry (DPV) was applied for quantitative determination of the RLX in pharmaceutical formulations. The DPV measurements showed that the anodic peak current of the RLX was linear to its concentration in the range of 0.2-50.0μM with a detection limit of 0.0750μM, relative standard deviation (RSD %) below 3.0%, and a good sensitivity. The proposed method was successfully applied for determination of the RLX in pharmaceutical and human plasma samples with a good selectivity and suitable recovery.

  20. Liquid chromatographic method for the determination of rizatriptan in human plasma.

    PubMed

    Chen, Jun; Jiang, Xinguo; Jiang, Wenming; Mei, Ni; Gao, Xiaoling; Zhang, Qizhi

    2004-06-01

    A high-performance liquid chromatographic (HPLC) method with fluorescence detection has been developed for the determination of rizatriptan in human plasma. Following a single-step liquid-liquid extraction with methyl tertiarybutyl ether, the analytes were separated using a mobile phase consisting of 0.05% (v/v) triethylamine in water (adjusting to pH 2.75 with 85% phosphoric acid) and acetonitrile (92:8, v/v). Fluorescence detection was performed at an excitation wavelength of 225nm and an emission wavelength of 360nm. The linearity for rizatriptan was within the concentration range of 0.5-50ng/ml. The intra- and inter-day precisions of the method were not more than 8.0%. The lower limit of quantification (LLOQ) was 0.5ng/ml for rizatriptan. The method was sensitive, simple and repeatable enough to be used in pharmacokinetic studies. PMID:15113554

  1. Malathion-induced inhibition of human plasma cholinesterase studied by the fluorescence spectroscopy method

    NASA Astrophysics Data System (ADS)

    Pavelkić, V. M.; Krinulović, K. S.; Savić, J. Z.; Ilić, M. A.

    2008-05-01

    The in vitro effect of technical grade malathion was assessed via the kinetic parameters of human plasma butyrylcholinesterase (BChE) using N-methylindoxyl acetate as a substrate for BChE. An inhibitor kinetics study demonstrated the existence of a biphasic inhibition curve, indicating high-and low-affinity binding sites of malathion. The IC 50 values as calculated from the experimental inhibition curves were 1.33 × 10-9 and 1.48 × 10-5 M for the high-and low-affinity binding sites, respectively; Hill’s analysis gave 1.29 × 10-9 and 1.38 × 10-6 M. The Cornish-Bowden plots and their secondary plots indicated that the nature of inhibition was of mixed type with the predominant competitive character of both affinity binding sites.

  2. Simultaneous quantification of 21 water soluble vitamin circulating forms in human plasma by liquid chromatography-mass spectrometry.

    PubMed

    Meisser Redeuil, Karine; Longet, Karin; Bénet, Sylvie; Munari, Caroline; Campos-Giménez, Esther

    2015-11-27

    This manuscript reports a validated analytical approach for the quantification of 21 water soluble vitamins and their main circulating forms in human plasma. Isotope dilution-based sample preparation consisted of protein precipitation using acidic methanol enriched with stable isotope labelled internal standards. Separation was achieved by reversed-phase liquid chromatography and detection performed by tandem mass spectrometry in positive electrospray ionization mode. Instrumental lower limits of detection and quantification reached <0.1-10nM and 0.2-25nM, respectively. Commercially available pooled human plasma was used to build matrix-matched calibration curves ranging 2-500, 5-1250, 20-5000 or 150-37500nM depending on the analyte. The overall performance of the method was considered adequate, with 2.8-20.9% and 5.2-20.0% intra and inter-day precision, respectively and averaged accuracy reaching 91-108%. Recovery experiments were also performed and reached in average 82%. This analytical approach was then applied for the quantification of circulating water soluble vitamins in human plasma single donor samples. The present report provides a sensitive and reliable approach for the quantification of water soluble vitamins and main circulating forms in human plasma. In the future, the application of this analytical approach will give more confidence to provide a comprehensive assessment of water soluble vitamins nutritional status and bioavailability studies in humans. PMID:26522745

