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Adsorption properties of hemodialyzers are traditionally retrieved from diffusive treatments and mainly focused on inflammatory markers and plasma proteins. The possible depurative enhancement of middle and high molecular weight solutes, as well as protein-bound uremic toxins by adsorption in convective treatments, is not yet reported. We used discarded plasma exchanges from uremic patients and out-of-date erythrocytes as a novel in vitro uremic precursor matrix to assess removal and adsorption patterns of distinct material and structure but similar surface hemodialyzers in hemodialysis and on-line hemodiafiltration treatments. We further related the obtained results to the possible underlying membrane pore blocking mechanisms. Convection improved removal but slightly enhanced adsorption in the cellulosic and synthetic dialyzers tested. The polymethylmethacrylate hemodialyzer obtained the highest extracted ([Formula: see text]) and adsorbed ([Formula: see text]) mass values when submitted to hemodiafiltration for all molecules analyzed including albumin ([Formula: see text] g, [Formula: see text] mg), whereas the polyamide membrane obtained substantial lower results even for this molecule ([Formula: see text] g, [Formula: see text] mg) under the same treatment parameters. Hemodiafiltration in symmetric and enlarged pore hemodialyzers enhances removal and adsorption by internal pore deposition (intermediate pore-blocking) for middle and high molecular weight toxins but leads to substantial and deleterious albumin depuration.

The impact of pH (6-9) and NaCl concentration (0-0.5 mol.L -1 ) on sunflower protein extraction was studied through design of experiments. The considered criteria were protein extraction yield (total proteins, helianthinin and albumins), chlorogenic acids covalently bound to proteins, and free chlorogenic acid concentration in the aqueous extract. Statistical analysis showed that the obtained by design of experiments the polynomial models of each extraction criteria were reliable for predicting the responses. They were employed in an original multi-objective optimization methodology. The optimal conditions revealed to be pH 7.3/0.3 mol.L -1 NaCl yielded 46.83% and 59.16% of total protein and albumin extraction yield, 1.730 and 1.998 mg.g -1 of chlorogenic acids covalently bound to helianthinin and albumins in aqueous extract, respectively. The sunflower protein isolate obtained after extraction in this condition had good solubility (40-80% at pH 5-8), functional properties (foaming and emulsifying) and a satisfying color., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Aliphatic di- and polyisocyanates are crucial chemical ingredients in many industrial processes and are a well-recognized cause of occupational asthma. Serologic detection of "chemical epitopes" in biological samples could serve as an exposure surveillance approach toward disease prevention, and thus we sought to generate aliphatic isocyanate-specific monoclonal antibodies (mAbs). Three hybridomas were generated from Balb/c mice immunized with a commercial product containing a combination of uretdione, homopolymer, and monomeric forms of hexamethylene diisocyanate (HDI). Three stable hybridomas were subcloned by limiting dilution, two secreting IgG1κ and one secreting IgMκ mAb that bind aliphatic di- and polyisocyanates (conjugated to albumin), but not aromatic toluene or methylene diphenyl diisocyanate (TDI or MDI). Each mAb demonstrates slight differences in epitope specificity, for example, recognition of hydrogenated MDI (HMDI) or different carrier proteins (transferrin, actin) reacted with vapor phase HDI, and is encoded by unique recombination of different germline antibody genes, with distinct complementary determining regions. By western blot, all three mAbs detect a molecule with characteristics of an albumin adduct uniquely in urine from mice skin exposed to a mixture of aliphatic di- and polyisocyanate. Together, the data define molecular determinants of humoral immune recognition of aliphatic di- and polyisocyanates through new mAbs, which will serve as useful research reagents and may be applicable to future exposure surveillance efforts.

Commercial fractionation of human plasma into immunoglobulin- and albumin-rich fractions is often initiated with sequential cold ethanol-based precipitation methods, which have changed little over the past 70 years. The required low temperature (-4 to -8°C) and high concentrations of ethanol 8-40%) necessitate large-scale fixed processing lines, and major capital investment and operating costs. The resulting fractions are then further purified by ethanol based precipitation or chromatographic procedures to obtain the purified final product. Aqueous polyacrylic acid (PAA) based precipitation, which readily interfaces with existing downstream processing, could offer advantages with respect to cost, safety, environmental impact, and flexibility. Sequential precipitation with 7%, 12%, and 20% (w/v) solutions of PAA 8000 in the presence of a kosmotropic salt (sodium citrate) gave fibrinogen-, immunoglobulin-, and albumin-rich fractions with 80-90% yield and 64%, 55%, and 82% purity, respectively. Further purification of the IgG-rich precipitate by caprylic acid precipitation and anion exchange chromatography, achieved a target purity of >99%. This was also achieved for the downstream processing of the albumin-rich precipitate using a two-step ion exchange chromatographic procedure. This work shows that PAA precipitation can be used in place of cold ethanol precipitation to generate crude IgG and albumin fractions which can be purified to final products of acceptable purity., (© 2020 Wiley Periodicals, Inc.)

A readily synthesizable fluorescent probe DMAT-π-CAP was evaluated for sensitive and selective detection of human serum albumin (HSA). DMAT-π-CAP showed selective turn-on fluorescence at 730 nm in the presence of HSA with more than 720-fold enhancement in emission intensity ([DMAT-π-CAP] = 10 μM), and rapid detection of HSA was accomplished in 3 seconds. The fluorescence intensity of DMAT-π-CAP was shown to increase in HSA concentration-dependent manner ( K d = 15.4 ± 3.3 μM), and the limit of detection of DMAT-π-CAP was determined to be 10.9 nM (0.72 mg/L). The 1:1 stoichiometry between DMAT-π-CAP and HSA was determined, and the displacement assay revealed that DMAT-π-CAP competes with hemin for the unique binding site, which rarely accommodates drugs and endogenous compounds. Based on the HSA-selective turn-on NIR fluorescence property as well as the unique binding site, DMAT-π-CAP was anticipated to serve as a fluorescence sensor for quantitative detection of the HSA level in biological samples with minimized background interference. Thus, urine samples were directly analyzed by DMAT-π-CAP to assess albumin levels, and the results were comparable to those obtained from immunoassay. The similar sensitivity and specificity to the immunoassay along with the simple, cost-effective, and fast detection of HSA warrants practical application of the NIR fluorescent albumin sensor, DMAT-π-CAP, in the analysis of albumin levels in various biological environments., Competing Interests: The authors declare no conflict of interest.

Introduction: Particles in exhaled air (PEx) provide samples of respiratory tract lining fluid from small airways containing, for example, Surfactant protein A (SP-A) and albumin, potential biomarkers of small airway disease. We hypothesized that there are differences between morning, noon, and afternoon measurements and that the variability of repeated measurements is larger between days than within days., Methods: PEx was obtained in sixteen healthy non-smoking adults on 11 occasions, within one day and between days. SP-A and albumin were quantified by ELISA. The coefficient of repeatability (CR), intraclass correlation coefficient (ICC), and coefficient of variation (CV) were used to assess the variation of repeated measurements., Results: SP-A and albumin increased significantly from morning towards the noon and afternoon by 13% and 25% on average, respectively, whereas PEx number concentration and particle mean mass did not differ significantly between the morning, noon and afternoon. Between-day CRs were not larger than within-day CRs., Conclusions: Time of the day influences the contents of SP-A and albumin in exhaled particles. The variation of repeated measurements was rather high but was not influenced by the time intervals between measurements., Competing Interests: I have read the journal's policy and the authors of this manuscript have the following competing interests: Björn Bake, Marianne Andersson and Gunilla Runström Eden are shareholders of PEXA® AB (www.PEXA.se) and Anna-Carin Olin is a board member (patent WO2009045163). This does not alter our adherence to PLOS ONE policies on sharing data and materials.

