Solid-liquid phase boundaries of lens protein solutions.

We report measurement of the solid-liquid phase boundary, or liquidus line, for aqueous solutions of three pure calf gamma-crystallin proteins: gamma II, gamma IIIa, and gamma IIIb. We also studied the liquidus line for solutions of native gamma IV-crystallin calf lens protein, which consists of 85% gamma IVa/15% gamma IVb. In all four proteins the liquidus phase boundaries lie higher in temperature than the previously determined liquid-liquid coexistence curves. Thus, over the range of concentration and temperature for which liquid-liquid phase separation occurs, the coexistence of a protein crystal phase with a protein liquid solution phase is thermodynamically stable relative to the metastable separated liquid phases. The location of the liquidus lines clearly divides these four crystallin proteins into two groups: those in which liquidus lines flatten at temperatures greater than 70 degrees C: gamma IIIa and gamma IV, and those in which liquidus lines flatten at temperatures less than 50 degrees C: gamma II and gamma IIIb. We have analyzed the form of the liquidus lines by using specific choices for the structures of the Gibbs free energy in solution and solid phases. By applying the thermodynamic conditions for equilibrium between the two phases to the resulting chemical potentials, we can estimate the temperature-dependent free energy change upon binding of protein and water into the solid phase.     

Effect of Polyphenylsulfone and Polysulfone Incompatibility on the Structure and Performance of Blend Membranes for Ultrafiltration

This study deals with the modification of polyphenylsulfone ultrafiltration membranes by introduction of an incompatible polymer polysulfone to the polyphenylsulfone casting solution to improve the permeability. The correlation between properties of the blend polyphenylsulfone/polysulfone solutions and porous anisotropic membranes for ultrafiltration prepared from these solutions was revealed. The blend polyphenylsulfone/polysulfone solutions were investigated using a turbidity spectrum method, optical microscopy and measurements of dynamic viscosity and turbidity. The structure of the prepared blend flat sheet membranes was studied using scanning electron microscopy. Membrane separation performance was investigated in the process of ultrafiltration of human serum albumin buffered solutions. It was found that with the introduction of polysulfone to the polyphenylsulfone casting solution in N-methyl-2-pyrrolidone the size of supramolecular particles significantly increases with the maximum at (40–60):(60:40) polyphenylsulfone:polysulfone blend ratio from 76 nm to 196–354 nm. It was shown that polyphenylsulfone/polysulfone blend solutions, unlike the solutions of pristine polymers, are two-phase systems (emulsions) with the maximum droplet size and highest degree of polydispersity at polyphenylsulfone/polysulfone blend ratios (30–60):(70–40). Pure water flux of the blend membranes passes through a maximum in the region of the most heterogeneous structure of the casting solution, which is associated with the imposition of a polymer-polymer phase separation on the non-solvent induced phase separation upon membrane preparation. The application of polyphenylsulfone/polysulfone blends as membrane-forming polymers and polyethylene glycol (M_n = 400 g·mol⁻¹) as a pore-forming agent to the casting solutions yields the formation of ultrafiltration membranes with high membrane pure water flux (270 L·m⁻²·h⁻¹ at 0.1MPa) and human serum albumin rejection of 85%.     

Antibody to the receptor for polymerized human serum albumin in acute and persistent infection with hepatitis B virus

The antibody against the receptor for polymerized human serum albumin was determined by radioimmunoassay. The method involved the inhibition by the test serum, absorbed with HBsAg particles without the receptor, on the binding of polymerized human serum albumin to HBsAg particles with the receptor fixed on a solid support. The amount of polymerized human serum albumin captured by the receptor on HBsAg was then determined by the radiolabeled monoclonal antibody directed to an epitope specific for polymerized human serum albumin. In acute infection, the antibody to the receptor for polymerized human serum albumin appeared in the early recovery phase while HBs antigenemia and elevated transaminase levels were still present, preceding the antibody to HBsAg (anti-HBs). The antibody was detected in 4 (1%) of 358 sera from asymptomatic carriers of HBsAg containing antibody to HBeAg, and in none of 67 sera containing HBeAg. Although the antibody was found in as many as 111 (74%) of 150 sera from blood donors who had presumably acquired anti-HBs after natural infection, it was not detected in any sera from 77 recipients of hepatitis B vaccine who had seroconverted for anti-HBs. On the basis of these observations, the determination of antibody to the receptor for polymerized human serum albumin helps in further understanding the immunity to hepatitis B virus.     