  3. Phenotyping of human complement component C4, a class-III HLA antigen.

    PubMed Central

    Sim, E; Cross, S J

    1986-01-01

    The plasma complement protein C4 is encoded at two highly polymorphic loci, A and B, within the class-III region of the major histocompatibility complex. At least 34 different polymorphic variants of human C4 have been identified, including non-expressed or 'null' alleles. The main method of identification of C4 polymorphic allotypes is separation on the basis of charge by agarose-gel electrophoresis of plasma. On staining by immunofixation with anti-C4 antibodies, each C4 type gives three major bands, but, since individuals can have up to five allotypes, the overlapping banding pattern is difficult to interpret. We show that digestion of plasma samples with carboxypeptidase B, which removes C-terminal basic amino acids, before electrophoresis, produces a single, sharp, distinct band for each allotype and allows identification of the biochemical basis of the multiple banding pattern previously observed in C4 phenotype determination. Images Fig. 1. Fig. 2. Fig. 3. Fig. 4. PMID:3103606

  4. Recombinant human lactoferrin-Fc fusion with an improved plasma half-life.

    PubMed

    Shiga, Yuki; Oshima, Yuta; Kojima, Yoshinori; Sugimoto, Akinori; Tamaki, Naomi; Murata, Daisuke; Takeuchi, Takashi; Sato, Atsushi

    2015-01-25

    Lactoferrin (LF), an 80-kDa iron-binding glycoprotein found in mammalian exocrine secretions, has potential therapeutic efficacy due to its extensive health-promoting effects. However, LF is rapidly cleared from the circulation (∼12.6min half-life for recombinant human LF [rhLF] in rats), which limits its therapeutic potential. Therefore, to improve plasma stability, we developed a recombinant human LF (hLF)-immunoglobulin G1 (IgG1) fragment crystallizable domain (Fc) fusion (hLF-hinge-CH2-CH3) expressed in a Chinese Hamster Ovary cell (CHO) expression system and evaluated the in vitro bioactivities and pharmacokinetic properties of the purified fusion. CHO DG44 cells were transfected with an expression vector coding for recombinant hLF-hinge-CH2-CH3. Iron binding, Caco-2 uptake, and thermal stability were investigated in vitro, and pharmacokinetic parameters were investigated in vivo. hLF-hinge-CH2-CH3 was significantly expressed in CHO cells (∼100mg/l culture), was readily purified, and exhibited 98.3% of the non-fused rhLF iron-binding activity. Caco-2 uptake and thermal stability were improved for hLF-Fc fusion relative to rhLF. Moreover, hLF-hinge-CH2-CH3 demonstrated a plasma half-life that was 9.1-fold longer than that of rhLF as well as longer than that of the PEGylated bovine LFs that we previously developed. Thus, CHO-derived hLF-hinge-CH2-CH3, with enhanced pharmacokinetic properties, is a promising candidate drug for potential parenteral administration.

  5. An UPLC-MS/MS method for the analysis of glimepiride and fluoxetine in human plasma.

    PubMed

    Qiu, Xiangjun; Wang, Hong-wei; Yuan, Ye; Wang, Ying-fei; Sun, Ming; Huang, Xue-sun

    2015-02-01

    A sensitive and rapid ultra performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) method was developed to determine glimepiride (GPD) and fluoxetine (FLU) in human plasma using diazepam as the internal standard (IS) simultaneously. The presented method used an Acquity UPLC BEH C18 column for chromatographic separation with tandem mass spectrometric detection on a QTrap5500 mass spectrometer operated in positive ESI mode. The mobile phase is a mixture of acetonitrile and 1% formic acid in water with gradient elution at a flow rate of 0.40mL/min. The GPD, FLU and IS were eluted at 1.46, 1.27 and 1.39min, respectively. The MRM transitions of m/z 491.3→126.3 and m/z 310.5→148.1 were used to quantify for GPD and FLU, respectively. The linearity of this method was found to be within the concentration range of 2.5-300ng/mL and 0.1-20ng/mL for GPD and FLU in human plasma, respectively. The intra- and inter-day precision (RSD%) were less than 10.3% and accuracy (RE%) was within ±7.3%. The matrix effect were 95.3-100.7% for GPD and FLU. GPD and FLU were sufficiently stable under all relevant analytical conditions. The method was also successfully applied to the clinical samples after a single oral dose of 2mg GLP and 40mg FLU in patients. PMID:25589252