The purpose of this paper was to study the extraction conditions of hemp proteins from undelipidated press-cakes. The effects of different hydration conditions on protein recovery yield and polypeptide profile were evaluated: pH (2 to 12), ionic strength (0 to 500 mM NaCl) and press-cake/liquid weight ratio (2% to 22%). pH was the most impacting factor. At acidic pH (2 to 7) the extraction yields were low and quite constant (<7%), corresponding mainly to hemp albumins solubilization. The extraction of globulins started to increase significantly from pH 8, with protein extraction yield varying from 8.3% at pH 8 to 67.1% at pH 12 for a 10% press-cake/liquid weight ratio. The addition of NaCl in press-cake suspensions did not increase the total nitrogen content in aqueous extracts at alkaline pH while the lowest press-cake/liquid weight ratios (5% to 10%) were revealed optimal regarding protein recovery rate. The intense coloration observed on the aqueous extracts above pH 8 was assigned to solubilization and oxidation of phenolic compounds whose concentration increased about sevenfolds from pH 2 to 12. At the highest applied pH (11 to 12), the formation of covalent complexes between phenolic compounds and some hemp polypeptides was hypothesized. Aqueous extraction at strong alkaline pH (>9) without salt addition and respecting a 10% press-cake/liquid weight ratio should be retained to optimize protein extraction yield. However, further purification steps are required to evaluate the nutritional, organoleptic, and techno-functional properties of hemp proteins extracted in such conditions. PRACTICAL APPLICATION: The traditional extraction process of hemp proteins by alkaline solubilization and isoelectric precipitation, mostly from delipidated hemp press-cake, leads to limited quantity and poor solubility of extracted proteins, and data related to extraction conditions are insufficiently available to optimize this process. This article aims to find optimal hydration conditions (pH, ionic strength, press-cake to liquid ratio) for protein extraction from undelipidated hemp press-cake, allowing high protein recovery and preserving protein quality. The results obtained represent very useful data for developing an economically viable and sustainable extraction process of proteins from raw hemp press-cake., (© 2019 Institute of Food Technologists®.)

The effects of ultrasonic-assisted extraction (UAE, 200 W, 20 min) on the yield and physicochemical properties of different walnut proteins (WNPs, including albumin, globulin, and glutelin) were investigated. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis indicated that UAE could result in protein molecular fragmentation of albumin, but did not affect the major bands of globulin and glutelin. The CD spectra demonstrated that different WNPs obtained by UAE had different changes in their secondary structure. Under UAE, there was an increase in surface hydrophobicity ( H 0 ) of albumin and gluten and no change in the fluorescence intensity, while decreases were observed in the H 0 and fluorescence intensity of globulin; and the contents of total and surface free sulfhydryl in albumin dramatically decreased. UAE reduced the size of the particles and the dimension of the microstructures in albumin and gluten, indicating that ultrasound could unfold protein aggregates. In addition, UAE increased the solubility, emulsifying activity (EA), foaming capacity (FC), and foam stability (FS) of the obtained proteins. The above results indicate that ultrasound extraction is a promising approach to improve the extraction yield and properties of walnut proteins.

Microalbuminuria is an important clinical marker of several cardiovascular, metabolic, and other diseases such as diabetes, hypertension, atherosclerosis, and cancer. The accurate detection of microalbuminuria relies on albumin quantification in the urine, usually via an immunoturbidity assay; however, like many antibody-based assessments, this method may not be robust enough to function in global health applications, point-of-care assays, or wearable devices. Here, we develop an antibody-free approach using synthetic molecular recognition by constructing a polymer to mimic fatty acid binding to the albumin, informed by the albumin crystal structure. A single-walled carbon nanotube, encapsulated by the polymer, as the transduction element produces a hypsochromic (blue) shift in photoluminescence upon the binding of albumin in clinical urine samples. This complex, incorporated into an acrylic material, results in a nanosensor paint that enables the detection of microalbuminuria in patient samples and comprises a rapid point-of-care sensor robust enough to be deployed in resource-limited settings.

The method described in the article aims at the quantification of both main storage proteins, globulins and albumins, in aqueous extract from rapeseed, as an alternative to the current reference methods, Kjeldahl and SDS-PAGE electrophoresis. The new method lies on the analytical separation of extracted compounds by Size-Exclusion High Performance Liquid Chromatography (SE-HPLC) (Biosep-SEC-s2000, 5 µm). The elution of rapeseed extracts with water/acetonitrile/trifluoroacetic acid (45/55/0.1% v/v) during 30 min yields two distinct peaks for the main proteins of rapeseed. Based on the protein extinction coefficients, a calibrationless methodology was developed for their quantification on the basis of the UV signal. The SE-HPLC method was successfully compared to references: Kjeldahl and SDS-PAGE densitometry for the determination of the proportion of each protein. Then, it was successfully applied on two other oleoproteagineous plants, linseed and sunflower., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Blood biomarkers are usually present in low concentration and can be masked by the high-abundance proteins, of which albumin is the predominant one. The purpose of this study was to compare four different albumin removal methods compatible with in-gel based proteomics, applicable for plasma, without requiring specific techniques and high financial input. Plasma underwent albumin depletion with ultrafiltration device Amicon Ultra, commercial ProteoPrep Blue Albumin and IgG Depletion Kit, acetonitrile precipitation method and precipitation with acetonitrile-methanol protocol. All samples were evaluated by 1-D and 2-D gel electrophoresis with subsequent mass spectrometry protein identification. Two of the tested methods (ProteoPrep BlueKit and acetonitrile-methanol precipitation) maintained sufficient protein content for further in-gel analyses. Their 2-D protein profiles were distinctively separated and overlapped with protein profile of crude plasma. Protein spot count showed significant increase in protein spots, compared to crude plasma, only with acetonitrile-methanol precipitation method. Precipitation with acetonitrile-methanol method significantly increased number of protein spots on 2-D protein profile and improved score of mass spectrometry identification. However, albumin was still present and found in number of protein spots.

Oxidative stress is a risk for and cause of various disease, however, measurements of oxidative stress are either time-consuming or non-specific. Here, we established a rapid method of using high performance liquid chromatography (HPLC) to measure serum oxidized albumin in a rat model. We optimized HPLC conditions for rat oxidized albumin. To validate our method, three-week-old male Sprague-Dawley rats were uninephrectomized and treated normal diet, high salt diet or high salt diet with Tempol, a superoxide dismutase (SOD) mimetic. After 4 weeks of treatment, we analyzed serum oxidized albumin. The main findings are listed as below. (i) Our method of oxidized albumin measurement only takes 16 minutes, with an intra-day and inter-day deviation within 1% and a detection limit concentration of 6.4 mg/ml. (ii) Oxidized albumin levels were significantly higher in the high salt diet group than in the normal salt diet group, and this effect was reversed by Tempol. (iii) Oxidized albumin levels also correlated with urinary protein and 8-isoprostane levels. In conclusion, we have established a simple method for evaluating rat serum oxidized albumin using HPLC. Our method is rapid and has an advantage over conventional methods and may be useful for animal models of oxidative stress.