Liquid-liquid separation in solutions of normal and sickle cell hemoglobin

We show that in solutions of human hemoglobin (Hb)--oxy- and deoxy-Hb A or S--of near-physiological pH, ionic strength, and Hb concentration, liquid-liquid phase separation occurs reversibly and reproducibly at temperatures between 35 and 40 degrees C. In solutions of deoxy-HbS, we demonstrate that the dense liquid droplets facilitate the nucleation of HbS polymers, whose formation is the primary pathogenic event for sickle cell anemia. In view of recent results that shifts of the liquid-liquid separation phase boundary can be achieved by nontoxic additives at molar concentrations up to 30 times lower than the protein concentrations, these findings open new avenues for the inhibition of the HbS polymerization.     

Observation of liquid-liquid phase separation for eye lens gammaS-crystallin

gammaS-crystallin (gammaS) is an important human and bovine eye lens protein involved in maintaining the transparency of the eye. By adding small amounts of polyethylene glycol (PEG) to the binary aqueous bovine gammaS solutions, we have observed liquid-liquid phase separation (LLPS) at -8 degrees C and revealed that, in the binary gammaS-water system, this phase transition would occur at -28 degrees C. We have measured both the effect of PEG concentration on the LLPS temperature and proteinPEG partitioning between the two liquid coexisting phases. We use our measurements of proteinPEG partitioning to determine the nature and the magnitude of the gammaS-PEG interactions and to quantitatively assess the effectiveness of PEG as a crystallizing agent for gammaS. We use our measurements of LLPS temperature as a function of protein and PEG concentration to successfully determine the location of the critical point for the binary gammaS-water system. This phase transition cannot be observed in the absence of PEG because it is inaccessible due to the freezing of the system. Our findings indicate that the effective interactions between gammaS molecules in the binary gammaS-water system are attractive. We compare the magnitude of the attraction found for gammaS with the results obtained for the other gamma-crystallins for which the critical temperature is located above the freezing point of the system. This work suggests that PEG can be used to reveal the existence of LLPS for a much wider range of binary protein-water systems than known previously.     

The use of albumin in clinical practice

The use of albumin in the clinical setting continues to generate controversy. Periodic shortages and the high cost of albumin have compelled many hospitals to develop guidelines regarding albumin administration. Our purpose is to review the human studies involving albumin. Particular emphasis will be placed on comparative trials involving albumin and the less expensive crystalloid solutions. It is hoped that this review will assist the clinician in making judgements concerning the appropriate use of albumin.     

IgE and IgG antibodies to beta-propiolactone and human serum albumin associated with urticarial reactions to rabies vaccine

We examined the antibody response to a rabies vaccine doubly inactivated with 0.025% beta-propiolactone and 0.1% tri(n)butyl phosphate and stabilized with 2.5% human serum albumin. Antibodies were measured by using the following four antigen preparations: complete doubly inactivated rabies vaccine, rabies vaccine inactivated only with tri(n)butyl phosphate, beta-propiolactone and human serum albumin, and human serum albumin alone. The fluid phase of the preparation of beta-propiolactone and human serum albumin completely inhibited IgE binding to solid-phase vaccine. Of 21 subjects with urticarial reactions to a booster, 19 had IgE to doubly inactivated vaccine and to beta-propiolactone and human serum albumin. None of 27 immunized subjects without urticaria had detectable IgE. In paired pre- and postimmunization sera, IgE appeared in six of seven of the subjects with urticaria and in one of seven nonreactors. These sera did not contain a significant level of IgE to singly inactivated vaccine or to human serum albumin alone.     

Cross-reaction of albumin with polyclonal LH antibody on western blots

When pituitary tissue was subjected to Western blot analysis utilizing polyclonal antibody NIDDK-rLH-S-10, bands at 17 and 19 Kd representing LH subunits were identified. In addition, a high molecular weight 66 Kd band was seen. Surprisingly this high molecular weight band was also seen in rat cerebral cortex, brain stem, hypothalamus, spinal cord, lung, liver, pancreas, spleen, kidney, testis, and serum. Antibody preabsorbed with iodination grade rat LH antigen no longer recognized the 17 and 19 Kd bands in pituitary, but recognized the 66 Kd bands in pituitary and the other tissues examined. Since 66 Kd is the molecular weight of albumin, we found that antisera to rat albumin recognized this same high molecular weight band in the tissues examined. Preabsorption of LH antibody with albumin reduced the ability of that antibody to recognize this 66 Kd. A monoclonal antibody to bovine LH beta-subunit recognized only the LH protein in anterior pituitary, but no high molecular weight band in either pituitary or the other tissues studied. Finally, 10, 100, and 1000 micrograms of rat albumin caused no substantial interference under conditions of RIA. We conclude that the polyclonal antibody, provided by the NIH, is excellent for conditions of RIA, but caution must be exercised when it is used for Western analysis where some lots of this antibody may recognize other unrelated proteins.     