  6. Persistent organohalogen contaminants in plasma from groups of humans with different occupations in Bangladesh.

    PubMed

    Zamir, R; Athanasiadou, M; Nahar, N; Mamun, M I R; Mosihuzzaman, M; Bergman, A

    2009-01-01

    The present study is aimed to assess persistent organic halogenated pollutants in humans living in Bangladesh. The results are compared to other similar studies in the region and globally. Human blood plasma were collected from groups of men and women with different occupations, i.e. being students, garment industry workers, employees at the Power Development Board (PDB), all groups in Dhaka, fishermen and fishermen wife's from Dhaka and another group from Barisal district. The plasma was analysed for hexachlorobenzene (HCB), the hexachlorocyclohexane isomers, alpha-HCH, beta-HCH, gamma-HCH and delta-HCH, the DDT group of chemicals, chlordane compounds, trans-chlordane, cis-chlordane, oxychlordane, trans-nonachlor, trans-heptachlorepoxide, methoxychlor and mirex. The most abundant contaminant, in all groups studied, p,p'-DDE is dominating, with p,p'-DDT/Sigma DDT ratios indicating recent and ongoing DDT exposure. Among the other pesticides analysed beta-HCH is the most abundant indicating the use of technical HCH products instead of Lindane (gamma-HCH). While the Sigma DDT is present in the low ppm range the beta-HCH is detected in up to approx. 400 ppb, lipid basis. The beta-HCH is most abundant in the groups of students. In contrast to the pesticides analysed very low concentrations of polychlorinated biphenyls (PCB) are present in all study groups, with e.g. CB-153 in the range of 5-30 ng g(-1) fat. The concentrations of the DDT group of chemical differ significantly between fishermen and fishermen's wives living and working in the Dhaka area versus those living and working in Barisal. Also, fishermen and their wives had significantly different concentrations of DDT compared to garment industry workers.

  7. Exhaustive and stable electromembrane extraction of acidic drugs from human plasma.

    PubMed

    Huang, Chuixiu; Gjelstad, Astrid; Seip, Knut Fredrik; Jensen, Henrik; Pedersen-Bjergaard, Stig

    2015-12-18

    The first part of the current work systematically described the screening of different types of organic solvents as the supported liquid membrane (SLM) for electromembrane extraction (EME) of acidic drugs, including different alcohols, ketones, and ethers. Seven acidic drugs with a wide logP range (1.01-4.39) were selected as model substances. For the first time, the EME recovery of acidic drugs and system-current across the SLM with each organic solvent as SLM were investigated and correlated to relevant solvent properties such as viscosity and Kamlet and Taft solvatochromic parameters. Solvents with high hydrogen bonding acidity (α) and dipolarity-polarizability (π*) were found to be successful SLMs, and 1-heptanol was the most efficient candidate, which provided EME recovery in the range of 94-110%. Both hydrogen bonding interactions, dipole-dipole interactions, and hydrophobic interactions were involved in stabilizing the deprotonated acidic analytes (with high hydrogen bonding basicity and high dipole moment) during mass transfer across the SLM. The efficiency of the extraction normally decreased with increasing hydrocarbon chain length of the SLM, which was mainly due to increasing viscosity and decreasing α and π* values. The system-current during EME was found to be dependent on the type and the volume of the SLM. In contact with human plasma, an SLM of pure 1-heptanol was unstable, and to improve stability, 1-heptanol was mixed with 2-nitrophenyl octyl ether (NPOE). With this SLM, exhaustive EME was performed from diluted human plasma, and the recoveries of five out of seven analytes were over 91% after 10min EME. This approach was evaluated using HPLC-UV, and the evaluation data were found to be satisfactory. PMID:26632516