Background: The first cases of food allergy to amaranth grain have recently been published. This pseudocereal is considered hypoallergenic, and there is scarce information about the allergenic potential of amaranth proteins, either before or after food processing. Objective: To evaluate, in a mouse model of food allergy, the sensitizing and allergenic potential of extruded and non-extruded albumin and globulin fractions from amaranth grains. Materials and Methods: Amaranth ( Amaranthus hypochondriacus ) flour was obtained and the albumin and globulin fractions isolated. These protein fractions were also obtained after flour extrusion. An intraperitoneal 28-day protocol was carried out to evaluate the sensitizing and allergenic potential of the proteins. The common and rarely allergenic proteins ovalbumin and potato acidic phosphatase were utilized as reference. Specific IgE and IgG antibodies were evaluated for all the proteins tested. Mast cell protease-1 (mMCP-1) responses were evaluated in serum samples collected after intragastric challenges with the proteins of interest. All serological evaluations were carried out using ELISA. Results: Mice were sensitized to the non-extruded albumin fraction from amaranth grains and to ovalbumin ( p = 0.0045). The extrusion process of amaranth proteins abrogated the IgE responses triggered under non-extruded conditions ( p = 0.0147). mMCP-1 responses were significantly detected in the group of mice sensitized to ovalbumin ( p = 0.0138), but not in others. Conclusions : The non-extruded albumin fraction from amaranth has the potential to sensitize BALB/c mice, but this sensitizing potential fails to induce detectable serum levels of the mast cell degranulation marker mMCP-1 after intragastric challenges. Furthermore, the extrusion process abolished the sensitization potential of the amaranth albumins., Competing Interests: The authors declare that they have no conflicts of interest.

Blood serum or plasma proteome is a gold mine of disease biomarkers. However, complexity and a huge dynamic range of their components, combined with multiple mechanisms of degradation and posttranslational modifications, further complicated by the presence of lipids, salts, and other metabolites, represent a real challenge for analytical sensitivity, resolution, and reproducibility. This problem exists particularly in the case of potential disease-specific markers, most typically represented by low-abundant proteins (LAPs), whose detection is usually impaired by the dominance of albumins, immunoglobulins, and other high-abundant serum/plasma proteins (HAPs). Hence, analysis of biomarker candidates in serum/plasma samples frequently requires separation of their components, usually including depletion of albumin in a fraction of interest. Such "preprocessing" of serum/plasma specimens is critical in proteomic analysis based on mass spectrometry. This approach is very potent; nevertheless a wide range of protein concentrations in serum/plasma represents a particular challenge, since high-abundant proteins (mostly albumin) dominate in a sample subjected to mass spectrometry and suppress peptide ions originating from low-abundant proteins, thus limiting probability and reliability of their detection. An emerging approach in serum-/plasma-based biomarker-oriented studies is the proteome component of exosomes - nanovesicles secreted by cells and involved in multiple aspects of intercellular communication. However, the presence of albumin, frequent contaminant of exosomes isolated from human serum/plasma, represents a real challenge also in this type of study. A similar problem is encountered in proteomic studies based on exosomes obtained in in vitro experiments where culture media are normally supplemented with fetal bovine serum containing growth factors and hormones. In this case exosomes are frequently contaminated with bovine serum albumin and other bovine serum proteins which should be removed before proteomic analysis of exosome cargo.

Inhibiting starch hydrolysis into sugar could reduce postprandial blood glucose elevation and contribute to diabetes prevention. Here, both buckwheat and wheat albumin that inhibited mammalian α-amylase in vitro suppressed blood glucose level elevation after starch loading in vivo, but it had no effect after glucose loading. In contrast to the non-competitive inhibition of wheat α-amylase inhibitor, buckwheat albumin acted in a competitive manner. Although buckwheat α-amylase inhibitor was readily hydrolysed by digestive enzymes, the hydrolysate retained inhibitory activity. Together with its thermal stability, this suggests its potential use in functional foods that prevent diabetes.

Drugs to protect against nerve agent toxicity are tested in animals. The current preferred small animal model is guinea pigs because their plasma bioscavenging capacity resembles that of NHP. We stained nondenaturing polyacrylamide slab gels with a variety of substrates, inhibitors, and antibodies to identify the esterases in heparinized guinea pig plasma. An intense band of carboxylesterase activity migrated behind albumin. Minor carboxylesterase bands were revealed after background activity from paraoxonase was inhibited by using EDTA. The major butyrylcholinesterase band was a disulfide-linked dimer. Incubation with the antihuman butyrylcholinesterase antibody B2 18-5 shifted the butyrylcholinesterase dimer band to slower migrating complexes. Carboxylesterases were distinguished from butyrylcholinesterase by their sensitivity to inhibition by bis- p -nitrophenyl phosphate. Acetylcholinesterase tetramers formed a complex with the antihuman acetylcholinesterase antibody HR2. Organophosphorus toxicants including cresyl saligenin phosphate, dichlorvos, and chlorpyrifos oxon irreversibly inhibited the serine esterases but not paraoxonase. Albumin pseudoesterase activity was seen in gels stained with α- or β-naphthyl acetate and fast blue RR. We conclude that guinea pig plasma has 2 types of carboxylesterase, butyrylcholinesterase dimers and 5 minor butyrylcholinesterase forms, a small amount of acetylcholinesterase tetramers, paraoxonase, and albumin pseudoesterase activity. A knockout mouse with no carboxylesterase activity in plasma is available and may prove to be a better model for studies of nerve agent toxicology than guinea pigs.

Wheat allergy specifically refers to the adverse reaction involving IgE antibody to one or more protein fraction of wheat such as albumin, globulin, gliadin and glutenin (gluten). The majority of IgE-mediated reactions to wheat involve albumin and globulin fraction while gluten (gliadin & glutenin) also cause allergy (Celiac disease). Allergic reactions to wheat may be caused by ingestion of wheat containing foods or inhalation of flour (Bakers asthma). The present study was an effort to explore the antibody response of different proteins present in wheat. ELISA results revealed that the antibody response for albumin varied from 0.92-1.78, whereas, for globulin ranged from 1.39-1.60. Antibody response against glutenin and gliadin ranged from 0.57-1.05 and 0.98-1.95 respectively, among the different varieties of wheat. All the tested wheat varieties showed the significant difference antibody response against the different fractions of protein.

Some molecular and functional properties of albumin (83.6% protein), globulin (95.5% protein), glutelin (81.3% protein) as well as protein isolate (80.7% protein) from cashew nut were investigated. These proteins were subjected to molecular (circular dichroism, gel electrophoresis, scanning electron microscopy) and functional (solubility, emulsification, foaming, water/oil holding capacity) tests. Cashew nut proteins represent an abundant nutrient with well-balanced amino acid composition and could meet the requirements recommended by FAO/WHO. SDS-PAGE pattern indicated cashew nut proteins were mainly composed of a polypeptide with molecular weight (MW) of 53 kDa, which presented two bands with MW of 32 and 21 kDa under reducing conditions. The far-UV CD spectra indicated that cashew proteins were rich in β-sheets. The surface hydrophobicity of the protein isolate was higher than that of the protein fractions. In pH 7.0, the solubility of protein fractions was above 70%, which was higher than protein isolate at any pH. Glutelin had the highest water/oil holding capacity and foaming properties. Protein isolate displayed better emulsifying properties than protein fractions. In summary, cashew nut kernel proteins have potential as valuable nutrition sources and could be used effectively in the food industry., Competing Interests: The authors declare no conflict of interest.

