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albumin human [keywords]
- A Digital Microfluidic Platform for Human Plasma Protein Depletion. [JOURNAL ARTICLE]
- Anal Chem 2014 Jul 24.
Many important biomarkers for disease diagnosis are present at low concentrations in human serum. These biomarkers are masked in proteomic analysis by highly abundant proteins such as human serum albumin (HSA) and immunoglobulins (IgGs) which account for up to 80% of the total protein content of serum. Traditional depletion methods using macro-scale LC-columns for highly abundant proteins involve slow separations which impart considerable dilution to the samples. Furthermore, most techniques lack the ability to process multiple samples simultaneously. We present a method of protein depletion using superparamagnetic beads coated in anti-HSA, Protein A and Protein G, manipulated by digital microfluidics (DMF). The depletion process was capable of up to 95% protein depletion efficiency for IgG and HSA in 10 minutes for four samples simultaneously, which resulted in an approximately 4-fold increase in signal-to-noise ratio in MALDI-MS analysis for a low abundance protein, hemopexin. This rapid and automated method has the potential to greatly improve the process of biomarker identification.
- Multiplexed LC-MS/MS Assay for Urine Albumin. [JOURNAL ARTICLE]
- J Proteome Res 2014 Jul 24.
Urinary excretion of albumin is a major diagnostic and prognostic marker of renal dysfunction and cardiovascular disease; therefore, accurate measurement of urine albumin is vital to clinical diagnosis. Although inter-method differences and analyte heterogeneity have been reported for urine albumin measurements, accuracy assessments of the available methods have been hindered by the lack of a reference system, including reference measurement procedures and reference materials, for this clinical analyte. To address the need for a reference measurement system for urine albumin, we have developed a candidate reference measurement procedure that utilizes isotope dilution-mass spectrometry (ID-MS) and multiple reaction monitoring (MRM) to quantify full-length urine albumin in a targeted mass spectrometric-based approach. The reference measurement procedure incorporates an isotopically-labeled (15N) full-length recombinant human serum albumin (15N-rHSA) material as the internal standard, which permits the absolute quantitation of albumin in urine. A total of 11 peptides with two transitions per peptide were selected from the tryptic digestion of human serum albumin on the basis of retention time reproducibility, peak intensity, and the degree of HSA sequence coverage. In addition to method validation, the generated calibration curves were used to determine the albumin content in pooled human urine samples to access the accuracy of the MS-based urine albumin quantitation method.
- Imatinib binding to human serum albumin modulates heme association and reactivity. [JOURNAL ARTICLE]
- Arch Biochem Biophys 2014 Jul 21.
Imatinib, an inhibitor of the Bcr-Abl tyrosine kinase, is approximately 95% bound to plasma proteins, α1-acid glycoprotein (AGP) being the primary carrier. However, human serum albumin (HSA) may represent the secondary carrier of imatinib in pathological states characterized by low AGP levels, such as pancreatic cancer, hepatic cirrhosis, hepatitis, hyperthyroidism, nephrotic syndrome, malnutrition, and cachexia. Here, thermodynamics of imatinib binding to full-length HSA and its recombinant Asp1-Glu382 truncated form (containing only the FA1, FA2, FA6, and FA7 binding sites; trHSA), in the absence and presence of ferric heme (heme-Fe(III)), and the thermodynamics of heme-Fe(III) binding to HSA and trHSA, in the absence and presence of imatinib, has been investigated. Moreover, the effect of imatinib on kinetics of peroxynitrite detoxification by ferric human serum heme-albumin (HSA-heme-Fe(III)) and ferric truncated human serum heme-albumin (trHSA-heme-Fe(III)) has been explored. All data were obtained at pH 7.0, and 20.0 °C and 37.0 °C. Imatinib binding to the FA7 site of HSA and trHSA inhibits allosterically heme-Fe(III) association to the FA1 site and vice versa, according to linked functions. Moreover, imatinib binding to the secondary FA2 site of HSA-heme-Fe(III) inhibits allosterically peroxynitrite detoxification. Docking simulations and local structural comparison with other imatinib-binding proteins support functional data indicating the preferential binding of imatinib to the FA1 and FA7 sites of HSA, and to the FA2 and FA7 sites of HSA-heme-Fe(III). Present results highlight the allosteric coupling of the FA1, FA2, and FA7 sites of HSA, and may be relevant in modulating ligand binding and reactivity properties of HSA in vivo.
