Showing papers on "Bovine serum albumin published in 1994"

Identification of the heme compound copurified with deoxyribonucleic acid (DNA) from bloodstains, a major inhibitor of polymerase chain reaction (PCR) amplification.

Abstract: The heme compound found in deoxyribonucleic acid (DNA) extracted from bloodstains, which is regarded as a major inhibitor of polymerase chain reaction (PCR), was characterized in comparison with alkaline and acid hematin, histidine and ammonia hemochromogens, and globin and serum albumin hemochromogens digested by proteinase K. Alkaline and acid hematin were almost completely removed by phenol/chloroform treatment and ethanol precipitation, so as not to be copurified with DNA from the specimens. Spectrophotometric results indicated that the contaminant was likely to be the product of proteinase K digestion of some heme-blood protein complex, which was not completely extracted by organic solvents and remained in the ethanol precipitates of DNA. The results of polyacrylamide gradient gel electrophoresis and intensity of the inhibition of PCR suggested that the ligand of the contaminant was a somewhat large molecule, resistant to the proteolysis by proteinase K. The addition of bovine serum albumin to the reaction mixture prevented the inhibition of PCR by the heme compounds, probably by binding to the heme. This showed that the inhibition was not due to the irreversible inactivation of the enzyme.

Effect of solution pH and ionic strength on the separation of albumin from immunoglobulins (IgG) by selective filtration.

Abstract: Although protein fractionation by selective membrane filtration has numerous potential applications in both the downstream processing of fermentation broths and the purification of plasma proteins, the selectivity for proteins with only moderately different molecular weights has generally been quite poor. We have obtained experimental data for the transport of bovine serum albumin (BSA) and immunoglobulins (IgG) through 100,000 and 300,000 molecular weight cutoff polyethersulfone membranes in a stirred ultrafiltration device at different solution pH and ionic strength. The selectivity was a complex function of the flux due to the simultaneous convective and diffusive solute transport through the membrane and the bulk mass transfer limitations in the stirred cell. Under phsioligical conditions (pH 7.0 and 0.15 M NaCI) the maximum selectivity for the BSA-IgG separation was only about 2.0 due primarily to the effects of protein adsorption. In contrast, BSA-IgG selectivities as high as 50 were obtained with the same membranes when the protein solution was at pH 4.8 and 0.0015 M NaCl. This enhanced selectivity was a direct result of the electrosatatic contributions to both bulk and membrane transport. The membrane selectivity could actually be reversed, with higher passage of the larger IgG molecules, by using a 300,000 molecular weight cutoff membrane at pH 7.4 and an ionic strength of 0.0015 M NaCl. These results clearly demonstrate that the effectiveness of selective protein filtration can be dramatically altered by appropriately controlling electrostatic interactions through changes in pH and/or ionic strength. (c) 1994 John Wiley & Sons, Inc.

Involvement of a lysine residue in the N-terminal Ni2+ and Cu2+ binding site of serum albumins. Comparison with Co2+, Cd2+ and Al3+.

Abstract: We report one-dimensional and two-dimensional 1H-NMR studies of the binding of Ni2+, Cu2+, Co2+, Cd2+ and Al3+ to defatted bovine and human serum albumins. The diamagnetic shifts induced by Ni2+, and paramagnetic effects due to Cu2+, were consistent with strong binding to a square-planar site formed by the three N-terminal amino acid residues (Asp-Thr-His for bovine, and Asp-Ala-His for human albumin). In contrast to previous studies on isolated 1-24 N-terminal peptide, a Lys residue also appeared to be involved in the binding site, and is assigned as Lys4. A second His residue is also close to the Cu2+/Ni2+ binding site in bovine serum albumin and is assigned to His59 (not present in human albumin). Co2+ caused specific perturbation of the resonances for the three N-terminal residues as well as those for Lys4. This is the first evidence for Co2+ binding to the N-terminal metal site of serum albumin. Neither Al3+ nor Cd2+ perturbed resonances for the N-terminal amino acids, but bind elsewhere in the protein.

