Interaction between beta 2-microglobulin and advanced glycation end products in the development of dialysis related-amyloidosis.

Dialysis related amyloidosis (DRA) is a progressive debilitating complication of long-term dialysis. beta 2-microglobulin (beta 2m) amyloid deposition occurs preferentially in older patients and initially is located in collagen-rich osteo-articular tissues. Since an age-dependent increase in the formation of advanced glycation end products (AGE) has been observed in collagen-containing structures, we hypothesized that AGE-modified beta 2m in the amyloid of DRA may be formed locally in osteo-articular structures as a subsequent event of its binding to collagen-AGE. Based on this hypothesis, we investigated the binding between beta 2m and AGE-modified collagen (collagen-AGE) in vitro. Significantly larger amounts of human beta 2m were bound to types I to IV of immobilized collagen-AGE than to unmodified collagens (P < 0.0001). The quantity of beta 2m bound to collagen-AGE was dependent on the concentrations of both beta 2m and of AGE contained in collagen (P < 0.01). Unmodified beta 2m was more avidly bound to collagen-AGE or collagen in comparison to AGE-modified beta 2m (P < 0.0001). beta 2m bound to collagen-AGE could be modified further by nonenzymatic glycosylation during three weeks of incubation with physiologic concentrations of glucose. Similar processes in vivo may be important in the pathobiology of DRA.

Detection and quantitation of beta-2-microglobulin glycosylated end products in human serum by matrix-assisted laser desorption/ionization mass spectrometry.

beta-2-Microglobulin (beta 2M) is a major protein component found in the amyloid deposits of dialysis-related amyloidosis (DRA) patients. Evidence has been shown that the advanced glycosylated end-products (AGEs) of beta 2M present in sera were related to DRA. We demonstrated that matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) is a useful tool to investigate the nature of glycosylation of beta 2M and detection of beta 2M or beta 2M-AGEs in human serum. The high-mass end of beta 2M-AGE distribution was found to extend to the neighborhood of 12,868 Da, corresponding to condensations with seven glucose molecules. We also have shown that both beta 2M and beta 2M-AGEs can be detected at low picomole levels directly in bovine serum. Based on these findings, the sera of DRA patients were studied to determine whether beta 2M-AGEs can be detected by MALDI-MS. In an attempt to investigate the possibility of quantitation with MALDI, human sera samples with different concentrations of beta 2M-AGE were examined. We were able to correlate the concentration of beta 2M-AGE with the number of detected AGE products, pointing to the feasibility of MALDI as a quantitative tool.

An evaluation of molecular models of the cytochrome P450 Streptomyces griseolus enzymes P450SU1 and P450SU2.

P450SU1 and P450SU2 are herbicide-inducible bacterial cytochrome P450 enzymes from Streptomyces griseolus. They have two of the highest sequence indentities to camphor hydroxylase (P450cam from Pseudomonas putida), the cytochrome P450 with the first known crystal structure. We have built several models of these two proteins to investigate the variability in the structures that can occur from using different modeling protocols. We looked at variability due to alignment methods, backbone loop conformations and refinement methods. We have constructed two models for each protein using two alignment algorithms, and then an additional model using an identical alignment but different loop conformations for both buried and surface loops. The alignments used to build the models were created using the Needleman-Wunsch method, adapted for multiple sequences, and a manual method that utilized both a dot-matrix search matrix and the Needleman-Wunsch method. After constructing the initial models, several energy minimization methods were used to explore the variability in the final models caused by the choice of minimization techniques. Features of cytochrome P450cam and the cytochrome P450 superfamily, such as the ferredoxin binding site, the heme binding site and the substrate binding site were used to evaluate the validity of the models. Although the final structures were very similar between the models with different alignments, active-site residues were found to be dependent on the conformations of buried loops and early stages of energy minimization. We show which regions of the active site are the most dependent on the particular methods used, and which parts of the structures seem to be independent of the methods.

Biocompatible polyurethane-based hydrogel.