- Comparison between the effects of intraoperative human albumin and normal saline on early graft function in renal transplantation. [JOURNAL ARTICLE]
- Int Urol Nephrol 2014 Jul 24.
The aim of the current study was to compare the effects of intraoperative infusion of 20 % human albumin versus 0.9 % normal saline on early and late graft function in renal transplantation.This prospective, randomized study was conducted on 44 patients with end-stage renal disease undergoing kidney transplantation. Patients were 32 males (72.7 %) and 12 females (27.3 %) with a mean age of 54.35 ± 11.15 years (range 20-58 years). Patients with cardiac disease and liver dysfunction were excluded from the study. Twenty-two of the 44 patients were given intraoperative intravenous infusion of 20 % human albumin with 0.9 % normal saline (albumin group), and the remaining 22 patients received intraoperative intravenous infusion of 0.9 % normal saline alone (saline group), as part of the intraoperative fluid hydration to keep central venous pressure between 10 and 15 mm of Hg.There was no statistically significant difference in mean intravenous fluid volume infused until the end of surgery between the saline group and the albumin group (P = 0.8326). Time of onset of diuresis and total intraoperative urine output were statistically insignificant between the two groups (P = 0.6255, P = 0.9231, respectively). Post-transplant serum creatinine on day 1, 3 and 5 between the albumin and saline groups were comparable (P = 0.8998, P = 0.7257, P = 0.8092, respectively). Post-transplant urine output on day 1, 3 and 5 between the albumin and saline groups were also comparable (P = 0.653, P = 0.9075, P = 0.946, respectively). Mean postoperative weight gain was higher in the saline group compared with the albumin group, but was not statistically significant (P = 0.6348).This study revealed that the use of 20 % human albumin as an intraoperative volume expander provides no more benefit than the use of 0.9 % normal saline in terms of immediate graft function in living donor renal transplantation.
- Characterization of a Novel BCHE "Silent" Allele: Point Mutation (p.Val204Asp) Causes Loss of Activity and Prolonged Apnea with Suxamethonium. [JOURNAL ARTICLE]
- PLoS One 2014; 9(7):e101552.
Butyrylcholinesterase deficiency is characterized by prolonged apnea after the use of muscle relaxants (suxamethonium or mivacurium) in patients who have mutations in the BCHE gene. Here, we report a case of prolonged neuromuscular block after administration of suxamethonium leading to the discovery of a novel BCHE variant (c.695T>A, p.Val204Asp). Inhibition studies, kinetic analysis and molecular dynamics were undertaken to understand how this mutation disrupts the catalytic triad and determines a "silent" phenotype. Low activity of patient plasma butyrylcholinesterase with butyrylthiocholine (BTC) and benzoylcholine, and values of dibucaine and fluoride numbers fit with heterozygous atypical silent genotype. Electrophoretic analysis of plasma BChE of the proband and his mother showed that patient has a reduced amount of tetrameric enzyme in plasma and that minor fast-moving BChE components: monomer, dimer, and monomer-albumin conjugate are missing. Kinetic analysis showed that the p.Val204Asp/p.Asp70Gly-p.Ala539Thr BChE displays a pure Michaelian behavior with BTC as the substrate. Both catalytic parameters Km = 265 µM for BTC, two times higher than that of the atypical enzyme, and a low Vmax are consistent with the absence of activity against suxamethonium. Molecular dynamic (MD) simulations showed that the overall effect of the mutation p.Val204Asp is disruption of hydrogen bonding between Gln223 and Glu441, leading Ser198 and His438 to move away from each other with subsequent disruption of the catalytic triad functionality regardless of the type of substrate. MD also showed that the enzyme volume is increased, suggesting a pre-denaturation state. This fits with the reduced concentration of p.Ala204Asp/p.Asp70Gly-p.Ala539Thr tetrameric enzyme in the plasma and non-detectable fast moving-bands on electrophoresis gels.
- Cytotoxicity and Comparative Binding Mechanism of Piperine with Human Serum Albumin and α-1-Acid Glycoprotein. [JOURNAL ARTICLE]
- J Biomol Struct Dyn 2014 Jul 23.:1-53.