  8. Atmospheric-Pressure Cold Plasma Induces Transcriptional Changes in Ex Vivo Human Corneas

    PubMed Central

    Rosani, Umberto; Tarricone, Elena; Venier, Paola; Brun, Paola; Deligianni, Velika; Zuin, Matteo; Martines, Emilio

    2015-01-01

    Background Atmospheric pressure cold plasma (APCP) might be considered a novel tool for tissue disinfection in medicine since the active chemical species produced by low plasma doses, generated by ionizing helium gas in air, induces reactive oxygen species (ROS) that kill microorganisms without substantially affecting human cells. Objectives In this study, we evaluated morphological and functional changes in human corneas exposed for 2 minutes (min) to APCP and tested if the antioxidant n-acetyl l-cysteine (NAC) was able to inhibit or prevent damage and cell death. Results Immunohistochemistry and western blotting analyses of corneal tissues collected at 6 hours (h) post-APCP treatment demonstrated no morphological tissue changes, but a transient increased expression of OGG1 glycosylase that returned to control levels in 24 h. Transcriptome sequencing and quantitative real time PCR performed on different corneas revealed in the treated corneas many differentially expressed genes: namely, 256 and 304 genes showing expression changes greater than ± 2 folds in the absence and presence of NAC, respectively. At 6 h post-treatment, the most over-expressed gene categories suggested an active or enhanced cell functioning, with only a minority of genes specifically concerning oxidative DNA damage and repair showing slight over-expression values (<2 folds). Moreover, time-related expression analysis of eight genes up-regulated in the APCP-treated corneas overall demonstrated the return to control expression levels after 24 h. Conclusions These findings of transient oxidative stress accompanied by wide-range transcriptome adjustments support the further development of APCP as an ocular disinfectant. PMID:26203910

  9. Elevated Plasma Soluble CD14 Levels Correlate with the Monocyte Response Status During Hantaan Virus Infection in Humans.

    PubMed

    Tang, Kang; Zhang, Chunmei; Zhang, Yusi; Zhang, Yun; Zhuang, Ran; Jin, Boquan; Ma, Ying

    2015-10-01

    Hantaan virus (HTNV) infection can cause severe hemorrhagic fever with renal syndrome (HFRS) in humans. CD14, a pattern recognition receptor recognizing lipopolysaccharide, is highly expressed on monocytes and can be shed as soluble CD14 (sCD14) upon monocyte activation. To understand the role of sCD14 in HFRS, the sCD14 plasma concentrations from 45 HFRS patients were quantified, and the relationships between the plasma sCD14 level and the monocyte response status and clinical parameters were analyzed. The plasma sCD14 levels were significantly higher in the HFRS patients and they correlated with monocyte expansion and activation, which were characterized by increased blood monocyte counts, the proportion of CD14(++)CD16(+) intermediate monocytes, as well as elevated plasma tumor necrosis factor-α (TNF-α) and soluble CD163 (sCD163) levels. Additionally, the high plasma sCD14 levels positively correlated with white blood cell counts and blood urea nitrogen levels and negatively correlated with platelet counts in the HFRS patients. Taken together, our data indicate that elevated plasma sCD14 levels are associated with the monocyte response status during HTNV infection in humans.

  10. [Development of online conventional array-based two-dimensional liquid chromatographic system for proteins separation in human plasma].

