Accessibility of an epitope common to all histone H3 variants in folded and unfolded chromatin as studied by a monoclonal antibody

In a recent publication the isolation and some characteristics of an anti-histone 3 monoclonal antibody, 1GB3 were described (Muller et al. FEBS Lett. 182: 459–464, 1985). We now report that the epitope recognized is phylogenetically conserved and located in the N-terminal part of H3, most likely between residues 40 and 50. Using the ELISA technique we found this region to be accessible in chromatin to the monoclonal antibody. The effect of non-ionic detergents on the adsorbtion of chromatin on microtiter plates was studied in this context.Immunological analysis of the reaction of the monoclonal antibody with chromatin by immunoinhibition and immunosedimentation shows that the H3 epitope is accessible in both folded and unfolded chromatin fibre as well as in high- and low-molecular weight oligonucleosomes.Abbreviations BSA Bovine srum albumin - mab Monoclonal antibody - PBS Phosphate buffered saline - PMSF Phenylmethyl sulfonyl fluoride     

HAM: A new epitope-tag for in vivo protein labeling

The ability to metabolically label proteins with ³⁵S-methionine is critical for the analysis of protein synthesis and turnover. Despite the importance of this approach, however, efficient labeling of proteins in vivo is often limited by a low number of available methionine residues, or by deleterious side-effects associated with protein overexpression. To overcome these limitations, we have created a methionine-rich variant of the widely used HA tag, called HAM, for use with ectopically expressed proteins. Here we describe the development of a series of vectors, and corresponding antisera, for the expression and detection of HAM-tagged proteins in mammalian cells. We show that the HAM tag dramatically improves the sensitivity of ³⁵S-methionine labeling, and permits the analysis of Myc oncoprotein turnover even when HAM-tagged Myc is expressed at levels comparable to that of the endogenous protein. Because of the improved sensitivity provided by the HAM tag, the vectors and antisera described here should be useful for the analysis of protein synthesis and destruction at physiological levels of protein expression.     

Engineering lysine‐rich caleosins as carrier proteins to render biotin as a hapten on artificial oil bodies for antibody production

It has been demonstrated that caleosin alone is sufficient to stabilize artificial oil bodies. A series of recombinant caleosins, mutated with 3, 5, 8, 11, 13, 15, and 17 extra Lys residues and over‐expressed in Escherichia coli, were used as carrier proteins to render biotin as a hapten on the surface of artificial oil bodies for antibody production. Biotinylation levels of the recombinant caleosins were step‐wisely elevated as the number of extra Lys residues increased, and the biotinylated Lys residues were identified by mass spectrometric analysis. Polyclonal antibodies against biotin were successfully generated in rats injected with artificial oil bodies constituted with each of the biotinylated caleosins. Moreover, those generated via the biotinylated caleosins with eight or more extra Lys residues no longer recognized caleosin. It appears that engineered Lys‐rich caleosins are suitable carrier proteins for the production of antibodies against small molecules. © 2011 American Institute of Chemical Engineers Biotechnol. Prog., 2011     

Targeting Interleukin-6: All the Way to Treat Autoimmune and Inflammatory Diseases

Interleukin (IL)-6, a cytokine featuring redundancy and pleiotropic activity, contributes to host defense against acute environmental stress, while dysregulated persistent IL-6 production has been demonstrated to play a pathological role in various autoimmune and chronic inflammatory diseases. Targeting IL-6 is thus a rational approach to the treatment of these diseases. Indeed, clinical trials of tocilizumab, a humanized anti-IL-6 receptor antibody have verified its efficacy and tolerable safety for patients with rheumatoid arthritis, Castleman''s disease and systemic juvenile idiopathic arthritis, resulting in approval of this innovative biologic for treatment of these diseases. Moreover, a considerable number of case reports and pilot studies of off-label use of tocilizumab point to the beneficial effects of tocilizumab for a variety of other phenotypically different autoimmune and chronic inflammatory diseases. Elucidation of the source of IL-6 and of mechanisms through which IL-6 production is dysregulated can thus be expected to lead to clarification of the pathogenesis of various diseases.     

The effects of manipulation of sedimentary iron and organic matter on sediment biogeochemistry and seagrasses in a subtropical carbonate environment

The microbial metabolism of organic matter (OM) in seagrass beds can create sulfidic conditions detrimental to seagrass growth; iron (Fe) potentially has ameliorating effects through titration of the sulfides and the precipitation of iron-sulfide minerals into the sediment. In this study, the biogeochemical effects of Fe availability and its interplay with sulfur and OM on sulfide toxicity, phosphorous (P) availability, seagrass growth and community structure were tested. The availability of Fe and OM was manipulated in a 2 × 2 factorial experiment arranged in a Latin square, with four replicates per treatment. The treatments included the addition of Fe, the addition of OM, the addition of both Fe and OM as well as no addition. The experiment was conducted in an oligotrophic, iron-deficient seagrass bed. Fe had an 84.5% retention efficiency in the sediments with the concentration of Fe increasing in the seagrass leaves over the course of the experiment. Porewater chemistry was significantly altered with a dramatic decrease in sulfide levels in Fe addition plots while sulfide levels increased in the OM addition treatments. Phosphorus increased in seagrass leaves collected in the Fe addition plots. Decreased sulfide stress was evidenced by heavier δ³⁴S in leaves and rhizomes from plots to which Fe was added. The OM addition negatively affected seagrass growth but increased P availability; the reduced sulfide stress in Fe added plots resulted in elevated productivity. Fe availability may be an important determinant of the impact that OM has on seagrass vitality in carbonate sediments vegetated with seagrasses.     