Abstract Human serum albumin (HSA) and α-1-acid glycoprotein (AGP) (acute phase protein) are the plasma proteins in blood system which transports many drugs. To understand the pharmacological importance of piperine molecule, here, we studied the anti-inflammatory activity of piperine on mouse macrophages (RAW 264.7) cell lines, which reveals that piperine caused an increase in inhibition growth of inflammated macrophages. Further, the fluorescence maximum quenching of proteins were observed upon binding of piperine to HSA and AGP through a static quenching mechanism. The binding constants obtained from fluorescence emission was found to be Kpiperine = 5.7 ± 0.2 × 10(5) M(-1) and Kpiperine= 9.3 ± 0.25 × 10 (4) M(-1) which corresponds to the free energy of -7.8 kcal M(-1) and -6.71 kcal M(-1)at 25 °C for HSA and AGP. Further, circular dichrosim studies revealed that there is a marginal change in the secondary structural content of HSA is due to partial destabilization of HSA-piperine complexes. Consequently, inference drawn from the site specific markers (phenylbutazone, site I marker) studies to identify the binding site of HSA was noticed that piperine binds at site I (IIA) which was further authenticated by molecular docking and molecular dynamic studies. The binding constants and free energy corresponds for experimental and computational analysis suggests that there are hydrophobic and hydrophilic interactions when piperine binds to HSA. Additionally, the molecular dynamics (MD) studies have showed that HSA-piperine complex reaches equilibration state at around 3 ns, which prove that the HSA-piperine complex is stable in nature.
- Soluble MHC-II proteins promote suppressive activity in CD4+ T-cells. [JOURNAL ARTICLE]
- Immunology 2014 Jul 22.
Soluble MHC-II (sMHCII) molecules are present in body fluids of healthy individuals and are considered to be involved in the maintenance of self tolerance, while they are also related to various diseases. Their concentration increases during in vivo antigen-specific tolerogenic stimulation and it was recently shown that exosome-mediated tolerance is MHC-II dependent. At the cellular level, sMHCII proteins compete with membrane MHCII for TCR binding on CD4+ T cells. Immunoaffinity purification techniques isolated sMHCII antigens from serum of human serum albumin (HSA)-tolerant mice as single highly glycozylated protein of ~60 kD, specifically interacting with anti-class II antibodies in WESTERN and ELISA techniques. Mass spectroscopy analysis showed these sMHCII proteins were loaded with the tolerogenic peptide as well as multiple self peptides. At the cellular level, sMHCII suppressed antigen-specific and to a lesser degree antigen-non-specific spleen cell proliferation and induced CD25 in naïve T cells. In T cells activated by antigen-seeded macrophages, sMHCII decreased CD28 and increased CTLA-4 protein expression, while decreasing IL-2 and increasing IL-10 production. In this case, sMHCII were shown to decrease ZAP70 and LAT phosphorylation. The results presented here for the first time provide evidence for the role of sMHCII proteins in immune response suppression and maintenance of tolerance, revealing novel regulatory mechanisms for immune system manipulation. This article is protected by copyright. All rights reserved.
- [Investigation of acid proteinase and phospholipase activity as virulence factors in clinical Aspergillus spp. isolates.] [JOURNAL ARTICLE]
- Mikrobiyol Bul 2014 Jul; 48(3):491-494.
Aspergillus spp. are widespread in nature and cause severe infections especially in immunocompromised patients. Aspergillus fumigatus complex is the most common species that causes infections in humans; however Aspergillus niger complex and Aspergillus flavus complex are the emerging agents that are isolated frequently from clinical specimens more recently. Besides the host factors such as immunosuppression, hematologic malignancy and corticosteroid use, fungal virulence factors such as elastase, acid protease and phospholipase enzymes are considered among the factors that affect the development of invasive aspergillosis. The aim of this study was to detect the acid proteinase and phospholipase enzyme activities in 30 A.fumigatus complex, nine A.flavus complex and four A.niger complex strains isolated from clinical specimens. Acid proteinase and phospholipase activities of the isolates were investigated by using bovine serum albumin agar (BSA), and egg yolk agar plates, respectively. Acid proteinase and phospholipase activity was detected in 76.7% (23/30) and 93.3% (28/30) of A.fumigatus complex isolates, respectively. None of the nine A.flavus complex isolates exhibited acid proteinase or phospholipase activity. Acid proteinase activity was not detected in any of the A.niger complex isolates (n= 4), however phospholipase activity was detected in one (25%) isolate. All of the acid proteinase positive A.fumigatus complex strains (n= 23) were also positive for phospholipase activity. In conclusion, further larger scale multicenter studies supported by clinical data, are needed to enlighten the roles of acid proteinase and phospholipase in the pathogenesis of infections due to Aspergillus spp.