The Fpg protein, a DNA repair enzyme, is inhibited by the biomediator nitric oxide in vitro and in vivo

Abstract: Nitric oxide has been shown to be a mediator molecule in the regulation of many physiological functions. However, this small diatomic molecule in the presence of O2 generates reactive intermediates which modify DNA bases and inactive enzymes at high concentrations (100 microM). We report that NO generated by 1,1-diethyl-2-hydroxy-2-nitrosohydrazine (DEA/NO, Et2NN(O)NO-Na+), a compound known to release NO in a predictable manner, caused irreversible damage at physiological concentrations to the zinc finger-containing DNA repair enzyme formamidopyrimidine-DNA glycolyase (Fpg protein). The inhibition of the enzyme activity was DEA/NO dose and time dependent with IC50s with respect to total NO released from this compound of approximately 110 and approximately 120 mumol/l respectively. This inhibitory effect by P3 was not reversible over time in the presence of reducing agents and/or Zn2+. Nitrite and diethylamine, the nitrogenous products of the decomposition of DEA/NO, did not inhibit the enzyme. The presence of 500 micrograms/ml bovine serum albumin did not protect the protein from the inhibitory effects of DEA/NO, however, the presence of 10 mM cysteine did dramatically abate the inhibition of the Fpg protein by DEA/NO. Other DNA glycosylases tested were not inhibited by exposure to these concentrations of NO. These results, together with reports of site-directed mutagenesis of this protein, suggest that the cysteine residues contained within the zinc finger motif of the Fpg protein are the primary sites of NO interaction. Our studies were then extended to intact cells. The Fpg protein activity was decreased following treatment in vivo when Escherichia coli MH321 (acr A-) cells were treated with DEA/NO. Furthermore, the Fapy-DNA glycosylase activity in H4 cells, a rat hepatoma line, was decreased when intact cells were incubated with DEA/NO.

Proteinase inhibition of the detection of Listeria monocytogenes in milk using the polymerase chain reaction

Abstract: The direct detection, using the polymerase chain reaction (PCR), of Listeria monocytogenes added to cows' milk was inhibited at some milk concentrations. This inhibitor was moderately heat-stable. Inhibition could be prevented by the addition of Bovine Serum Albumin (BSA) or proteinase inhibitors to the PCR and the evidence suggests that the inhibitor was plasmin.

Prevention of Proteinuria by the Administration of Anti-interleukin 8 Antibody in Experimental Acute Immune Complex-induced Glomerulonephritis

Abstract: Glomerular infiltration by neutrophils is a hallmark of acute glomerulonephritis. The pathophysiological role of interleukin 8 (IL-8), a potent neutrophil chemotactic cytokine (chemokine), was explored in an animal model of acute immune complex-mediated glomerulonephritis by administering a neutralizing antibody against IL-8. Repeated injection of bovine serum albumin (BSA) into rabbits caused the deposition of immune complexes consisting of BSA and rabbit IgG in glomeruli. Histological analyses revealed a small but significant number of neutrophils in glomeruli and the fusion of epithelial cell foot processes. Concomitantly, urinary levels of protein and albumin increased markedly (3.20 +/- 0.97 and 1.39 +/- 0.53 mg/h, respectively) compared with those of untreated animals (0.77 +/- 0.21 and 0.01 +/- 0.01 mg/h, respectively). Anti-IL-8 antibody treatment decreased the number of neutrophils in glomeruli by 40% and dramatically prevented the fusion of epithelial cell foot process. Furthermore, treatment with anti-IL-8 antibody completely normalized the urinary levels of protein and albumin (0.89 +/- 0.15 and 0.02 +/- 0.01 mg/h, respectively). These results indicated that IL-8 participated in the impairment of renal functions in experimental acute immune complex-mediated glomerulonephritis through activating as well as recruiting neutrophils.

A simple, specific, and highly sensitive blocking enzyme-linked immunosorbent assay for detection of antibodies to bovine herpesvirus 1.