A polyurethane polymer developed at W. R. Grace has been evaluated for a variety of biomedical applications. The primary property of the polymer exploited for these applications is its ability to prevent protein adsorption when coated on a surface. The prepolymer consists of a trifunctional poly(ethyleneoxide-propylene oxide) triol end capped with isophorone diisocyanate. The prepolymer is reactive with water and can be converted to a hydrogel, a thin coating, or a soluble conjugate with another compound. Each category lends itself to separate biomedical applications which are described in detail. The non-toxic nature of the polymer was demonstrated in a number of systems and suggests its utility in biomedical applications.

Functional peptide-polyurethane conjugates with extended circulatory half-lives.

Peptides containing the RGD sequence were covalently attached to an isocyanate-containing polyurethane prepolymer and the biological properties of the complexes were evaluated. For the pentapeptide H2N-Tyr-Arg-Gly-Asp-Ser-OH (single-letter code, YRGDS), polymer conjugation lead to an increased half-life in the blood circulation of mice to > 10 h. The effect of covalent attachment of polymer on biological activity was examined in a related peptide, H2N-Gly-Arg-Gly-Asp-Ser-Pro-Ala-Cys- OH (GRGDSPAC). Inhibition of B16-F10 melanoma cell attachment and spreading on plates coated with fibronectin by an RGD-containing peptide and polymer-conjugated GRGDSPAC was observed to occur at similar concentrations. We conclude that the conjugation of peptides containing the Arg-Gly-Asp motif to this polymer resolves previous problems of their rapid loss from the circulation, while still allowing retention of full biological inhibitory activity.

3 Nsec molecular dynamics simulation of the protein ubiquitin and comparison with X-ray crystal and solution NMR structures.

Mainly due to computational limitations, past protein molecular dynamics simulations have rarely been extended to 300 psec; we are not aware of any published results beyond 350 psec. The present work compares a 3000 psec simulation of the protein ubiquitin with the available x-ray crystallographic and solution NMR structures. Aside from experimental structure availability, ubiquitin was studied because of its relatively small size (76 amino acids) and lack of disulfide bridges. An implicit solvent model was used except for explicit treatment of waters of crystallization. We found that the simulated average structure retains most of the character of the starting x-ray crystal structure. In two highly surface accessible regions, the simulation was not in agreement with the x-ray structure. In addition, there are six backbone-backbone hydrogen bonds that are in conflict between the solution NMR and x-ray crystallographic structures; two are bonds that the NMR does not locate, and four are ones that the two methods disagree upon the donor. Concerning these six backbone-backbone hydrogen bonds, the present simulation agrees with the solution NMR structure in five out-of-the six cases, in that if a hydrogen bond is present in the x-ray structure and not in the NMR structure, the bond breaks within 700 psec. Of the two hydrogen bonds that are found in the NMR structure and not in the x-ray structure, one forms at 1400 psec and the other forms rarely. The present results suggest that relatively long molecular dynamics simulations, that use protein x-ray crystal coordinates for the starting structure and a computationally efficient solvent representation, may be used to gain an understanding of conformational and dynamic differences between the solid-crystal and dilute-solution states.

A new hydrophilic polymer for biomaterial coatings with low protein adsorption.

BIOPOL polyurethane polymers, an extension of the HYPOL Polymer series of foamable hydrophilic polymers, have been developed which exhibit improved performance for selected biomedical applications. Members of the BIOPOL polyurethane polymer series, with molecular weights in the range of 7000 to 30,000, are larger molecules than HYPOL polymers (MW less than 3000) and produce hydrogels, rather than foams, when mixed with water. The prototype material in this series, BIOPOL XP-5, is a liquid prepolymer which chain extends in water and forms a hydrogel which can contain greater than 85% water. The time required for polymerization with water was dependent on the prepolymer: water ratio. This prepolymer was coated onto silica and medical grade tubing and then cured in place with water to form a stable coating which was resistant to non-specific protein binding. In addition, soluble, isocyanate-free forms of the prepolymer were tested for toxicity and shown to produce no adverse effects when injected intravenously into mice or when applied to a chicken chorioallantoic membrane. BIOPOL polymers can be useful in applications where protein adsorption is an undesirable event.

Monoclonal antibody production in hollow fiber bioreactors using serum-free medium.