    PubMed

    Huang, Zhi; Hong, Guangfeng; Gao, Mingxia; Zhang, Xiangmin

    2014-04-01

    Human plasma is one of the proteins-containing samples most difficult to characterize on account of the wide dynamic concentration range of its intact proteins. Herein, we developed a high-throughput conventional array-based two-dimensional liquid chromatographic system for proteins separation in human plasma in online mode. In the system, a conventional strong-anion exchange chromatographic column was used as the first separation dimension and eight parallel conventional reversed-phase liquid chromatographic columns were integrated as the second separation dimension. The fractions from the first dimension were sequentially transferred into the corresponding reversed-phase liquid chromatographic precolumns for retention and enrichment using a 10-port electrically actuated multi-position valve. The second dimensional solvent flow was directly and identically split into 8 channels. The fractions were concurrently back-flushed from the precolumns into the 8 conventional RP columns and were separated simultaneously. An 8-channel fraction collector was refitted to collect the reversed-phase liquid chromatographic fractions for further investigation. Bicinchoninic acid (BCA) dyein solution was conveniently used for high-abundance protein location. Two separation dimensions were relatively independent parts, as well as each channel of the second dimensional array separation. Therefore, the new system could improve the separation throughput and total peak capacity. The system was successfully applied for the separation of human plasma intact proteins. The results indicated the established system is an effective method for removing high abundance proteins in plasma and in-depth research in plasma proteomics.

  11. Liquid chromatography-tandem mass spectroscopy assay for quercetin and conjugated quercetin metabolites in human plasma and urine.

    PubMed

    Wang, Liang; Morris, Marilyn E

    2005-07-25

    A sensitive and specific method was developed and validated for the quantitation of quercetin in human plasma and urine. The application of liquid chromatography-tandem mass spectrometry (LC/MS/MS) with a TurboIonspray (TIS) interface in negative mode under multiple reactions monitoring was investigated. Chromatographic separation was achieved on a C12 column using a mobile phase of acetonitrile/water with 0.2% formic acid (pH 2.4) (40/60, v/v). The detection limit was 100 pg/ml and the lower limit of quantification was 500 pg/ml for plasma samples; the detection limit was 500 pg/ml and the lower limit of quantification was 1 ng/ml for urine samples. The calibration curve was linear from 1 to 800 ng/ml for plasma samples and was linear from 1 to 200 and 50 to 2000 ng/ml for urine samples. All the intra- and inter-day coefficients of variation were less than 11% and intra- and inter-day accuracies were within +/-15% of the known concentrations. This represents a LC/MS/MS assay with the sensitivity and specificity necessary to determine quercetin in human plasma and urine. This assay was used to determine both parent quercetin and the quercetin after enzymatic hydrolysis with beta-glucuronidase/sulfatase in human plasma and urine samples following the ingestion of quercetin 500 mg capsules.

  12. Effects of atmospheric pressure cold plasma on human hepatocarcinoma cell and its 5-fluorouracil resistant cell line

    NASA Astrophysics Data System (ADS)

    Yang, H.; Lu, R.; Xian, Y.; Gan, L.; Lu, X.; Yang, X.

    2015-12-01

    Atmospheric pressure cold plasma showed selective killing efficiency on cancer cells in vitro and in vivo, which makes plasma a potential option for cancer therapy. However, the plasma effects on chemotherapeutic drugs-resistant cells are rarely to be found. In this paper, the effects of plasma on human hepatocellular carcinoma Bel7402 cells and 5-fluorouracil (5-FU) resistant Bel7402/5FU cells were intensively investigated. The results showed that plasma induced superior toxicity to Bel7402 cells compared with Bel7402/5FU cells. Incubation with plasma-treated medium for 20 s induced more than 85% death rate in Bel7402 cells, while the same death ratio was achieved when Bel7402/5FU cells were treated for as long as 300 s. The hydrogen peroxide in the medium played a leading role in the cytotoxicity effects. Further studies implicated that when the treatment time was shorter than 60 s, the depolarization of mitochondrial membrane potential and apoptosis occurred through the intracellular reactive oxygen species accumulation in Bel7402 cells. Molecular analysis showed an increase in the transcription factor activity for AP-1, NF-кB, and p53 in Bel7402 cells. No obvious damage could be detected in plasma-treated Bel7402/5FU cells due to the strong intracellular reactive oxygen stress scavenger system.