Abstract: By using a monoclonal antibody directed against an epitope located on glycoprotein B of bovine herpesvirus 1 (BHV1), a simple, convenient blocking enzyme-linked immunosorbent assay (ELISA) which combines a high sensitivity with a low false-positive rate has been developed. The test can be performed at low variance on undiluted bovine serum samples. The epitope on glycoprotein B appears to be conserved, because it could be detected by immunostaining in all of 160 BHV1 isolates originating from 10 countries. In testing 215 anti-BHV1 antibody-negative and 179 anti-BHV1 antibody-positive serum samples, specificity and sensitivity were 0.96 and 0.99, respectively. This blocking ELISA is superior to a commercially available indirect ELISA and to the 24-h virus neutralization test in detecting low antibody levels in serum. In addition, this blocking ELISA is able to detect specific antibodies in serum as early as 7 days postinfection. To minimize any risk of introducing latent BHV1 carriers among noninfected cattle, this blocking ELISA would be, in our opinion, the test of choice.

SPARC (secreted protein acidic and rich in cysteine) regulates endothelial cell shape and barrier function.

Abstract: SPARC (secreted protein acidic and rich in cysteine) can be selectively expressed by the endothelium in response to certain types of injury and induces rounding in adherent endothelial cells in vitro. To determine whether SPARC might influence endothelial permeability, we studied the effect of exogenous SPARC on the movement of 14C-labeled bovine serum albumin across postconfluent bovine pulmonary artery endothelial cells. SPARC increased (P or = 0.5 microgram/ml. At a fixed dose (15 micrograms/ml), exposure times > or = 1 h augmented (P < 0.005) albumin flux by 1.3- to 3.6-fold; this increase was blocked by anti-SPARC antibodies but not by inhibition of protein synthesis. Barrier dysfunction was not associated with loss of cell viability. Monolayers exposed to SPARC exhibited a rounded morphology and intercellular gaps. Prior stabilization of F-actin with phallicidin protected against the changes in barrier function (P = 0.0001) that were otherwise induced by SPARC. Bovine aortic and retinal microvascular endothelia also responded to SPARC. We propose that SPARC regulates endothelial barrier function through F-actin-dependent changes in cell shape, coincident with the appearance of intercellular gaps, that provide a paracellular pathway for extravasation of macromolecules.

Albumin based hydrogel

Abstract: Novel bioartificial hydrogels consisting of a three-dimensional crosslinked mixture of: (a) a bifunctionalized polyethylene oxide, activated with an activating agent, dissolved in an aqueous solution; and (b) albumin type protein. The novel hydrogels are based on the crosslinking of albumin type protein of various sources including, for example, bovine serum albumin, lactalbumin or ovalbumin, with a bifunctionalyzed polyethylene oxide, most proferably polyethylene glycol, or a mixture of bifunctionalyzed polyethylene oxides preferably polyethylene glycol, of various molecular masses (Mr 2,000 to 35,000), dissolved in aqueous solution in adequate proportions. Also provided is a method and conditions for preparing the novel hydrogels. Also divulged are a variety of biomedical applications for the novel hydrogels. Additionally, it has been found that the mechanical properties of the novel hydrogels can be improved by adding to the casting solution unreactive polyethylen glycol or other inert polymer of high molecular masses (Mr > 100,000). In general terms the novel hydrogels possess advantageous properties such as shape retention and shape memory, high water content (more than 94 % (w/w) based on the dry weight of the hydrogel), good mechanical and optical properties. The hydrogels exhibit additional charcteristics which may render them extremely useful in the pharmaceutic and medical areas due to other advantageous properties such as biocompatibility, resistance to proteases action, slow release of various drugs, hydrophillic surface, good oxygen permeability, controlled porosity.

