Alpha 2-macroglobulin binding to cultured fibroblasts: identification by affinity chromatography of high-affinity binding sites

Binding sites having the properties of high-affinity receptors for activated alpha 2-macroglobulin (alpha 2M) have been purified over 100-fold from membranes of spontaneously transformed NIH-3T3 cells (J. A. Hanover, S.-y. Cheng, M. C. Willingham, and I. H. Pastan [1983] J. Biol. Chem. 258, 370-377). To identify the molecular species involved in high-affinity binding, the solubilized receptor has been purified 500-fold by conventional procedures and further purified by affinity chromatography. After radioiodination of the 500-fold-purified preparation, the detergent-solubilized extract was applied to alpha 2M-Sepharose and an 85,000 +/- 5000 Mr species was selectively retained by the column. Binding of the 85,000 +/- 5000 Mr species to the affinity resin was inhibited by EDTA and by excess alpha 2M. Elution from the affinity column could be accomplished with bacitracin, a competitive inhibitor of alpha 2M binding, or with EDTA. Consistent with the previously reported characteristics of the high-affinity alpha 2M receptor, the 85,000 Mr species bound much more efficiently to methylamine-activated alpha 2M-Affigel than to alpha 2M-Affigel which had not been amine-activated. The present data suggest that a protein with a subunit Mr of 85,000 +/- 5000 may represent a component of the high-affinity alpha 2M receptor present on cultured fibroblasts.     

Glycosyl acceptors in intact and permeabilized normal and cystic fibrosis fibroblasts

Using cell permeabilization, a technique which allows addition of exogenously supplied radiolabeled sugar nucleotides to serve as direct glycosyl donors, oligosaccharide biosynthesis was examined in fibroblasts obtained from normal and cystic fibrosis (CF) subjects. Incubation of logarithmically growing cells with either radiolabeled leucine or xylose has indicated that there was a difference in the synthetic rate between the cell types. Protein synthesis in normal cells made permeable with 50 m?g/ml lysolecithin (LL) was demonstrated to be absent, and could not be induced to take place by adding exogenous components, including energy sources and amino acids, normally required for protein synthesis. Thus radiolabeled sugars were being added to peptide acceptors which were already present at the time of LL addition. Both permeable and intact fibroblasts were exposed to labeled UDP-xylose, UDP-galactose, and UDP-glucuronic acid, all donors of mucopolysaccharide precursors. The uptake of xylose into protein was the same for both normal and CF cells, but permeable CF fibroblasts incorporated statistically greater amounts of sugar from UDP-galactose and UDP-glucuronic acid. Intact CF cells were also labeled using these two sugar nucleotides. Trypan blue exclusion indicated CF and normal fibroblasts were equally intact. This and the fact that preincubation of CF cells with the appropriate cold sugar nucleotide eliminated the differences in incorporation between the normal and CF cells suggested that CF fibroblasts had more cell surface acceptor than the normal cells.     

Preparation of monoclonal antibody-ferritin conjugates of high specific activity

125I-monoclonal IgG anti-gamma chain antibodies were conjugated to ferritin using glutaraldehyde as a bifunctional reagent. The molar ratio of IgG:ferritin:glutaraldehyde resulting in the highest yield was determined. Free IgG was separated from IgG bound to ferritin by sucrose density gradient ultracentrifugation; free ferritin was separated from antibody-ferritin conjugates by differential salt precipitation. The IgF:ferritin molar ratio of the resulting product was 1:1.4, containing over 90% ferritin-IgG "monomers"; 70-90% of the 125I activity bound immunospecifically to sepharose-IgG or aggregated human globulin (AHG). The product was used as an immunologic EM marker for AHG. Monoclonal antibody-ferritin conjugates prepared by this method should prove useful for quantitative ultrastructural analysis of surface antigens.     

Intra- and extracellular forms of alpha-glucosidase from Aspergillus niger.