Aeolotopic interactions of globular proteins

Protein crystallization, aggregation, liquid-liquid phase separation, and self-assembly are important in protein structure determination in the industrial processing of proteins and in the inhibition of protein condensation diseases. To fully describe such phase transformations in globular protein solutions, it is necessary to account for the strong spatial variation of the interactions on the protein surface. One difficulty is that each globular protein has its own unique surface, which is crucial for its biological function. However, the similarities amongst the macroscopic properties of different protein solutions suggest that there may exist a generic model that is capable of describing the nonuniform interactions between globular proteins. In this paper we present such a model, which includes the short-range interactions that vary from place to place on the surface of the protein. We show that this aeolotopic model [from the Greek aiolos ("variable") and topos ("place")] describes the phase diagram of globular proteins and provides insight into protein aggregation and crystallization.     

Quantitation of Soluble HLA Class I Antigen in Human Albumin and Immunoglobulin Preparations for Intravenous Use by Solid-Phase Immunoassay

Soluble HLA class I antigens in human plasma preparations possibly play a role in HLA sensitization and modulation of the immune response. We therefore have determined their concentration in albumin and immunoglobulin preparations from several commercial sources and compared these values to the concentration in normal human sera. For this purpose we used a newly developed solid-phase enzyme immunoassay employing rabbit anti-mouse antibody, monomorphic HLA class I monoclonal antibody and a polyclonal enzyme-linked beta 2-microglobulin-specific antiserum. Soluble HLA antigen concentration in 14 albumin batches from 6 manufacturers and in 16 immunoglobulin batches from 11 manufacturers ranged from 0 to 9.6 and from 0 to 20.9 ng/ml. The concentration in normal human sera 1,328 +/- 954 ng/ml (n = 54). We conclude that the concentration of soluble HLA concentration in albumin and immunoglobulin preparations is more than 50 times lower than in normal human serum, but considerable differences exist between products of various manufacturers.     

Antibody-dependent Cell-mediated Cytotoxicity (ADCC) of Penicillin-treated Human Red Blood Cells

S ummary . Penicillin-treated human red blood cells (RBC) were lysed by the cooperation of autologous nonsensitized peripheral blood mononuclear cells and human anti-penicillin serum. Using a rapid (3 h) assay of antibody-dependent cell-mediated cytotoxicity (ADGC), lysis was proportional to serum (anti-penicillin antibody) concentration, to incubation time and to the concentration of attacking cells, which were obtained from normal human peripheral blood by Ficoll-Hypaque separation. Incubation of these lymphoid effector cells on a nylon column prior to the tests depleted the number of phagocytic (latex positive) cells in the effluent; there was a concomitant drop in cytotoxic activity. Enrichment of mononuclear phagocytes in the attacking cell population by albumin gradient separation led to an increase in cytotoxicity. Granulocytes separated by Ficoll-Hypaque were not active in this system. Using specific antisera the antibody was found to be of the IgG1 sub-class. Anti-penicillin antibody activated the complement system in vitro , but failed to induce lysis of penicillin-treated RBC in the presence of complement without attacking cells.
These results suggest that ADCC may participate in the destruction of RBC in penicillin-induced haemolysis in vivo.     

Pilot-Plant Scale Preparation of Human Serum Albumin

Pilot-plant scale experiments using production scale fractionation equipment have been carried out to prepare human serum albumin according to a two-step simple method reported previously. Results have indicated that scale-up is feasible and the resultant albumin is of at least the same quality but with better yield when compared to that obtained by the conventional ethanol fractionation method. Filtration to replace centrifugation in one of the steps for liquid-solid separation was also attempted. It proved to be superior to centrifugation in terms of time and labor savings, but a small percentage of albumin was not recoverable. However, because of the simplicity and higher yield of this fractionation method the final albumin yield is still higher than that obtained by the conventional ethanol method even if the filtration step is introduced. Diafiltration and ultrafiltration are used for solvent removal and concentration, instead of lyophilization and reconstitution to prepare the final albumin solutions.     