Murine hybridoma cells that produce monoclonal antibody directed against human fibronectin have been cultured in VITAFIBER II and VITAFIBER V hollow fiber bioreactors using defined, serum-free WRC 935 medium. During a two-week growth period, following inoculation of the bioreactors, the cells proliferated to an extent where the bioreactor was filled with cultured cells. Using a 5 sq. ft. VITAFIBER V bioreactor, over 15 grams of antibody were produced during the 40 days of the experiment. This antibody was greater than 95% IgG. During the production period, this packed mass of cells produced 579 +/- 15 mg IgG per day. Because the medium is formulated for air equilibration and high cell densities, WRC 935 medium is especially useful for production of gram quantities of monoclonal antibodies using continuous feed hollow fiber bioreactor cell culture systems.

A new serum-free medium for monoclonal antibody production.

A new serum-free, defined-protein, medium for the growth of murine hybridoma cells and the production of monoclonal antibodies has been developed. Designated WRC 935 medium, this formulation supports the growth of hybridoma cells in higher numbers, and promotes better cell viabilities and increased monoclonal antibody levels compared to growth in DMEM supplemented with 10% fetal bovine serum or in a DMEM/F-12 serum-free mixture. In suspension cultures, WRC 935 medium typically promoted cell growth to densities over two million cells per milliliter. This medium also promoted the rapid growth of cells following their transfer from liquid nitrogen storage. WRC 935 medium is especially useful for high density cell culture production methods using hollow-fiber bioreactors. Hollow-fiber bioreactors using this medium produced antibody at an average rate of 11 mg/day, and the antibody concentration ranged from 10 to 40 mg/ml.

A solid-phase, enzyme-linked immunosorbent assay for a human lung tumor-associated antigen.

A solid-phase, enzyme-linked immunosorbent assay (ELISA) for a human lung tumor-associated antigen (LTA) was based on immobilized LTA that was detected with the use of an antiserum raised in a goat against a highly purified antigen preparation. Bound goat antibodies were detected in a series of steps that included incubation with a) biotinylated rabbit antibodies to goat immunoglobulins, b) glucose oxidase conjugated to avidin, and c) peroxidase and the substrates glucose and 2,2'-azino-di-(3-ethylbenzthiazoline sulfonic acid). The absorbance of the final product was measured at 405 nm, and its formation was dependent on substrate incubation time and antibody concentration. The antigen was immobilized and highly purified, and the goat antiserum was bound to and eluted from an immobilized crude antigen column before use. The ELISA could detect less than 1 ng antigen and was able to discriminate extracts of normal lung tissue from those of lung tumor. As was found earlier with a radioimmunoassay for the same antigen, normal human serum could inhibit in the ELISA but only when used at high concentration, indicating levels of antigen or antigen-like activity in the 100-200 ng/ml range. With the use of this assay, 3 lung cancer patients were monitored 6-12 months prior to death. In all 3 patients, LTA levels rose dramatically 2-4 months before the patients died; in 2 patients the levels exceeded 3,000 ng/ml just before death. In contrast, in 2 of these patients, carcinoembryonic antigen levels remained essentially unchanged, with no more than a twofold increase prior to death.

Serum levels of a human lung tumor-associated antigen using an improved radioimmunoassay.

A human lung tumor-associated antigen, previously purified to apparent homogeneity from an extract of a small cell tumor, was radioiodinated with Bolton-Hunter reagent for use in a competitive protein-binding radioimmunoassay. A panel of 215 sera was assembled from normal individuals and pretreatment patients with lung cancer, benign lung disease, and nonlung cancers, and lung tumor antigen in each was quantitated using the radioimmunoassay. The mean of normals was 0.92 +/- 0.43 (S.D.) microgram/ml (n = 88), and values greater than 2 standard deviations above the mean (1.78 micrograms/ml) were considered positive. Positive rates in lung cancers of the following histological types were found: adenocarcinoma, 60% (9 of 15); squamous cell, 42% (13 of 31); large cell, 17% (3 of 18); and small cell, 19% (3 of 16). In addition, 13% (3 of 23) of other cancers, 0% (0 of 24) of benign lung disease, and 2% (2 of 88) of normals were positive. Approximately one-third of Stage 1 patients in the squamous cell and adenocarcinoma groups were positive while two-thirds of patients with more advanced Stage III disease in these categories showed elevations.

