Reduction of product-related species during the fermentation and purification of a recombinant IL-1 receptor antagonist at the laboratory and pilot scale

Through a parallel approach of tracking product quality through fermentation and purification development, a robust process was designed to reduce the levels of product-related species. Three biochemically similar product-related species were identified as byproducts of host-cell enzymatic activity. To modulate intracellular proteolytic activity, key fermentation parameters (temperature, pH, trace metals, EDTA levels, and carbon source) were evaluated through bioreactor optimization, while balancing negative effects on growth, productivity, and oxygen demand. The purification process was based on three non-affinity steps and resolved product-related species by exploiting small charge differences. Using statistical design of experiments for elution conditions, a high-resolution cation exchange capture column was optimized for resolution and recovery. Further reduction of product-related species was achieved by evaluating a matrix of conditions for a ceramic hydroxyapatite column. The optimized fermentation process was transferred from the 2-L laboratory scale to the 100-L pilot scale and the purification process was scaled accordingly to process the fermentation harvest. The laboratory- and pilot-scale processes resulted in similar process recoveries of 60 and 65%, respectively, and in a product that was of equal quality and purity to that of small-scale development preparations. The parallel approach for up- and downstream development was paramount in achieving a robust and scalable clinical process.     

An improved ontological representation of dendritic cells as a paradigm for all cell types

Background  

Recent increases in the volume and diversity of life science data and information and an increasing emphasis on data sharing and interoperability have resulted in the creation of a large number of biological ontologies, including the Cell Ontology (CL), designed to provide a standardized representation of cell types for data annotation. Ontologies have been shown to have significant benefits for computational analyses of large data sets and for automated reasoning applications, leading to organized attempts to improve the structure and formal rigor of ontologies to better support computation. Currently, the CL employs multiple is_a relations, defining cell types in terms of histological, functional, and lineage properties, and the majority of definitions are written with sufficient generality to hold across multiple species. This approach limits the CL's utility for computation and for cross-species data integration.     

Production and characterization of a transgenic bread wheat line over-expressing a low-molecular-weight glutenin subunit gene

The end-use properties, and thus the value, of wheat flours are determined to a large extent by the proteins that make up the polymeric network called gluten. Low molecular weight glutenin subunits (LMW-GS) are important components of gluten structure. Their relative amounts and/or the presence of specific components can influence dough visco-elasticity, a property that is correlated with the end-use properties of wheat flour. For these reasons, manipulation of gluten dough strength and elasticity is important. We are pursuing this goal by transforming the bread wheat cultivar Bobwhite with a LMW-GS gene driven by its own promoter. Particle bombardment of immature embryos produced several transgenic lines, one of which over-expressed the LMW-GS transgene. Southern blots confirmed that the transgene was integrated into the wheat genome, although segregation analyses showed that its expression was sometimes poorly transmitted to progeny. We have determined that the transgene-encoded LMW-GS accumulates to very high levels in seeds of this line, and that it is incorporated into the glutenin polymer, nearly doubling its overall amount. However, SDS sedimentation test values were lower from the transgenic material compared to a non transgenic flour. These results suggest that the widely accepted correlation between the amount of the glutenin polymers and flour technological properties might not be valid, depending on the components of the polymer.     

Selective inhibition of prostaglandin biosynthesis by gold salts and phenylbutazone

Gold salts and phenylbutazone selectively inhibit the synthesis of PGF_2α and PGE₂ respectively. Lowered production of one prostaglandin species is accompanied by an increased production of the other. Selective inhibition by these drugs was observed in the presence of adrenaline, reduced glutathione and copper sulphate under conditions when most anti-inflammatory compounds inhibited PGE₂ and PGF_2α syntheses equally. It is postulated that selective inhibitors may have a different mode of action and beneficial effects may be related to the endogenous ratio of PGE to PGF required for normal function.     