An intracellular α-glucosidase (α-glu₁) of Aspergillus niger was purified and its properties were compared to those of a secreted α-glucosidase (α-glu_E). The estimated molecular weight of α-glu_I was 95,000 by gel filtration (α-glu_E = 63,000); it is a glycoprotein possessing 29 mol of mannose, 6 mol of glucosamine, and 14 mol of glucose (α-glu_E has 5–6 and 2 mol of mannose and glucosamine, respectively). The K_m′s of α-glu₁ for p-nitrophenyl-α-d-glucopyranoside and maltose were 1.49 and 1.04, respectively, slightly lower than those of α-glu_E. In addition, at 65 °C α-glu_I enzymatic activity decayed fivefold faster than that of α-glu_E, and anti-α-glu_E antibody did not recognize α-glu_I. While some of these distinctions between the enzymes could be ascribed to conformational differences, the great dissimilarity in molecular weight (approximately 32,000) and lack of reactivity with anti-α-glu_E argue against α-glu_I being related to α-glu_E. The antibody covalently coupled to horseradish peroxidase (Ab-Px) was used as a probe to determine the cellular location of α-glu_E by electron microscopic immunocytology. It was found on both sides of the plasma membrane (pm) and in the outer of the two layers of the cell wall. This may mean that α-glu_E is synthesized at the inner surface of the pm, is extruded through the pm, becomes associated with the outer layer of the cell wall (perhaps as enzyme—substrate complex), and is eventually released into the growth medium.     

Glycoprotein enzymes secreted by Aspergillus fumigatus: purification and properties of a second beta-glucosidase.

A second extracellular beta-glucosidase (betalarge) of Aspergillus fumigatus was purified to homogeneity and shown to be a glycoprotein, as determined by polyacrylamide gel electrophoresis followed by staining for protein and for carbohydrate. Its molecular weight was approximately 340,000 by gel filtration, while sodium dodecyl sulfate-polyacrylamide gel electrophoresis gave an apparent molecular weight of 170,000, suggesting that the enzyme has two subunits. The glucosidase contained covalently bound sugars consisting of about 2 mol of glucosamine and 16 mol of mannose per mol of protein. The carbohydrate was found to be attached to the peptide via glucosaminyl leads to peptide linkage, possibly to asparagine residues. At pH 4.5 this enzyme readily hydrolyzed p-nitrophenyl-beta-D-glucopyranoside (Km = 0.88 mM) and cleaved two glucose disaccharides: gentiobiose (beta,1 leads to 6; Km = 0.75 mM) and cellobiose (beta,1 leads to 4; Km = 0.84 mM). Although its activity is similar to that of a previously purified beta-glucosidase (betasmall), the two enzymes differ with respect to their pH activity profiles, substrate specificities, and molecular weights. Also double diffusion tests with anti-betasmall antiserum and both purified beta-glucosidases revealed a nonidentical cross-reaction. Microcomplement fixation of native and periodate-oxidized betasmall suggested that the oligosaccharide chain(s) was not a major antigenic site.     

Macromolecular events following refeeding of starved Tetrahymena

Tetrahymena were starved in inorganic buffer for 24 h and then refed with enriched nutrient medium. An early increase of protein synthesis which is correlated with the rapid reformation of polysomes occurs prior to the onset of increased RNA synthesis. Inhibition of RNA synthesis with actinomycin D during refeeding did not stop the initiation of increased protein synthesis nor did it stop the reformation of polysomes. The occurrence of increased protein synthesis prior to increased RNA synthesis is a different sequence than has occurred in other studies on this cell and indicates that unlike in bacteria the macromolecular response of this cell to a new environment is not fixed but is dependent upon the cell's previous environmental history.     

Use of polyethyleneimine polymer in cell culture as attachment factor and lipofection enhancer

Background  

Several cell lines and primary cultures benefit from the use of positively charged extracellular matrix proteins or polymers that enhance their ability to attach to culture plates. Polyethyleneimine is a positively charged polymer that has gained recent attention as a transfection reagent. A less known use of this cationic polymer as an attachment factor was explored with several cell lines.     