In this study, Single walled carbon nanotube (SWNT)-streptavidin complexes were used to capture and purify biotinylated proteins, including bio-GFP and bio-DBS using a pull-down method. The purification conditions were systematically studied, including surface blocking of SWNT using chicken egg albumin (CEA), the ratio of SWNT-streptavidin complexes to the cell lysate, as well as the centrifugation speed. Optimization of the protein purification using SWNT-streptavidin complexes shows the possibility of carbon nanotubes as a promising candidate for protein purification applications. The SWNT-streptavidin could be used as a scaffold to analyze protein structure directly by cryo-transmission electron microscopy, which provides better understanding in protein–protein interactions and biological processes.

Large middle molecules (LMM) are an important subclass of uremic toxins. Many of them have been linked with poor outcomes in hemodialysis (HD) patients. The onset of high-flux membranes and convective techniques allowed to dramatically improve their clearance but without a clear and undebatable reduction of mortality in HD patients. Despite the real effect on the removal of selected toxins, little is known about the influence of modern HD techniques on the global removal of uremic toxins. Mostly explained by a lack of knowledge and selective assays, LMM removal is not evaluated appropriately. The development of highly sensitive and widespread detection techniques such as mass spectrometry could increase our knowledge about the real state of their removal in HD. Nevertheless, these techniques remain cost effective and are difficult to handle. On the contrary, the improvement of LMM removal raises the question of a tolerable albumin removal. Indeed, increasing membrane permeability can significantly increase LMM removal accompanied by a higher albumin loss. However, in chronic kidney disease and particularly in HD patients, albumin can be modified, and it subsequently exerts detrimental effects. This could be avoided by the clearance of the modified forms in HD, but future efforts should be done to investigate the real impact of their removal., (© 2017 S. Karger AG, Basel.)

The electro-osmotic flow, a significant factor in capillary electrophoretic separations, is very sensitive to small changes in structure and surface roughness of the inner surface of fused silica capillary. Besides a number of negative effects, the electro-osmotic flow can also have a positive effect on the separation. An example could be fused silica capillaries with homogenous surface roughness along their entire separation length as produced by etching with supercritical water. Different strains of methicillin-resistant and methicillin-susceptible Staphylococcus aureus were separated on that type of capillaries. In the present study, fused-silica capillaries with a gradient of surface roughness were prepared and their basic behavior was studied in capillary zone electrophoresis with UV-visible detection. First the influence of the electro-osmotic flow on the peak shape of a marker of electro-osmotic flow, thiourea, has been discussed. An antifungal agent, hydrophobic amphotericin B, and a protein marker, albumin, have been used as model analytes. A significant narrowing of the detected zones of the examined analytes was achieved in supercritical-water-treated capillaries as compared to the electrophoretic separation in smooth capillaries. Minimum detectable amounts of 5 ng/mL amphotericin B and 5 μg/mL albumin were reached with this method., (© 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Introduction: There is a growing interest in new therapeutic strategies for the treatment of Alzheimer disease (AD) which focus on reducing the beta-amyloid peptide (Aβ) burden in the brain by sequestering plasma Aβ, a large proportion of which is bound to albumin and other proteins. This review discusses the concepts of interaction between Aβ and albumin that have given rise to AMBAR (Alzheimer's Disease Management by Albumin Replacement) project, a new multicentre, randomised, controlled clinical trial for the treatment of AD., Development: Results from preliminary research suggest that Albutein(®) (therapeutic albumin, Grifols) contains no quantifiable levels of Aβ. Studies also show that Albutein(®) has Aβ binding capacity. On the other hand, AD entails a high level of nitro-oxidative stress associated with fibrillar aggregates of Aβ that can induce albumin modification, thus affecting its biological functions. Results from the phase ii study confirm that using therapeutic apheresis to replace endogenous albumin with Albutein(®) 5% is feasible and safe in patients with AD. This process resulted in mobilisation of Aβ and cognitive improvement in treated patients. The AMBAR study will test combination therapy with therapeutic apheresis and haemopheresis with the possible leverage effect of Albutein(®) with intravenous immunoglobulin replacement (Flebogamma(®) DIF). Cognitive, functional, and behavioural changes in patients with mild to moderate AD will be assessed., Conclusions: the AMBAR study represents a new therapeutic perspective for AD., (Copyright © 2014 Sociedad Española de Neurología. Publicado por Elsevier España, S.L.U. All rights reserved.)

Biomarkers in exhaled breath are useful for respiratory disease diagnosis in human volunteers. Conventional methods that collect non-volatile biomarkers, however, necessitate an extensive dilution and sanitation processes that lowers collection efficiencies and convenience of use. Electret filter emerged in recent decade to collect virus biomarkers in exhaled breath given its simplicity and effectiveness. To investigate the capability of electret filters to collect protein biomarkers, a model that consists of an atomizer that produces protein aerosol and an electret filter that collects albumin and carcinoembryonic antigen-a typical biomarker in lung cancer development- from the atomizer is developed. A device using electret filter as the collecting medium is designed to collect human albumin from exhaled breath of 6 volunteers. Comparison of the collecting ability between the electret filter method and other 2 reported methods is finally performed based on the amounts of albumin collected from human exhaled breath. In conclusion, a decreasing collection efficiency ranging from 17.6% to 2.3% for atomized albumin aerosol and 42% to 12.5% for atomized carcinoembryonic antigen particles is found; moreover, an optimum volume of sampling human exhaled breath ranging from 100 L to 200 L is also observed; finally, the self-designed collecting device shows a significantly better performance in collecting albumin from human exhaled breath than the exhaled breath condensate method (p<0.05) but is not significantly more effective than reported 3-stage impactor method (p>0.05). In summary, electret filters are potential in collecting non-volatile biomarkers in human exhaled breath not only because it was simpler, cheaper and easier to use than traditional methods but also for its better collecting performance.

The objective of this study was to elucidate the mixing state of proteins and amino acid excipients concentrated in the amorphous non-ice region of frozen solutions. Thermal analysis of frozen aqueous solutions was performed in heating scans before and after a heat treatment. Frozen aqueous solutions containing a protein (e.g., recombinant human albumin, gelatin) or a polysaccharide (dextran) and an amino acid excipient (e.g., L-arginine, L-arginine hydrochloride, L-arginine monophosphate, sodium L-glutamate) at varied mass ratios showed single or double T g ' (glass transition temperature of maximally freeze-concentrated solutes). Some mixture frozen solutions rich in the polymers maintained the single T g ' of the freeze-concentrated amorphous solute-mixture phase. In contrast, amino acid-rich mixture frozen solutions revealed two T g 's that suggested transition of concentrated non-crystalline solute-mixture phase and excipient-dominant phase. Post-freeze heat treatment induced splitting of the T g ' in some intermediate mass ratio mixture solutions. The mixing state of proteins and amino acids varied depending on their structure, salt types, mass ratio, composition of co-solutes (e.g., NaCl) and thermal history. Information on the varied mixing states should be valuable for the rational use of amino acid excipients in lyophilized protein pharmaceuticals.