  13. Effects of atmospheric pressure cold plasma on human hepatocarcinoma cell and its 5-fluorouracil resistant cell line

    SciTech Connect

    Yang, H.; Gan, L.; Yang, X. E-mail: yangxl@mail.hust.edu.cn; Lu, R.; Xian, Y.; Lu, X. E-mail: yangxl@mail.hust.edu.cn

    2015-12-15

    Atmospheric pressure cold plasma showed selective killing efficiency on cancer cells in vitro and in vivo, which makes plasma a potential option for cancer therapy. However, the plasma effects on chemotherapeutic drugs-resistant cells are rarely to be found. In this paper, the effects of plasma on human hepatocellular carcinoma Bel7402 cells and 5-fluorouracil (5-FU) resistant Bel7402/5FU cells were intensively investigated. The results showed that plasma induced superior toxicity to Bel7402 cells compared with Bel7402/5FU cells. Incubation with plasma-treated medium for 20 s induced more than 85% death rate in Bel7402 cells, while the same death ratio was achieved when Bel7402/5FU cells were treated for as long as 300 s. The hydrogen peroxide in the medium played a leading role in the cytotoxicity effects. Further studies implicated that when the treatment time was shorter than 60 s, the depolarization of mitochondrial membrane potential and apoptosis occurred through the intracellular reactive oxygen species accumulation in Bel7402 cells. Molecular analysis showed an increase in the transcription factor activity for AP-1, NF-kB, and p53 in Bel7402 cells. No obvious damage could be detected in plasma-treated Bel7402/5FU cells due to the strong intracellular reactive oxygen stress scavenger system.

  14. Comprehensive and quantitative proteomic analyses of zebrafish plasma reveals conserved protein profiles between genders and between zebrafish and human

    PubMed Central

    Li, Caixia; Tan, Xing Fei; Lim, Teck Kwang; Lin, Qingsong; Gong, Zhiyuan

    2016-01-01

    Omic approaches have been increasingly used in the zebrafish model for holistic understanding of molecular events and mechanisms of tissue functions. However, plasma is rarely used for omic profiling because of the technical challenges in collecting sufficient blood. In this study, we employed two mass spectrometric (MS) approaches for a comprehensive characterization of zebrafish plasma proteome, i.e. conventional shotgun liquid chromatography-tandem mass spectrometry (LC-MS/MS) for an overview study and quantitative SWATH (Sequential Window Acquisition of all THeoretical fragment-ion spectra) for comparison between genders. 959 proteins were identified in the shotgun profiling with estimated concentrations spanning almost five orders of magnitudes. Other than the presence of a few highly abundant female egg yolk precursor proteins (vitellogenins), the proteomic profiles of male and female plasmas were very similar in both number and abundance and there were basically no other highly gender-biased proteins. The types of plasma proteins based on IPA (Ingenuity Pathway Analysis) classification and tissue sources of production were also very similar. Furthermore, the zebrafish plasma proteome shares significant similarities with human plasma proteome, in particular in top abundant proteins including apolipoproteins and complements. Thus, the current study provided a valuable dataset for future evaluation of plasma proteins in zebrafish. PMID:27071722

  15. Comprehensive and quantitative proteomic analyses of zebrafish plasma reveals conserved protein profiles between genders and between zebrafish and human.

    PubMed

    Li, Caixia; Tan, Xing Fei; Lim, Teck Kwang; Lin, Qingsong; Gong, Zhiyuan

    2016-01-01

    Omic approaches have been increasingly used in the zebrafish model for holistic understanding of molecular events and mechanisms of tissue functions. However, plasma is rarely used for omic profiling because of the technical challenges in collecting sufficient blood. In this study, we employed two mass spectrometric (MS) approaches for a comprehensive characterization of zebrafish plasma proteome, i.e. conventional shotgun liquid chromatography-tandem mass spectrometry (LC-MS/MS) for an overview study and quantitative SWATH (Sequential Window Acquisition of all THeoretical fragment-ion spectra) for comparison between genders. 959 proteins were identified in the shotgun profiling with estimated concentrations spanning almost five orders of magnitudes. Other than the presence of a few highly abundant female egg yolk precursor proteins (vitellogenins), the proteomic profiles of male and female plasmas were very similar in both number and abundance and there were basically no other highly gender-biased proteins. The types of plasma proteins based on IPA (Ingenuity Pathway Analysis) classification and tissue sources of production were also very similar. Furthermore, the zebrafish plasma proteome shares significant similarities with human plasma proteome, in particular in top abundant proteins including apolipoproteins and complements. Thus, the current study provided a valuable dataset for future evaluation of plasma proteins in zebrafish. PMID:27071722