LTA: a human lung tumor-associated antigen common to primary lung tumors and cultured lung tumor cell lines.

A human lung tumor-associated antigen (LTA) previously purified from a primary lung tumor has been identified in the sera of lung cancer patients. Frequencies of LTA elevations in lung cancer were: adenocarcinoma, 60%; squamous cell carcinoma, 42%; large cell carcinoma, 17%; and small cell carcinoma, 19%; normals, 2%; benign lung disease, 0%; and non-lung malignancies, 13%. The antigen was also shown to be produced by seven of the eight human lung tumor cell lines that were examined. A preliminary small-scale purification was attempted on an extract from one of these lines, ChaGo, which yielded a smaller and more basic form of LTA but which possessed similar, if not identical, antigenic activities as primary tumor LTA.

Characterization of a human lung tumor-associated antigen and development of a radioimmunoassay.

A human lung tumor-associated antigen (LTA), previously isolated from a small cell carcinoma, was further studied after labeling with N-succinimidyl-3-(4-hydroxy-5-[125I]iodophenyl)propionate. Two immunoreactive forms of the labeled antigen were observed and isolated after electrophoresis in 7% polyacrylamide gels. These components, referred to as LTA-I and LTA-II in order of mobility, were judged homogeneous by gel electrophoresis, G-200 gel filtration, size exclusion high-performance liquid chromatography, and sedimentation velocity analysis. The latter three techniques produced identical profiles for both forms of the LTA. Sephadex chromatography and high-performance liquid chromatography analyses indicated the mass of the antigens to be approximately 140,000 to 150,000 daltons with a D20,w of 4.2 to 4.3 x 10(-7) sq cm/sec. The S20,w values for both were 4.5 to 4.6S. Sodium dodecyl sulfate gel electrophoresis of LTA-II gave a single component with a molecular weight of 81,700, while LTA-I had a major component identical in size to LTA-II and two minor components with molecular weights of 50,000 and 27,700, respectively. The isoelectric point of LTA-II (peaks at pH 2.6 and 3.2) generally was more acidic than LTA-I (major component centered at pH 4.7, minor component centered at pH 3.1). A radioimmunoassay, with a useful detection range of from 1 to 100 ng/ml, was developed with LTA-I. This assay was used to determine the concentration of LTA in the sera of normal and lung cancer patients. Fifteen normal sera had a mean of 17 +/- 22 (S.D.) ng/ml, with none greater than 83 ng/ml (+3 S.D.). Thirteen lung cancer patients with Stage I (i.e., localized disease) had a mean of 187 +/- 219 ng/ml, with the means for 7 of 13 patients being greater than 83 ng/ml; 15 lung cancer patients with Stage III, more extensive disease, had a mean of 277 +/- 252 ng/ml, with the means for 12 of 15 patients being greater than 83 ng/ml. This antigen may be useful for the early detection or monitoring of lung cancer.

Identification and purification of a human lung tumor-associated antigen from a primary lung tumor.

A human lung tumor-associated protein has been purified from an extract of a human small cell carcinoma of the lung and shown by Ouchterlony double diffusion analysis to be antigenically identical to a component which was previously demonstrated in 84 of 98 lung tumor extracts of all histological types but absent from extracts of normal adult and fetal lung, other normal tissues, and tumors of other organs. These studies utilized xenoantisera raised against a pool of lung tumor extracts which were exhaustively adsorbed with normal serum and tissue extracts. A radial immunodiffusion assay developed for the antigen permitted its quantitation throughout the course of isolation. Purification was accomplished by ion-exchange chromatography, gel filtration, and affinity immunoadsorption. By ion-exchange chromatography, the proteins appeared to be quite heterogeneous, with immunological reactivity detected in three different peaks. However, all the active components were immunologically identical. Gel filtration of the major antigenic component from diethylaminoethyl cellulose similarly demonstrated a further fractionation into several active, immunologically identical forms. These results suggest a charge-size isomeric relationship among the various forms, all of which possess a common and identical antigenic site. The major component was isolated throughout the purification scheme. The final product represented 9% of the input activity, produced a single, although broad, protein-staining region on 7% polyacrylamide gels which was coincident with antigenic activity, and exhibited immunological identity with the antigen in the crude extract as well as with that in an extract from another lung tumor.