Relationship of aerobic and anaerobic parameters with 400 m front crawl swimming performance

The aims of the present study were to investigate the relationship of aerobic and anaerobic parameters with 400 m performance, and establish which variable better explains long distance performance in swimming. Twenty-two swimmers (19.1±1.5 years, height 173.9±10.0 cm, body mass 71.2±10.2 kg; 76.6±5.3% of 400 m world record) underwent a lactate minimum test to determine lactate minimum speed (LMS) (i.e., aerobic capacity index). Moreover, the swimmers performed a 400 m maximal effort to determine mean speed (S400m), peak oxygen uptake (V.O2PEAK) and total anaerobic contribution (C_ANA). The C_ANA was assumed as the sum of alactic and lactic contributions. Physiological parameters of 400 m were determined using the backward extrapolation technique (V.O2PEAK and alactic contributions of C_ANA) and blood lactate concentration analysis (lactic anaerobic contributions of C_ANA). The Pearson correlation test and backward multiple regression analysis were used to verify the possible correlations between the physiological indices (predictor factors) and S400m (independent variable) (p < 0.05). Values are presented as mean ± standard deviation. Significant correlations were observed between S400m (1.4±0.1 m·s^-1) and LMS (1.3±0.1 m·s^-1; r = 0.80), V.O2PEAK (4.5±3.9 L·min^-1; r = 0.72) and C_ANA (4.7±1.5 L·O₂; r= 0.44). The best model constructed using multiple regression analysis demonstrated that LMS and V.O2PEAK explained 85% of the 400 m performance variance. When backward multiple regression analysis was performed, C_ANA lost significance. Thus, the results demonstrated that both aerobic parameters (capacity and power) can be used to predict 400 m swimming performance.     

Pheno2Geno - High-throughput generation of genetic markers and maps from molecular phenotypes for crosses between inbred strains

Background

Genetic markers and maps are instrumental in quantitative trait locus (QTL) mapping in segregating populations. The resolution of QTL localization depends on the number of informative recombinations in the population and how well they are tagged by markers. Larger populations and denser marker maps are better for detecting and locating QTLs. Marker maps that are initially too sparse can be saturated or derived de novo from high-throughput omics data, (e.g. gene expression, protein or metabolite abundance). If these molecular phenotypes are affected by genetic variation due to a major QTL they will show a clear multimodal distribution. Using this information, phenotypes can be converted into genetic markers.

Results

The Pheno2Geno tool uses mixture modeling to select phenotypes and transform them into genetic markers suitable for construction and/or saturation of a genetic map. Pheno2Geno excludes candidate genetic markers that show evidence for multiple possibly epistatically interacting QTL and/or interaction with the environment, in order to provide a set of robust markers for follow-up QTL mapping.We demonstrate the use of Pheno2Geno on gene expression data of 370,000 probes in 148 A. thaliana recombinant inbred lines. Pheno2Geno is able to saturate the existing genetic map, decreasing the average distance between markers from 7.1 cM to 0.89 cM, close to the theoretical limit of 0.68 cM (with 148 individuals we expect a recombination every 100/148=0.68 cM); this pinpointed almost all of the informative recombinations in the population.

Conclusion

The Pheno2Geno package makes use of genome-wide molecular profiling and provides a tool for high-throughput de novo map construction and saturation of existing genetic maps. Processing of the showcase dataset takes less than 30 minutes on an average desktop PC. Pheno2Geno improves QTL mapping results at no additional laboratory cost and with minimum computational effort. Its results are formatted for direct use in R/qtl, the leading R package for QTL studies. Pheno2Geno is freely available on CRAN under “GNU GPL v3”. The Pheno2Geno package as well as the tutorial can also be found at: http://pheno2geno.nl.

Electronic supplementary material

The online version of this article (doi:10.1186/s12859-015-0475-6) contains supplementary material, which is available to authorized users.