Spectroscopic characterization of albumin and myoglobin entrapped in bulk sol-gel glasses

Abstract: The immobilization of proteins by entrapment in optically clear, porous glasses prepared by sol-gel techniques appears to be a promising approach to optical biosensor development. However, little is known about the physical environment of the immobilized protein or the mechanism(s) of entrapment. In this study, absorbance and fluorescence spectroscopies have been used to characterize the properties of two model proteins, bovine serum albumin (BSA) and horse heart myoglobin (Mb), entrapped in wet sol-gel glass bulks. The fluorescence behavior of dissolved and entrapped BSA in the presence of acid, a chemical denaturant, and a collisional quencher was examined. The results show that a large fraction of the BSA added to the sol is entrapped within the gelled glass in a native conformation. However, the reversible conformational transitions that BSA undergoes in solution are sterically restricted in the gel. In contrast, the native properties of Mb are largely lost upon entrapment, as judged by the changes in the visible absorbance spectra of dissolved and entrapped Mb in acidic solutions. Fluorescence studies of dissolved and entrapped apomyoglobin support this conclusion.

Effect of nutrients, hormones and serum on survival of rat islet beta cells in culture.

Abstract: This study quantifies the survival of purified single rat beta cells under different culture conditions. Less than 10% of the cells survive 9 days of culture in Ham's F10 medium without supplements. Addition of fetal calf serum (5%) increases cell survival to 54% in the absence and to 78% in the presence of isobutylmethylxanthine (50 μmol/l). The effect of serum is explained, at least partly, by the presence of albumin and of low molecular weight constituents. In serum-free Ham's F10 with 50 μmol/l isobutylmethylxanthine, 75% of cells survive after the addition of bovine serum albumin (1%) and of ultroser (0.2%), a commercial serum substitute. Survival of at least 75% of cells is also maintained in Ham's F10 with isobutylmethylxanthine plus albumin, and supplemented by metabolizable nutrients or by the peptides glucagon (10−8 mol/l) or growth hormone (1 μg/ml) plus insulin like growth factor-I (50 ng/ml). d-Glucose increases beta-cell survival in a dosedependent manner up to 10 mmol/l; a beneficial effect is also observed with other metabolizable compounds (leucine and glutamine) but not with non-metabolizable monosaccharides. Glucose-induced survival of islet beta cells can be attributed to its dose-dependent recruitment of cells into metabolic activities; however, a 9-day exposure to excessively high nutrient concentrations (> 20 mmol/l glucose) is deleterious to the cells. These results define culture media, with or without serum, wherein at least 75% of single rat islet beta cells can survive for a minimum of 9 days. This will allow for studies on beta-cell toxic conditions and potentially protective agents. The data also serve as basis for developing media with better survival of beta cells in cultured aggregates.

Intermolecular electrostatic interactions and their effect on flux and protein deposition during protein filtration.

Abstract: Although membrane filtration is used extensively to process protein solutions containing a variety of electrolytes, there is currently little fundamental understanding of the effect of the solution environment (and in particular, the solution pH) on the filtrate flux in these systems. We have obtained data for the flux and sieving coefficients during the batch (stirred cell) filtration of solutions of bovine serum albumin, immunoglobulins, hemoglobin, ribonuclease A, and lysozyme through 0.16-micron microfiltration membranes at different pH values. The flux declined significantly for all five proteins due to the formation of a protein deposit on the upper surface of the membrane. The quasi-steady ultrafiltrate fluxes at the individual protein isoelectric pH's were essentially identical, despite the large differences in molecular weight and physicochemical characteristics of these proteins. The flux increased at pH's away from the isoelectric point, with the data well-correlated with the protein surface charge density. These results were explained in terms of a simple physical model in which the protein deposit continues to grow, and thus the flux continues to decline, until the drag force on the proteins associated with the filtrate flow is no longer able to overcome the intermolecular repulsive interactions between the proteins in the bulk solution and those in the protein deposit on the surface of the membrane.

Antibody binding of circulating ergot alkaloids in cattle grazing tall fescue.