There is a rising need for low-cost and scalable platforms for sensitive medical diagnostic testing. Fabric weaving is a mature, scalable manufacturing technology and can be used as a platform to manufacture microfluidic diagnostic tests with controlled, tunable flow. Given its scalability, low manufacturing cost (<$0.25 per device), and potential for patterning multiplexed channel geometries, fabric is a viable platform for the development of analytical devices. In this paper, we describe a fabric-based electrophoretic platform for protein separation. Appropriate yarns were selected for each region of the device and weaved into straight channel electrophoretic chips in a single step. A wide dynamic range of analyte molecules ranging from small molecule dyes (<1 kDa) to macromolecule proteins (67-150 kDa) were separated in the device. Individual yarns behave as a chromatographic medium for electrophoresis. We therefore explored the effect of yarn and fabric parameters on separation resolution. Separation speed and resolution were enhanced by increasing the number of yarns per unit area of fabric and decreasing yarn hydrophilicity. However, for protein analytes that often require hydrophilic, passivated surfaces, these effects need to be properly tuned to achieve well-resolved separations. A fabric device tuned for protein separations was built and demonstrated. As an analytical output parameter for this device, the electrophoretic mobility of a sedimentation marker, Naphthol Blue Black bovine albumin in glycine-NaOH buffer, pH 8.58 was estimated and found to be -2.7 × 10(-8) m(2) V(-1) s(-1). The ability to tune separation may be used to predefine regions in the fabric for successive preconcentrations and separations. The device may then be applied for the multiplexed detection of low abundance proteins from complex biological samples such as serum and cell lysate.

We recently demonstrated glycation of monoclonal IgA and the presence of IgA-albumin complexes, but the significance of the complexes was not clear. We describe a non-diabetic patient with IgA type M-protein whose serum fructosamine and glycoalbumin levels were elevated. On electrophoresis of the serum protein of the patient, the albumin band shifted to the cathode side. The abnormal precipitin arc of IgA-albumin complexes was detected by immunoelectrophoresis. To elucidate the mechanism of IgA-albumin complexes, we analyzed their properties using immunoelectrophoresis, Western blotting, and two-dimensional gel electrophoresis. The macromolecularized albumin spots were demonstrated by two-dimensional Western blotting with antiserum to human albumin of the patient's serum. Moreover, the IgA-albumin complexes were dissociated on treatment with 2-mercaptoethanol. It can be considered that albumin is bound to the monoclonal IgA molecule by covalent disulfide bonds, and that the albumin binding site is located near the hinge region (311Cys) of the IgA molecule and involves the free SH group, thought to be present in the α-chain.

Removal of highly abundant proteins in plasma is often carried out using immunoaffinity depletion to extend the dynamic range of measurements to lower abundance species. While commercial depletion columns are available for this purpose, they generally are not applicable to limited sample quantities (<20 μL) due to low yields stemming from losses caused by nonspecific binding to the column matrix and concentration of large eluent volumes. Additionally, the cost of the depletion media can be prohibitive for larger-scale studies. Modern LC-MS instrumentation provides the sensitivity necessary to scale-down depletion methods with minimal sacrifice to proteome coverage, which makes smaller volume depletion columns desirable for maximizing sample recovery when samples are limited, as well as for reducing the expense of large-scale studies. We characterized the performance of a 346 μL column volume microscale depletion system, using four different flow rates to determine the most effective depletion conditions for ∼6-μL injections of human plasma proteins and then evaluated depletion reproducibility at the optimum flow rate condition. Depletion of plasma using a commercial 10-mL depletion column served as the control. Results showed depletion efficiency of the microscale column increased as flow rate decreased, and that our microdepletion was reproducible. In an initial application, a 600-μL sample of human cerebrospinal fluid (CSF) pooled from multiple sclerosis patients was depleted and then analyzed using reversed phase liquid chromatography-mass spectrometry to demonstrate the utility of the system for this important biofluid where sample quantities are more commonly limited.

The present study reports the characterisation of a novel ~12-kDa heterodimeric protein, designated as putrin, from the seeds of Putranjiva roxburghii. The purification of putrin to homogeneity was accomplished using DEAE-sepharose where protein was unbound, CM-sepharose and Cibacron blue 3GA where it was bound and appeared as single peak on a size-exclusion chromatography column. A 15 % sodium dodecyl sulphate polyacrylamide electrophoresis gel, under reducing condition, demonstrated that putrin is made of two polypeptide chains of approximately 4.5 and 7.5 kDa. Circular dichroism studies demonstrated the helical nature and conformational stability of protein at increasing temperatures. Putrin exhibited both RNase and DNase activities and exerted antifungal activity but possessed relatively weak translation-inhibitory activity in cell-free system. The cloning and sequence analysis revealed a 414 bp open reading frame encoding a preproprotein of 137 amino acid residues. The amino acid sequence comparisons and phylogenetic analysis of putrin showed significant homology to 2S seed storage family proteins. The results demonstrated that putrin belongs to 2S albumin family and exhibits a spectrum of biotechnologically exploitable functions.

Albumins, globulins, gliadins, and glutenins were isolated from wheat flour and the effects of those proteins on retrogradation of wheat starch were investigated. The results showed that only glutenins retarded retrogradation of wheat starch and other 3 proteins promoted it. The results of IR spectra proved that no S-S linkage formed during retrogradation of wheat starch blended with wheat proteins. Combination of wheat starch and globulins or gliadins through glucosidic bonds hindered the hydrolysis of wheat starch by α-amylase. The melting peak temperatures of retrograded wheat starch attached to different proteins were 128.46, 126.14, 132.03, 121.65, and 134.84 °C for the control with no protein, albumins, glutenins, globulins, gliadins groups, respectively, and there was no second melting temperature for albumins group. Interaction of wheat proteins and starch in retrograded wheat starch greatly decreased the endothermic enthalpy (△H) of retrograded wheat starch. Retrograded wheat starch bound to gliadins might be a new kind of resistant starch based on glycosidic bond between starch and protein., (© 2014 Institute of Food Technologists®)

Hierarchical imprinting was developed to prepare the protein imprinted materials, as the artificial antibody, for the selective depletion of HSA from the human serum proteome. Porcine serum albumin (PSA) was employed as the dummy template for the fabrication of the recognition sites. To demonstrate the advantages of the hierarchical imprinting, molecularly imprinted polymers prepared by hierarchical imprinting technique (h-MIPs) were compared with those obtained by bulk imprinting (b-MIPs), in terms of the binding capacity, adsorption kinetics, selectivity and synthesis reproducibility. The binding capacity of h-MIPs could reach 12 mg g(-1). And saturation binding could be reached in less than 20 min for the h-MIPs. In the protein mixture, h-MIPs exhibit excellent selectivity for PSA, with imprinting factors as about 3.6, much higher than those for non-template proteins. For the proteomic application, the identified protein group number in serum treated by h-MIPs was increased to 422, which is 21% higher than that obtained from the original serum, meanwhile the identified protein group number for the Albumin Removal kit was only 376. The results demonstrate that protein imprinted polymers prepared by hierarchical imprinting technique, might become the artificial antibodies for the selective depletion of high abundance proteins in proteome study.