EPR properties of mixed-valent μ-oxo and μ-hydroxo dinuclear iron complexes produced by radiolytic reduction at 77 K

 Radiolytic reduction at 77 K of oxo-/hydroxo-bridged dinuclear iron(III) complexes in frozen solutions forms kinetically stabilized, mixed-valent species in high yields that model the mixed-valent sites of non-heme, diiron proteins. The mixed-valent species trapped at 77 K retain ligation geometry similar to the initial diferric clusters. The shapes of the mixed-valent EPR signals depend strongly on the bridging ligands. Spectra of the Fe(II)OFe(III) species reveal an S=1/2 ground state with small g-anisotropy as characterized by the uniaxial component (g _z –g _av /2<0.03) observable at temperatures as high as ∼100 K. In contrast, hydroxo-bridged mixed-valent species are characterized by large g-anisotropy (g _z –g _av /2>0.03) and are observable only below 30 K. Annealing at higher temperatures causes structural relaxation and changes in the EPR characteristics. EPR spectral properties allow the oxo- and hydroxo-bridged, mixed-valent diiron centers to be distinguished from each other and can help characterize the structure of mixed-valent centers in proteins. Received: 27 June 1998 / Accepted: 25 February 1999     

Analytical characterization and purification of plasma membrane from cultured hepatoma cells (HTC cells)

The plasma membrane of the hepatoma cell line, HTC cells, has been characterized and purified by cell fractionation techniques. In the absence of true 5′-nucleotidase in HTC cells, alkaline phosphodiesterase I has been used as a marker enzyme, following conclusions gained from differential and isopycnic centrifugation studies (Lopez Saura, P., Trouet A. and Tulkens P. (1978) Biochim. Biophys. Acta 543, 430–449). To confirm this localization, HTC cells were exposed to anti-plasma membrane IgG at 4°C and fractionated. Alkaline phosphodiesterase I and IgG showed super imposable distribution patterns in linear sucrose gradients. Alkaline phosphodiesterase I is, however, only poorly resolved from enzyme markers of other organelles, especially NADPH-cytochrome $\text{c}$ reductase (endoplasmic reticulum) and galactosyltransferase (Golgi complex). Maximal purification from the homogenate is only 13-fold, on a protein basis, even when using a microsomal fraction (67 and 13% of alkaline phosphodiesterase I and protein, respectively) as the starting material. Improved resolution can be obtained after the addition of small quantities of digitonin (equimolar with respect to the cholesterol content). Digitonin increases the buoyant density of alkaline phosphodiesterase I by approx. 0.05 g/cm³, whereas the buoyant densities of galactosyltransferase and NADPH-cytochrome $\text{c}$ reductase are increased only by 0.03 and 0.015 g/cm³, respectively. Accordingly, a procedure has been designed which yields a fraction containing 22.8% of alkaline phosphodiesterase I with a purification of 21-fold on a protein basis. The content of NADPH-cytochrome $\text{c}$ reductase and galactosyltransferase is 1.2 and 2.1%, respectively. Electron microscopy shows smooth surface membrane elements and vesicles, with only occasional other recognizable elements.     

Hormone-dependent processing of the avian progesterone receptor

Avian progesterone receptor exists as two forms, A and B, with molecular weights of 79,000 and 110,000 daltons, respectively. The origin and significance of these two forms is an area of active investigation and debate. Monoclonal antibodies produced against these two forms were used to examine receptor stability in cytosol and changes in the receptor forms induced by hormone binding. The lability of hormone binding at elevated temperatures is well documented. Analysis by Western blotting showed the receptor was stable in freshly prepared oviduct cytosol for 2 hr at 37°C, while hormone binding was lost within 30 min. However, loss of receptor through degradation was seen when cytosol was prepared from frozen tissue or when homogenization was excessive. Progesterone was injected into diethylstilbestrol-stimulated chicks to examine, in vivo, effects of hormone treatment on receptor forms in the cytosol and nuclear fractions. Progesterone treatment caused a time- and dose-dependent conversion of the A receptor to a form (A′) with a slower electrophoretic mobility. The cytosolic progesterone receptor was divided equally between the B and A forms, while the nuclear receptor was predominantly A′. The amount of nuclear receptor was consistently less than cytosolic receptor. Receptor phosphorylation was analyzed by incubating tissue minces with [³²P]orthophosphate with or without progesterone followed by immune isolation of receptor forms. Progesterone treatment caused a time-dependent increase in cytosol receptor phosphorylation which was evident after 5 min of treatment. This phosphorylation was observed with both the A and B receptor forms. The results indicate that receptor phosphorylation is a very early event during progesterone action.