Abstract: Direct evidence linking alkaloids found in endophyte-infected tall fescue forage with the livestock disorder known as fescue toxicosis is lacking Physiologic effects of fescue toxicosis include reduced serum prolactin concentration in cattle A monoclonal antibody specific to the lysergic moiety of ergot alkaloids was developed in mice after creating an immunogen by linking lysergol to human serum albumin The antibody was specific to the lysergic moiety and, therefore, it cross-reacted with ergot alkaloids, lysergic acid, and lysergol The antibody did not cross-react with alkaloid derivatives that had bromated or hydrogenated lysergic ring moieties Fescue toxicosis conditions were elicited in yearling Angus steers by permitting them to graze endophyte-infected tall fescue containing > 650 micrograms/kg of ergovaline for 60 days Passive immunization of steers by infusion of the monoclonal antibody increased serum prolactin concentration by 7 ng/ml, beginning immediately after infusion Control steers did not respond to treatment with bovine serum albumin Active immunization of yearling Angus heifers with immunogens containing lysergol or ergonovine linked to human serum albumin resulted in an antibody response

Effect of beta -alanyl-l-histidinato zinc on protein components in osteoblastic mc3t3-el cells : increase in osteocalcin, insulin-like growth factor-i and transforming growth factor-beta

Abstract: The effect of beta-alanyl-L-histidinato zinc (AHZ) on protein components in osteoblastic MC3T3-E1 cells was investigated. Cells were cultured for 3 days at 37 degrees C in CO2 incubator in plastic dishes containing alpha-modified minimum essential medium supplemented with 10% fetal bovine serum. After the cultures, the medium was exchanged for that containing 0.1% bovine serum albumin plus various concentrations of AHZ or other reagents, and the cells were cultured further 3 or 6 days. The homgenate of cells was analyzed with SDS-polyacrylamide gel electrophoresis (SDS-PAGE). The presence of AHZ (10(-7) to 10(-5) M) caused an appreciable increase of many protein components in cells. Especially, the 67 killo-dalton (kDa) and 44 kDa proteins which are the major components from control cells were clearly increased by the presence of AHZ. Furthermore, the concentrations of osteocalcin, insulin-like growth factor-I and transforming growth factor-beta in the culture medium secreted from osteoblastic cells were markedly increased by the presence of AHZ (10(-6) and 10(-5) M). The effect of AHZ was a greater than that of zinc sulfate (10(-6) and 10(-5) M). The present findings suggest that AHZ can increase many proteins which are involved in the stimulation of bone formation and cell proliferation in osteoblastic cells.

A new structural transition of serum albumin dependent on the state of Cys34. Detection by 1H-NMR spectroscopy.

Abstract: 1 Reactions of fatty-acid-free bovine serum albumin and recombinant human albumin with a range of antiarthritic gold(I) complexes [auranofin, deacetylated auranofin, triethylphosphinegold(I) chloride] and related thiols (thioglucose, tetraacetylthioglucose, glutathione, dithiothreitol) have been investigated using 1H-NMR spectroscopy 2 In reactions of albumin with auranofin, tetraacetylthioglucose and dithiothreitol, release of cystine was detected, whereas for deacetylated auranofin, thioglucose and glutathione, mixed disulphides with cysteine were produced It has been previously proposed that Cys34 of human and bovine serum albumins is partly blocked by disulphide formation with cysteine and glutathione The above reactions lead to deblocking by thiol–disulphide interchange reactions No release of glutathione from albumin was detected 3 Changes in the His Hɛ1 regions of the 1H-NMR spectra show that albumin exists in two structural forms dependent on whether the side-chain of Cys34 is a free thiolate, or blocked by gold(I)triethylphosphine, by disulphide formation with cysteine or by another form of oxidation We propose that Cys34 is either in a buried or in an exposed environment; the possible molecular basis of the structural change is discussed 4 The relationship between reactions at Cys34, cysteine release, and the observed structural transition are discussed in terms of chrysotherapy, albumin metabolism and the use of gold(I) as a heavy atom derivative in X-ray crystallographic studies of albumins