  16. ANGPTL3 blockade with a human monoclonal antibody reduces plasma lipids in dyslipidemic mice and monkeys1[S

    PubMed Central

    Gusarova, Viktoria; Alexa, Corey A.; Wang, Yan; Rafique, Ashique; Kim, Jee Hae; Buckler, David; Mintah, Ivory J.; Shihanian, Lisa M.; Cohen, Jonathan C.; Hobbs, Helen H.; Xin, Yurong; Valenzuela, David M.; Murphy, Andrew J.; Yancopoulos, George D.; Gromada, Jesper

    2015-01-01

    Angiopoietin-like protein 3 (ANGPTL3) is a circulating protein synthesized exclusively in the liver that inhibits LPL and endothelial lipase (EL), enzymes that hydrolyze TGs and phospholipids in plasma lipoproteins. Here we describe the development and testing of a fully human monoclonal antibody (REGN1500) that binds ANGPTL3 with high affinity. REGN1500 reversed ANGPTL3-induced inhibition of LPL activity in vitro. Intravenous administration of REGN1500 to normolipidemic C57Bl/6 mice increased LPL activity and decreased plasma TG levels by ≥50%. Chronic administration of REGN1500 to dyslipidemic C57Bl/6 mice for 8 weeks reduced circulating plasma levels of TG, LDL-cholesterol (LDL-C), and HDL-cholesterol (HDL-C) without any changes in liver, adipose, or heart TG contents. Studies in EL knockout mice revealed that REGN1500 reduced serum HDL-C through an EL-dependent mechanism. Finally, administration of a single dose of REGN1500 to dyslipidemic cynomolgus monkeys caused a rapid and pronounced decrease in plasma TG, nonHDL-C, and HDL-C. REGN1500 normalized plasma TG levels even in monkeys with a baseline plasma TG greater than 400 mg/dl. Collectively, these data demonstrate that neutralization of ANGPTL3 using REGN1500 reduces plasma lipids in dyslipidemic mice and monkeys, and thus provides a potential therapeutic agent for treatment of patients with hyperlipidemia. PMID:25964512

  17. Association of CD2 with fibrinogen in human plasma: depletion of the soluble E-receptor in blood clotting.

    PubMed

    Smorodin, Eugeniy P; Kurtenkov, O A; Shevchuk, I N

    2007-01-01

    The soluble E-receptor (SER) of lymphocytes that is related to CD2 was detected in human plasma and serum using immunoelectrophoresis with sheep antiserum. All plasma samples (n=18) demonstrated reactivity with antiserum, whereas the reactivity of the corresponding sera remained low or undetectable. The depletion of SER in clotting is associated with fibrinogen, as shown by crossed-affinity immunoelectrophoresis with antisera to plasma proteins. The SER-associated fibrinogen was purified and analysed by the SDS-polyacrylamide gel electrophoresis and immunoblotting. A band close to 66 kDa was detected with monoclonal antibodies to CD2. The association of CD2 and other soluble receptors with fibrinogen via domains is suggested. It is recommended that the fresh plasma, not serum, should be used to study circulating receptors because coagulation may appreciably diminish their physiological level in blood samples.