- Effect of alcohol fermented feed on lactating performance, blood metabolites, milk Fatty Acid profile and cholesterol content in holstein lactating cows. [Journal Article]
- Asian-Australas J Anim Sci 2012 Nov; 25(11):1546-52.
A feeding experiment with 40 lactating Holstein cows and 4 dietary treatments was conducted to investigate supplementation with different levels of alcohol fermented feed to the TMR on lactating performance, blood metabolites, milk fatty acid profile and cholesterol concentration of blood and milk. Forty Holstein lactating cows (106±24 d post-partum; mean±SD) were distributed into four groups and randomly assigned to one of four treatments with each containing 10 cows per treatment. The treatment supplemented with TMR (DM basis) as the control (CON), and CON mixed with alcohol-fermented feeds (AFF) at a level of 5%, 10% and 15% of the TMR as T1, T2 and T3, respectively. Dry matter intake and milk yield were not affected by supplementation of AFF. An increased 4% FCM in the milk occurred in cows fed T3 diet compared with CON, while T1 and T2 diets decreased 4% FCM in a dose dependent manner. Supplementation of AFF increased the concentration of albumin, total protein (TP), ammonia, and high density lipoprotein-cholesterol in serum compared with CON. In contrast, supplementation with AFF clearly decreased concentration of blood urea nitrogen (BUN) and total cholesterol (TC) compare with CON. AFF supplementation increased the proportion of C18:1n9 and C18:2n6 compared to CON. A decrease in the concentration of saturated fatty acid (SFA) for T1, T2 and T3 resulted in an increased unsaturated fatty acid (USFA) to SFA ratio compared to CON. Concentration of cholesterol in milk fat was reduced in proportion to the supplemental level of AFF. Feeding a diet supplemented with a moderate level AFF to lactating cows could be a way to alter the feed efficiency and fatty acid profile of milk by increasing potentially human consumer healthy fatty acid without detrimental effects on feed intake and milk production. A substantially decreased cholesterol proportion in milk induced by supplementation AFF suggests that alcohol fermented feed may improve milk cholesterol levels without any negative effects in lactating cows.
- Spectrometric characteristics and tumor-affinity of a novel photosensitizer: mono-l-aspartyl aurochlorin e6 (Au-NPe6). [JOURNAL ARTICLE]
- Photodiagnosis Photodyn Ther 2004 Dec; 1(4):295-301.
Photodiagnosis and photodynamic therapy with photosensitizers can be indicated only for tumors of the superficial type, because these approaches utilizing visible light are limited by said light penetrability. To overcome this disadvantage, we innovated a novel photosensitizer, mono-l-aspartyl aurochlorin e6 (Au-NPe6), by incorporating a gold atom in the center of tetrapyrrole ring of NPe6 with a coordination bond. The gold atom in Au-NPe6 plays a role as an X-ray interceptor to detect deeply sited tumors. In this study, the absorption spectrum of novel Au-NPe6 in the diagnosis of deeply sited tumors was investigated, and the results were compared with the parent photosensitizer NPe6. Furthermore, the tumor-affinity of Au-NPe6 was evaluated using atomic absorption spectrometry. Despite the fact that both photosensitizers display a difference in the absorption spectrum, waveform changes of either photosensitizer with human serum albumin established a saturation point at a molar ratio of 1:1. The results indicate that it is highly possible that Au-NPe6 bound with albumin at a molar ratio (1:1) similar to NPe6. The accumulation rate of gold in tumor tissues was always significantly (p<0.05) higher than that in normal muscle tissues during the observation terms. Moreover, absorption spectra of tumor-tissue homogenates obtained from tumor-bearing mice after Au-NPe6 administration revealed a common peak with a wavelength equivalent to that of albumin-bond Au-NPe. This result suggests that the gold atom and NPe6 probably remained bonded even when Au-NPe6 was incorporated in tumor tissues.