This study describes a green, facile and low cost approach for imprinting protein on the surface of multi-walled carbon nanotubes (MWNTs) using papain as the template, dopamine as the functional monomer. By simply mixing MWNTs, dopamine, template protein in weak alkaline aqueous solution, a thin adherent polydopamine (PDA) film imprinted with protein was spontaneously obtained on the surface of MWNTs to produce the imprinted nanomaterials (MWNTs@MIPs). The obtained MWNTs@MIPs were characterized with Fourier transform infrared spectrometer (FT-IR), Raman spectroscopy, X-ray photoelectron spectroscopy (XPS), scanning electron microscopy (SEM) and transmission electron microscopy (TEM). The adsorption process of the MWNTs@MIPs towards template protein was investigated in detail. The effects of the concentration of the monomer and template, polymerization time, extraction process were optimized. The prepared MWNTs@MIPs show fast binding kinetics, high binding capacity and acceptable specific recognition behavior towards template proteins. Furthermore, the stability and regeneration were also investigated, which indicated that the MWNTs@MIPs had good reusability. The good recognizing behavior coupled to the low cost and facile one-step preparation make the MWNTs@MIPs attractive for separation and specific protein recognition., (© 2013 Published by Elsevier B.V.)

Background: Protein composition, amino acid profile and nutritional value of the lotus seed and its Osborne fractions were investigated. The seed was rich in protein with 19.85%, and showed well balanced amino acid composition compared with FAO/WHO pattern, Its nutritive properties were similar to those observed in the reference soybean protein. Phenylalanine, tyrosine, leucine and lysine were the limiting amino acids in the seed proteins. The albumin and globulin were the main protein fraction, the amino acid profile and nutritional value were close to the seed protein., Results: Changes in transition temperature and thermal stability were observed through different solvent extractions. Albumin possessed the predominant thermal stability (81.4 °C) followed by globulin (74.49 °C), prolamin (69 °C) and glutelin (65.6 °C). So, solvent compositions influence the profile of AAs and their nutritive value, and aqueous solvent with 0.1 mol L⁻¹ NaCl was an efficient protein solubiliser., Conclusion: The results indicated that the extraction processes influenced the lotus seed protein quality and thermal stability. Overall, the study revealed that the lotus seed protein was nutritionally well-balanced protein and might be of significant importance in the formulation of diets for humans., (© 2012 Society of Chemical Industry.)

Analysis of serum and plasma proteomes is a common approach for biomarker discovery, and the removal of high-abundant proteins, such as albumin and immunoglobins, is usually the first step in the analysis. However, albumin binds peptides and proteins, which raises concerns as to how the removal of albumin could impact the outcome of the biomarker study while ignoring the possibility that this could be a biomarker subproteome itself. The first goal of this study was to test a new commercially available affinity capture reagent from Protea Biosciences and to compare the efficiency and reproducibility to four other commercially available albumin depletion methods. The second goal of this study was to determine if there is a highly efficient albumin depletion/isolation system that minimizes sample handling and would be suitable for large numbers of samples. Two of the methods tested (Sigma and ProteaPrep) showed an albumin depletion efficiency of 97% or greater for both serum and cerebrospinal fluid (CSF). Isolated serum and CSF albuminomes from ProteaPrep spin columns were analyzed directly by LC-MS/MS, identifying 128 serum (45 not previously reported) and 94 CSF albuminome proteins (17 unique to the CSF albuminome). Serum albuminome was also isolated using Vivapure anti-HSA columns for comparison, identifying 105 proteins, 81 of which overlapped with the ProteaPrep method., (© 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Numerous studies have shown that consumption of soybean products decrease the risk of cancers in humans. Experiments at the molecular level have demonstrated that in most cases proteins and peptides are responsible for the anticancer properties of soybeen. Special attention should be paid to lunasin - a peptide described for the first time 16 years ago. Due to its structure it causes i.a., inhibition of cancer cell proliferation. A novel procedure for the isolation and purification of low-molecular-mass 2S soybean albumin protein is described in the present paper. A fraction of four peptides one of them corresponding to molecular mass and isoelectric point characteristic for lunasin. The obtained peptides decreased on the rate of HeLa cell proliferation.

The intrinsic advantages of microcapsules with regard to nanocapsules as intravenous drug carrier systems are still not fully exploited. Especially, in clinical situations where a long-term drug release within the vascular system is desired, if large amounts of drug have to be administered or if capillary leakage occurs, long-circulating microparticles may display a superior alternative to nanoparticles. Here, microcapsules were synthesised and parameters such as in vitro tendency of agglomeration, protein adsorption and in vivo performance were investigated. Biocompatible poly(ethylene glycol) (PEG)-coated poly(DL-lactide-co-glycolide) (PLGA) as wall material, solid and perfluorodecalin (PFD)-filled PEG-PLGA microcapsules (1.5 µm diameter) were manufactured by using a modified solvent evaporation method with either 1% poly(vinyl alcohol) (PVA) or 1.5% cholate as emulsifying agents. Compared to microcapsules manufactured with cholate, the protein adsorption (albumin and IgG) was clearly decreased and agglomeration of capsules was prevented, when PVA was used. The intravenous administration of these microcapsules, both solid and PFD-filled, in rats was successful and exhibited a circulatory half-life of about 1 h. Our data clearly demonstrate that PEG-PLGA microcapsules, manufactured by using PVA, are suitable biocompatible, long-circulating drug carriers, applicable for intravenous administration.

A new composite protein-imprinted macroporous cryogel was prepared for depletion of albumin from human serum prior to use in proteom applications. Polyhydroxyethyl-methacylate-based molecularly imprinted polymer (MIP) composite cryogel was prepared with high gel fraction yields up to 83%, and its morphology and porosity were characterized by Fourier transform infrared, scanning electron microscopy, swelling studies, flow dynamics, and surface area measurements. Selective binding experiments were performed in the presence of competitive proteins human transferrin (HTR) and myoglobin (MYB). MIP composite cryogel exhibited a high binding capacity and selectivity for human serum albumin (HSA) in the presence of HTR and MYB. The competitive adsorption amount for HSA in MIP composite cryogel is 722.1 mg/dL in the presence of competitive proteins (HTR and MYB). MIP composite cryogel column was successfully applied in the fast protein liquid chromatography system for selective depletion of albumin in human serum. The depletion ratio was highly increased by embedding beads into cryogel (85%). Finally, MIP composite cryogel can be reused many times with no apparent decrease in HSA adsorption capacity., (Copyright © 2012 John Wiley & Sons, Ltd.)

Epithelial cells in the proximal tubule of the kidney reclaim and metabolize protein from the glomerular filtrate. Proteinuria, an overabundance of protein in the urine, affects tubular cell function and is a major factor in the progression of chronic kidney disease. By developing experimental systems to study tubular protein handling in a setting that simulates some of the environmental conditions of the kidney tubule in vivo, we can better understand how microenviromental conditions affect cellular protein handling to determine if these conditions are relevant in disease. To this end, we used two in vitro microfluidic models to evaluate albumin handling by renal proximal tubule cells. For the first system, cells were grown in a microfluidic channel and perfused with physiological levels of shear stress to evaluate the effect of mechanical stress on protein uptake. In the second system, a porous membrane was used to separate an apical and basolateral compartment to evaluate the fate of protein following cellular metabolism. Opossum kidney (OK) epithelial cells were exposed to fluorescently labeled albumin, and cellular uptake was determined by measuring the fluorescence of cell lysates. Confocal fluorescence microscopy was used to compare uptake in cells grown under flow and static conditions. Albumin processed by the cells was examined by size exclusion chromatography (SEC) and SDS-PAGE. Results showed that cellular uptake and/or degradation was significantly increased in cells exposed to flow compared to static conditions. This was confirmed by confocal microscopy. Size exclusion chromatography and SDS-PAGE showed that albumin was broken down into small molecular weight fragments and excreted by the cells. No trace of intact albumin was detectable by either SEC or SDS-PAGE. These results indicate that fluid shear stress is an important factor mediating cellular protein handling, and the microfluidic bioreactor provides a novel tool to investigate this process., (Copyright © 2011 Wiley Periodicals, Inc.)