APOBEC3G expression is restricted to neurons in the brains of pigtailed macaques

The Vif protein of human immunodeficiency virus-1 (HIV-1) has been shown to interact with members of the APOBEC family of cytidine deaminases, particularly APOBEC3G/F. In this study, we isolated RNA from 12 regions of the brain from two pigtailed macaques that were exsanguinated and perfused with saline. Our results indicate that APOBEC3G was detected in all regions of the brain analyzed. Immunoblot analysis using lysates prepared from these same regions of the brain and a monoclonal antibody to APOBEC3G confirmed the RT-PCR findings. To determine which cell types express APOBEC3G, immunohistochemical studies were performed using this monoclonal antibody on whole brain sections. Our results clearly show that the pyramidal neurons within the gray matter of cerebral and cerebellar cortices express APOBEC3G. However, APOBEC3G expression in the pyramidal neurons appeared to be nuclear or associated with nuclei. In contrast to our findings in the cerebral cortex, immunohistochemical analysis of the spleen and kidney tissues revealed that APOBEC3G expression in the cells of these tissues was predominantly cytoplasmic. We further investigated the expression of APOBEC3G in astrocytes. Immunohistochemical staining of serial sections was performed using antibodies to glial fibrillary acidic protein (GFAP) and APOBEC3G. As expected, the cortical and cerebellar white matter showed extensive immunostaining of astrocytes with the antibody against GFAP but a lack of reactivity to the antibody to APOBEC3G. Additionally, Immunoblot analysis of lysates prepared from primary human fetal astrocytes revealed a lack of APOBEC3G expression. Taken together, these results indicate that APOBEC3G expression is restricted to neurons in the brain and that astrocytes and microglia probably do not express this protein or express it at levels undetectable by immunohistochemistry. These finding have implications for the brain as a potential reservoir for Vif-defective viruses.     

Mechanism of the Albumin Agglutination Phenomenon

Summary. The albumin agglutination phenomenon is due to antibodies which cause agglutination of all human red cells when these cells are suspended in an albumin medium. Two sera with this property were studied. We suggest that the reaction is due to non-specific adsorption of antigen-antibody complex onto red cells. The antibody is a gamma globulin directed at albumin which has been altered by the addition of acetyl tryptophanate or caprylate. These chemicals are added as stabilizers in the manufacture of albumin to prevent denaturation when the albumin is heated.
Since unaltered (native) albumin does not react with these antibodies, blood or plasma transfusion to patients with this serologic abnormality should present no unusual hazard. A non-reactive albumin should be used in crossmatching blood for such patients. Therapeutic human serum albumin and plasma products containing stabilized albumin are probably contraindicated in these patients.     

Protein losing enteropathy due to systemic lupus erythematosus.

We report the case of a 29 year old woman with a protein losing enteropathy caused by systemic lupus erythematosus presenting with periorbital oedema. Only three other cases of protein losing enteropathy due to systemic lupus erythematosus have been described, two of which were thought to be because of a primary enteropathy, although the exact pathogenesis was unknown. We suggest that both the protein losing enteropathy and periorbital oedema in this patient were because of increased capillary permeability to serum albumin, as a result of products of plasma C3 conversion which were present in large amounts. It is also of interest that the antigen/antibody system in this patient was RNP/anti-RNP and that DNA antibodies were not detected. This patient falls into a subset of systemic lupus erythematosus in which anti-DNA antibodies are not present, some of which appear to have a more favourable prognosis.     

Chronic treatment of Ménétrier's disease with Erbitux: clinical efficacy and insight into pathophysiology.

BACKGROUND & AIMS: Ménétrier's disease is a rare premalignant hypertrophic gastropathy characterized by large rugal folds, foveolar hyperplasia with glandular atrophy, hypochlorhydria, and hypoalbuminemia. Patients with severe disease often exhibit refractory nausea and vomiting and require gastrectomy. Evidence from both mice and human beings suggests a critical role for epidermal growth factor receptor (EGFR) signaling in the pathogenesis of this disease. We previously reported significant clinical and biochemical improvement of a single patient treated for 1 month with Erbitux, a monoclonal antibody that blocks ligand binding to EGFR. METHODS/RESULTS: We describe 2 patients who were given longer-term treatment with Erbitux as an alternative to gastrectomy. The first patient presented with nausea, hypoalbuminemia, and peripheral edema that required total parenteral nutrition (TPN) and infusions of albumin. On institution of Erbitux, there was rapid improvement in nausea and vomiting and stabilization of serum albumin with discontinuation of TPN and albumin infusions. Serum albumin remained stable during a 1-year course of Erbitux without supplemental protein. Application before and after Erbitux of the radiopaque dye ruthenium red to biopsies of the gastric oxyntic gland mucosa demonstrated prompt and persistent closure of tight junctions by electron microscopy. The second patient presented with chronic gastric bleeding that required bimonthly blood transfusions. During a 4-month course of Erbitux, his hematocrit stabilized, and transfusion requirements were eliminated. CONCLUSIONS: The present report demonstrates the efficacy of prolonged Erbitux therapy in patients with different presentations of severe Ménétrier's disease and also provides insight into the pathophysiology of the protein-losing gastropathy.     