  18. Danger signal-dependent activation of human dendritic cells by plasma-derived factor VIII products.

    PubMed

    Miller, L; Weissmüller, S; Ringler, E; Crauwels, P; van Zandbergen, G; Seitz, R; Waibler, Z

    2015-08-01

    Treatment of haemophilia A by infusions of the clotting factor VIII (FVIII) results in the development of inhibitors/anti-drug antibodies in up to 25 % of patients. Mechanisms leading to immunogenicity of FVIII products are not yet fully understood. Amongst other factors, danger signals as elicited upon infection or surgery have been proposed to play a role. In the present study, we focused on effects of danger signals on maturation and activation of dendritic cells (DC) in the context of FVIII application. Human monocyte-derived DC were treated with FVIII alone, with a danger signal alone or a combination of both. By testing more than 60 different healthy donors, we show that FVIII and the bacterial danger signal lipopolysaccharide synergise in increasing DC activation, as characterised by increased expression of co-stimulatory molecules and secretion of pro-inflammatory cytokines. The degree and frequency of this synergistic activation correlate with CD86 expression levels on immature DC prior to stimulation. In our assay system, plasma-derived but not recombinant FVIII products activate human DC in a danger signal-dependent manner. Further tested danger signals, such as R848 also induced DC activation in combination with FVIII, albeit not in every tested donor. In our hands, human DC but not human B cells or macrophages could be activated by FVIII in a danger signal-dependent manner. Our results suggest that immunogenicity of FVIII is a result of multiple factors including the presence of danger, predisposition of the patient, and the choice of a FVIII product for treatment.

  19. Expression of c-myc and induction of DNA synthesis by platelet-poor plasma in human diploid fibroblasts

    SciTech Connect

    Ferrari, S.; Calabretta, B.; Battini, R.; Cosenza, S.C.; Owen, T.A.; Soprano, K.J.; Baserga, R. )

    1988-01-01

    When WI-38 human diploid fibroblasts become confluent, they stop synthesizing DNA and dividing. Addition of serum causes the quiescent cell to reenter the cell cycle. Prolonged quiescence after confluence decreases and delays the response to serum. For a few days after reaching confluence. WI-38 cells also respond to platelet-poor plasma. During this period, although not cycling, WI-38 cells still express c-myc and other growth-regulated genes, as measured by steady-state RNA levels. If the quiescence is prolonged further, c-myc expression (and that of two other growth-regulated genes) is no longer detectable, and its disappearance coincides with a loss of response to platelet-poor plasma. These results suggest that, also under physiological conditions, the expression of c-myc and other growth-regulated genes can cooperate with platelet-poor plasma in inducing cellular DNA synthesis in human diploid fibroblasts.

  20. Quantitative determination of terbutaline and orciprenaline in human plasma by gas chromatography/negative ion chemical ionization/mass spectrometry.

    PubMed

    Leis, H J; Gleispach, H; Nitsche, V; Malle, E

    1990-06-01

    A method for the determination of unconjugated terbutaline and orciprenaline in human plasma is described. The assay is based on stable isotope dilution gas chromatography/negative ion chemical ionization/mass spectrometry. An inexpensive and rapid method for preparation of stable isotope labelled analogues as well as their use in quantitative gas chromatography/mass spectrometry is shown. A highly efficient sample work-up procedure with product recoveries of more than 95% is presented. The method developed permits quantitative measurement of terbutaline and orciprenaline in human plasma down to 100 pg ml-1, using 1 ml of sample. Plasma levels of terbutaline after oral administration of 5 mg of terbutaline sulphate were estimated. PMID:2357489

  1. Rational design of urea-based glutamate carboxypeptidase II (GCPII) inhibitors as versatile tools for specific drug targeting and delivery.

    PubMed

    Tykvart, Jan; Schimer, Jiří; Bařinková, Jitka; Pachl, Petr; Poštová-Slavětínská, Lenka; Majer, Pavel; Konvalinka, Jan; Šácha, Pavel

    2014-08-01

    Glutamate carboxypeptidase II (GCPII), also known as prostate specific membrane antigen (PSMA), is an established prostate cancer marker and is considered a promising target for specific anticancer drug delivery. Low-molecular-weight inhibitors of GCPII are advantageous specific