Ammonium sulfate precipitation (ASP) was explored as a method for depleting some highly abundant proteins from blood plasma, in order to reduce the dynamic range of protein concentration and to improve the detection of low abundance proteins by 2D-PAGE. 40% ammonium sulfate saturation was chosen since it allowed depleting 39% albumin and 82% α-1-antitrypsin. ASP-depletion showed high reproducibility in 2D-PAGE analysis (4.2% variation in relative abundance of albumin), similar to that offered by commercial affinity-depletion columns. Besides, it allowed detecting 59 spots per gel, very close to the number of spots detected in immuno-affinity-depleted plasma. Thus, ASP at 40% saturation is a reliable depletion method that may help in proteomic analysis of blood plasma. Finally, ASP-depletion seems to be complementary to hydrophobic interaction chromatography (HIC)-depletion, and therefore an ASP-step followed by a HIC-step could probably deplete the most highly abundant plasma proteins, thus improving the detection of low abundance proteins by 2D-PAGE., (Copyright © 2011 Elsevier B.V. All rights reserved.)

A fused-silica capillary modified with Fe³⁺-immobilized magnetic nanoparticles (Fe³⁺-IMAN) has been investigated for the capillary electrophoretic (CE) separation of phosphoproteins and non-phosphoproteins. The Fe³⁺-IMAN capillary was achieved by covalently immobilising epoxy-based magnetic silica nanoparticles (160 nm) on the prederivatized 3-aminopropyl-trimethoxysilane (APTMS) fused-silica capillary (75 μm id), followed by disodium iminodiacetate and Fe³⁺. The buildup process was examined by measuring the streaming potentials of the bare capillary, APTMS capillary, epoxy-based nanoparticle capillary and Fe³⁺-IMAN capillary by varying the buffer pH. An inverted fluorescence microscope was used to determine the surface features of the Fe³⁺-IMAN capillary derivatized with morin. Further experimental results confirmed that Fe³⁺-IMAN bonded on the inner wall of the APTMS capillary could provide sufficient solute-bonded phase interactions to allow for the CE separation of phosphoproteins and non-phosphoproteins at concentration levels down to 50 μg/mL. The highest number of theoretical plates obtained was about 233,000/m, and the relative standard deviation (RSD) for migration times was <2.57% for eight consecutive runs, respectively. Additionally, the Fe³⁺-IMAN modifing method was also applied to the analyses of bovine milk proteins. With simplicity, high resolving power, and high repeatability, the proposed method has shown great potential for phosphoproteomics applications., (Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Background: Baru (Dipteryx alata Vog.) is a fruit distributed throughout the Brazilian savanna and contains a seed with a high protein content, whose properties have been rarely explored. The purpose of this study was to characterize this protein, especially by isolation and quantifying its fractions and measuring some of its molecular properties., Results: Baru seeds contain 244 g kg(-1) protein on a dry weight basis. Solubility profiles showed a preponderance of globulins. This fraction dominated the seed composition, with 61.7 wt% of the total soluble proteins. Albumins and glutelins accounted for 14 and 3.3 wt%, respectively. SDS-PAGE resolution of albumin and globulin showed main bands with molecular weights of 84 kDa and 64, 66 and 73 kDa, respectively. The total protein of the flour and the globulin showed values of in vitro digestibility of 85.59% and 90.54%, relative to casein. Total globulin produced only one chromatographic peak, both on Sepharose CL-6B gel filtration and on DEAE-cellulose ion-exchange columns, eluted at a concentration of 0.12 mol L(-1) NaCl., Conclusion: The baru seed had high protein content with large quantities of storage proteins. The chromatographic and solubility profiles indicate the predominance of a fraction with characteristics of a legumin-type protein., (Copyright © 2011 Society of Chemical Industry.)

Cibacron Blue F3GA was covalently attached onto magnetic poly(vinyl alcohol) (mPVAL) beads (100-150 μm in diameter) for human serum albumin (HSA) adsorption from human plasma. Despite low nonspecific adsorption of HSA on mPVAL beads, Cibacron Blue F3GA attachment significantly increased the HSA adsorption. The maximum HSA adsorption was observed at pH 5.0. Higher HSA adsorption was observed from human plasma. Desorption of HSA from mPVAL beads was achieved by medium containing 1.0 M KSCN at pH 8.0. To test the efficiency of albumin adsorption from human serum, before and after albumin adsorption was demonstrated with sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analyses. HSA molecules could be reversibly adsorbed and desorbed 10 times with the magnetic beads without noticeable loss in their HSA adsorption capacity.

Workflows in urinary proteomics studies are often complex and require many steps to enrich, purify, deplete, and separate the complex mixture. Many of these methods are laborious, are time-consuming, and have the potential for error. Although individual steps of these methods have been previously studied, their downstream compatibilities with fractionation technologies such as off-gel electrophoresis have not been investigated. We developed a one-step sample preparation workflow that simultaneously (i) concentrates proteins, (ii) purifies by removing salts and other low molecular weight compounds, and (iii) depletes (albumin) from urine samples. This simple and robust workflow can be multiplexed and is compatible with a diverse range of downstream multidimensional separation technologies. Additionally, because of its high reproducibility and flexibility in processing samples with different volumes and concentrations, it has the potential to be used for standardization of urinary proteomics studies, as well as for studying other body fluids of similar complexity.

This study was designed to investigate the effect and molecular mechanisms of Haishengsu (HSS), a protein extract from a shellfish Tegillarca granosaL., on a drug resistant leukemia cell line. Cultured K562/Adriamycin (ADM) cells were treated with HSS at 10, 20 and 40 microg/mL, respectively. The apoptosis and expression of p-glycoprotein was evaluated by flow cytometry. Expressions of caspase-3 and Bcl-2 were also evaluated. There was a significant dose-dependent increase in the apoptosis in the HSS treated K562/ADM cells (P < 0.05 and 0.01, respectively). The p-glycoprotein expression in the 40 microg/mL HSS group (14.8%) was lower than in the control (16.9%, P < 0.05) and the 10 microg/mL HSS group (7.3%, P < 0.05), but it was similar to the HSS 20 microg/mL group (10.7%, P > 0.05). The expressions of apoptosis-stimulating protein caspase-3 protein were increased, whereas the expressions of apoptosis-suppressing Bcl-2 were decreased in the HSS groups, as compared with the levels in the control group (P < 0.05). We conclude that HSS induces apoptosis of the Adriamycin-resistant K562/ADM cells. The enhanced expressions in caspase-3 and the reduced expressions in Bcl-2 protein may have contributed to the apoptosis-stimulating effect of HSS. The inhibition of p-glycoprotein suggests that HSS may diminish the resistance to Adriamycin and potentially enhance the therapeutic effects.