Toward the application of proteomics to human thyroid tissue.

CONTEXT: In this paper we describe for the first time a systematic approach to proteome analysis of human thyroid tissue. OBJECTIVE AND DESIGN: We report different methods to decrease the complexity of the human thyroid tissue proteome by applying different solubilization strategies and correcting for thyroglobulin protein abundance; to increase the protein resolution by prefractionation and by the use of narrow-range pH gradients; to detect proteins using sensitive and quantitative stains; and to identify soluble and membrane-bound thyroid tissue proteins by mass spectrometry analysis. MAIN OUTCOME/RESULTS: We found that buffers containing high contents of urea and detergents allow the best solubilization of human thyroid tissue proteins; highly variable abundance of thyroglobulin is a major pitfall of human thyroid proteome analysis, which in contrast to centrifugal ultrafiltration, size-exclusion chromatography and microdissection, can be countered best by adapting the protein amount to the thyroglobulin content per sample; prefractionation leads to a significant enrichment of proteins and allows subcellar localization of thyroid proteins; application of narrow-range immobilized pH gradient (IPG) strips allows further improvement of spot detection and separation; and protein detection with the fluorescent stain ruthenium II Tris bathophenanthroline disulfonate (RuBPs) is a highly sensitive and reliable tool for quantitative proteome analysis. Finally, in a pilot study of four patients with benign nodular thyroid disease we found that the described procedures allow a highly reproducible detection and identification of alterations in protein expression between nodular and corresponding normal thyroid tissues. CONCLUSIONS: Application of the described methods provides the basis for a highly sensitive and reproducible proteome analysis of the human thyroid, providing an additional novel tool to elucidate complex proteins changes in human thyroid biology as well as pathophysiology of human thyroid disease.     

Increased survival in advanced primary biliary cirrhosis patients with regular albumin infusions?

Primary biliary cirrhosis (PBC) is a chronic cholestatic liver disease. Frequently, it can slowly progress to cirrhosis and, finally, death. Liver transplantation is the only way to avoid this endpoint. For patients not accepted for liver transplantation, only symptomatic treatment remains. In advanced PBC, one of the main concerns is a decreasing albumin level. As albumin is an important protein in the human body, we wondered whether regular albumin infusions may result in a higher quality of life and prolong survival. In this case report we describe three patients who survived for a remarkably long time on this treatment regimen before finally succumbing to the sequela of advanced liver disease. Some features of albumin are mentioned which may help explain this phenomenon. We conclude that regular albumin infusions may be a means of decreasing morbidity and prolonging survival in a subgroup of patients with advanced PBC. However, prospective evaluation of regular albumin infusions in a group of PBC patients (e.g. those rejected for liver transplantation) seems mandatory.     

Organic anion transport in HepG2 cells: absence of the high-affinity, chloride-dependent transporter.

In previous studies, we identified a 55 kD organic anion-binding protein in liver cell sinusoidal plasma membrane subfractions. Other investigators identified another 55 kD bromosulfophthalein/bilirubin binding protein on the surface of rat hepatocytes and HepG2 cells and suggested that this protein served as a transporter for these ligands. In this study, transport of 35S-sulfobromophthalein by the human hepatoma cell line, HepG2, was quantified in the presence and absence of bovine serum albumin to further clarify the possible function of these plasma membrane binding proteins. In contrast to results in normal rat hepatocytes, virtually no uptake of 35S-sulfobromophthalein by HepG2 cells in the presence of bovine serum albumin was found. In the absence of albumin, HepG2 cells expressed temperature-dependent uptake of 35S-sulfobromophthalein. However, the high-affinity Cl(-)-dependent sulfobromophthalein transport that characterizes normal rat hepatocytes was absent, as indicated by an approximately 95-fold lower affinity and 170-fold higher capacity of HepG2 cells for sulfobromophthalein compared with previous results with rat hepatocytes. These results suggest that 55 kD sulfobromophthalein/bilirubin-binding protein on the liver cell surface differs from organic anion-binding protein and is not responsible for sulfobromophthalein extraction in the presence of albumin, although it may play some role in lower affinity transport by cells. Immunoblot analysis and metabolic labeling of HepG2 cells demonstrated synthesis of organic anion-binding protein. However, light microscopic immunocytochemistry and immunoprecipitation of surface iodinated rat hepatocytes and HepG2 cells with antibody to a recombinant organic anion-binding protein fusion protein indicated absence of organic anion-binding protein on the surface of HepG2 cells.(ABSTRACT TRUNCATED AT 250 WORDS)