Studies on a purified human lung tumor-associated antigen.

A human lung tumor-associated antigen has been identified by Ouchterlony analysis in 86% of lung tumor extracts of all histologic types. It was not detected by this method in extracts of normal adult or fetal lung, 12/13 other tumors, normal tissues or in normal serum. The antigen was purified from the extract of an undifferentiated small cell lung carcinoma by DEAE-cellulose. Sephadex G-200, and antibody affinity chromatography. The purified antigen exhibited size and charge heterogeneity, yet all isolated forms produced precipitin lines of identity with each other, as well as with an extract of an unrelated squamous cell carcinoma of the lung. The major form had a Mr of 150 000 by gel filtration and 81 700 by detergent gel electrophoresis. By radioimmunoassay, elevated levels of antigen were detected in 0/15 normal sera, 7/13 stage I and 12/15 stage III lung cancer sera. The urine of a different patient with a small cell lung carcinoma contained a related antigenic activity, but the major component was 30 000-50 000 daltons. The antigen has also been detected in extracts of a continuous cell line from a large cell carcinoma of the lung (ChaGo). The antigen is being evaluated for its usefulness as a lung tumor marker.

Cytostasis of tumor cell lines by granulocytes from cancer patients and normal human donors.

Granulocytes of normal human donors were previously shown to have cytostatic activity in vitro against a variety of tumor cell lines. In the present study, we have compared the levels of granulocyte-mediated cytostatic activity in cancer patients and normal donors. In an initial study of 25 tumor-bearing patients and 21 individuals with benign or no disease, decreased cytostatic activity was observed in 84% of the cancer patients. Nine cancer patients with no evidence of disease had reactivity in the normal range. Granulocytes separated by a one-step method on a double Ficoll-Percoll gradient showed decreased reactivity. This procedure eliminated the differences previously detected between tumor-bearing patients and controls. Addition of either pooled normal AB human serum or autologous serum to the assay restored the reactivity. Only with autologous serum and not with allogeneic serum, were the differences between tumor-bearing patients and controls again seen. Therefore, in a subsequent study, we examined the effect of serum on cytostasis by normal granulocytes that were isolated on double gradients. We observed lowered serum restorative activity (SRA) in 41 of the 46 (89%) tumor-bearing patients tested. Fractionation of sera by Sephadex G-200 chromatography indicated that SRA of both cancer patients and normal donors was in the 100,000 molecular weight region.

Biosynthesis of the yeast cell wall. I. Preparation and properties of beta-(1 leads to 3)glucan synthetase.

The synthesis of the major linkage found in yeast cell wall structural polysaccharides, glucosyl-beta-(1 leads to 3)-glucosyl, was studied with a membrane preparation from Saccharomyces cerevisiae. The sugar donor was UDP-glucose, and the reaction required addition of glycerol bovine serum albumin, and ATP or GTP for maximal activity. Under optimal conditions, extremely efficient glucose transfer was obtained, with 20 to 50% of the substrate utilized in 20 min at 30 degrees C. The polysaccharide formed in the reaction was insoluble in water and soluble in alkali; it was characterized enzymatically and chemically as a beta-(1 leads to 3)-linked linear glucan of chain length 60 to 80. The terminal reducing group was found to be labeled with 14C, as was the substrate used; therefore, the polysaccharide is synthesized de novo. For each glucosyl group transferred, one equivalent of UDP was formed. No evidence was found for a lipid-linked intermediate. When yeast protoplast lysates were subjected to fractionation by centrifugation in Renografin gradients, glucan synthetase was found in the plasma membrane fraction, with the same distribution and sidedness as chitin synthetase. Because of the spatially restricted growth of the cell wall during cell division in budding yeasts, this result suggests localized and reversible activation of the enzyme during the cell cycle.