Isoelectric focusing (IEF) is the first step for two-dimensional (2D) gel electrophoresis and plays an important role in sample purification for proteomics. However, biases in protein size and pI resolution, as well as limitations in sample volume, gel capacity, sample loss, and experimental time, remain challenges. In order to address some of the limitations of traditional IEF, we present a microfluidic free flow IEF (FF-IEF) device for continuous protein separation into 24 fractions. The device reproducibly establishes a nearly linear pH gradient from 4 to 10. Optimized dynamic coatings of 4% poly(vinyl alcohol) (PVA) minimize peak broadening by transverse electrokinetic flows. Even though the device operates at high electric fields (up to 370 V/cm), efficient cooling maintains solution temperature inside the separation channel controllably in the range 2-25 degrees C. Protein samples with a dynamic concentration range from microg/mL to mg/mL can be loaded into the microdevice at a flow rate of 1 mL/h and residence time of approximately 12 min. By using a protein complex of nine proteins and 13 isoforms, we demonstrate improved separation with the FF-IEF system over traditional 2D gel electrophoresis. Device-to-device reproducibility is also illustrated through the efficient depletion of the albumin and hemoglobin assays. Post-device sample concentrations result in a 10-20-fold increase, which allow for isolation and detection of low abundance proteins. The separation of specific proteins from a whole cell lysate is demonstrated as an example. The microdevice has the further benefits of retaining high molecular weight proteins, providing higher yield of protein that has a broader range in pI, and reducing experimental time compared to conventional IEF IGP gel strip approaches.

Significant effort has been made world-wide to boost biofuels with the expectation of a positive contribution to renewable fuel and greenhouse gas reduction. Jatropha curcas L. has proved to be an opportunistic crop in tropical areas, particularly in unfavorable environments. For this reason, analyses of toxicity and allergy caused by its seeds and pollen are necessary. A 12kDa, allergenic 2S albumin, denoted Jat c 1, was isolated from Physic nut (J. curcas) seeds. Jat c 1 binds IgE attached to rat mast cells, inducing histamine release. It also showed strong cross-reactivity with the major allergens from castor bean, Ric c 1 and Ric c 3.

Purified plasma derived human albumin has been available as a therapeutic product since World War II. However, cost effective recombinant production of albumin has been challenging due to the amount needed and the complex folding pattern of the protein. In an effort to provide an abundant source of recombinant albumin, a herd of transgenic cows expressing high levels of rhA in their milk was generated. Expression cassettes efficiently targeting the secretion of human albumin to the lactating mammary gland were obtained and tested in transgenic mice. A high expressing transgene was transfected in primary bovine cell lines to produce karyoplasts for use in a somatic cell nuclear transfer program. Founder transgenic cows were produced from four independent cell lines. Expression levels varying from 1-2 g/l to more than 40 g/l of correctly folded albumin were observed. The animals expressing the highest levels of rhA exhibited shortened lactation whereas cows yielding 1-2 g/l had normal milk production. This herd of transgenic cattle is an easily scalable and well characterized source of rhA for biomedical uses.

Advanced oxidation protein products (AOPPs) are carried by oxidized plasma proteins, especially albumin and accumulate in subjects with renal disease and coronary artery disease. AOPPs represent an excellent novel marker of oxidative stress and their roles in the development of cardiovascular disease might be of great importance. Here, we show that in vitro-generated AOPP-albumin binds with high affinity to the high-density lipoprotein (HDL) receptor scavenger receptor class B type I (SR-BI). Already an equimolar concentration of AOPP-albumin to HDL blocked HDL association to SR-BI and effectively inhibited SR-BI-mediated cholesterol ester (CE) uptake. Interestingly, albumin extensively modified by advanced glycation end products (AGE-albumin), which is an established SR-BI ligand known to accumulate in renal disease, only weakly interfered with HDL binding to SR-BI. Furthermore, AOPP-albumin administration increased the plasma half-life of [3H]CE-HDL in control mice 1.6-fold (P=0.01) and 8-fold (P=0.0003) in mice infected with adenoviral vectors encoding human SR-BI. Moreover, albumin isolated from hemodialysis patients, but not albumin isolated from healthy controls, markedly inhibited SR-BI-mediated HDL-CE transfer in vitro dependent on the AOPP content of albumin. These results indicate that AOPP-albumin effectively blocks SR-BI in vitro and in vivo. Thus, depressed plasma clearance of HDL-cholesterol may contribute to the abnormal composition of HDL and the high cardiovascular risk observed in patients with chronic renal failure.

Poly(ethylene oxide)-stabilized silver nanoparticles (PEO-SSNPs) were synthesized and investigated for their potential use in capillary electrophoretic separation of proteins. It was found that the PEO-SSNPs were relatively stable against changes of the buffer ionic strength, while they began to aggregate with increase of buffer pH. Adding 0.024 g/L PEO-SSNPs to 30 mmol/L phosphate buffer (pH 2.66) led to obvious increase in the peak heights of albumin bovine and albumin egg. Also, the separation efficiency for all the standards enhanced due to the suppressed wall-adsorption of proteins in the presence of the nanoparticles. The above experiments suggest that the PEO-SSNPs are effective pseudostationary phase for CE separation of proteins.

Objectives: Extracting proteins from conventional hydrogel (CH) and silicone hydrogel (SH) contact lens materials using a mixture of trifluoroacetic acid/acetonitrile (TFA/ACN) is a well-established procedure for quantifying individual and total protein deposited on contact lenses. The purpose of this study was to determine the efficacy of TFA/ACN in extracting albumin from SH and a CH group IV lens material using an in vitro model., Methods: One CH group IV lens material (etafilcon A) and five different SH lens materials (lotrafilcon A, lotrafilcon B, balafilcon A, galyfilcon A, and senofilcon A) were incubated in both simple albumin solution and a complex artificial tear protein solution containing 125I-labeled albumin. All the lens materials were incubated for 14 days at 37 degrees C with constant rotations. Following the incubation period, radioactive counts were determined and the lenses were placed in an appropriate volume of the extraction solvent. After the specified time, the lenses were removed and radioactive counts were determined again to calculate the amount of albumin remaining on the lenses post-extraction., Results: Extraction efficiencies for albumin from the artificial tear protein solution were 97.2% +/- 2 for etafilcon A, 77.3% +/- 6.2 for lotrafilcon A, 73.5% +/- 5.6 for lotrafilcon B, 81.5% +/- 5.8 for balafilcon, 91.2% +/- 3.4 for galyfilcon A, and 89.2% +/- 3.4 for senofilcon A. Results were similar for the albumin extracted after incubating in the simple albumin solution., Conclusions: Although TFA/ACN is efficient at extracting albumin deposited on etafilcon lenses, it does not extract all the albumin that is deposited on SH lenses and alternative extraction procedures should be sought.

An MEKC method combining field-amplified sample injection (FASI) has been developed for the analysis of albumin (Alb) and transferrin (TRF) in human urines. Various parameters affecting FASI preconcentration were optimized systematically. Under the optimum conditions, using electrokinetic sample injection for 90 s, the sensitivity was improved 77.6- and 45.3-fold for TRF and Alb, respectively, without loss of separation efficiency when compared with hydrodynamical sample injection for 5 s (0.5 psi). The detection limits of TRF and Alb were found to be 0.31 and 0.14 mg/L, respectively. The proposed method has been successfully applied to the determination of TRF and Alb in desalted urines from patients with kidney diseases, and the recoveries of TRF and Alb were 91.1-101.7%.