Disruption of spectrin-like cytoskeleton in differentiating keratinocytes by PKCδ activation is associated with phosphorylated adducin

Spectrin is a central component of the cytoskeletal protein network in a variety of erythroid and non-erythroid cells. In keratinocytes, this protein has been shown to be pericytoplasmic and plasma membrane associated, but its characteristics and function have not been established in these cells. Here we demonstrate that spectrin increases dramatically in amount and is assembled into the cytoskeleton during differentiation in mouse and human keratinocytes. The spectrin-like cytoskeleton was predominantly organized in the granular and cornified layers of the epidermis and disrupted by actin filament inhibitors, but not by anti-mitotic drugs. When the cytoskeleton was disrupted PKCδ was activated by phosphorylation on Thr505. Specific inhibition of PKCδ(Thr505) activation with rottlerin prevented disruption of the spectrin-like cytoskeleton and the associated morphological changes that accompany differentiation. Rottlerin also inhibited specific phosphorylation of the PKCδ substrate adducin, a cytoskeletal protein. Furthermore, knock-down of endogenous adducin affected not only expression of adducin, but also spectrin and PKCδ, and severely disrupted organization of the spectrin-like cytoskeleton and cytoskeletal distribution of both adducin and PKCδ. These results demonstrate that organization of a spectrin-like cytoskeleton is associated with keratinocytes differentiation, and disruption of this cytoskeleton is mediated by either PKCδ(Thr505) phosphorylation associated with phosphorylated adducin or due to reduction of endogenous adducin, which normally connects and stabilizes the spectrin-actin complex.     

Characterisation and chromosomal localisation of C-type low-molecular-weight glutenin subunits in the bread wheat cultivar Chinese Spring

Low-molecular-weight glutenin subunits are classically divided into the B, C and D groups. Most attention has been paid to the characterisation of the B and D groups, whereas C subunits, although represented by a large number of protein components, have not been thoroughly characterised, mainly because they tend to separate with the gliadins in many fractionation procedures. Here we describe a procedure for obtaining a fraction strongly enriched in C subunits that has allowed us to determine the chromosomal location of these subunits in the bread wheat cultivar Chinese Spring. This analysis has shown that these subunits are coded on chromosome groups 1 and 6. Comparison between N-terminal amino acid sequencing of B and C subunits has shown that, whereas the former group includes mainly subunits with typical LMW-GS type sequences (76%), the C subunit group is made up almost completely of subunits with gliadin-like sequences (95%), including the alpha-type. These results indicate that the LMW-GSs are likely to be coded not only by the typical Glu-3 loci, but also by loci tightly linked to, and possibly included within, the Gli-1 and Gli-2 loci.     

Facultative heterochromatization in parahaploid male mealybugs: involvement of a heterochromatin-associated protein

The behavior of chromosomes during development of the mealybug Planococcus citri provides one of the most dramatic examples of facultative heterochromatization. In male embryos, the entire haploid paternal chromosome set becomes heterochromatic at mid-cleavage. Male mealybugs are thus functionally haploid, owing to heterochromatization (parahaploidy). To understand the mechanisms underlying facultative heterochromatization in male mealybugs, we have investigated the possible involvement of an HP-1-like protein in this process. HP-1 is a conserved, nonhistone chromosomal protein with a proposed role in heterochromatinization in other species. It was first identified in Drosophila melanogaster as a protein enriched in the constitutive heterochromatin of polytene chromosome. Using a monoclonal antibody raised against the Drosophila HP-1 in immunoblot and immunocytological experiments, we provide evidence for the presence of an HP-1-like in Planococcus citri males and females. In males, the HP-1-like protein is preferentially associated with the male-specific heterochromatin. In the developing male embryos, its appearance precedes the onset of heterochromatization. In females, the HP-1-like protein displays a scattered but reproducible localization pattern along chromosomes. The results indicate a role for an HP-1-like protein in the facultative heterochromatization process.     

Lead inhibits growth and induces apoptosis in normal rat fibroblasts

The effects on normal rat fibroblasts of lead supplementation (as lead acetate) in the medium were examined. The amount of lead acetate ranged from 0.078 microM to 320 microM, at 14 concentrations. The normal level of lead in the medium was 0.060 microM, and the normal concentration of lead in the fibroblasts was 3.1 +/- 0.1 ng/10(7) cells: these represented the control conditions. On studying fibroblast proliferation and survival after incubation for 48 hours, a lead acetate dose-dependent inhibition of cell proliferation was observed, the results being shown to be significant by ANOVA (p < 0.01), and suggesting a significant dose-response relationship. Apoptosis, evaluated by quantifying cytoplasmic DNA fragments, differs significantly between the lead levels tested. The distribution in the cell cycle, evaluated by using a fluorescence-activated cell sorter, showed a dose-dependent accumulation of cells in the G0/G1 phase, with a compensatory decrease in the percentage of cells in the S phase. Moreover, the occurrence of a subdiploid peak confirmed that apoptosis was more evident when the medium was supplemented with lead acetate at concentrations of 5-20 microM. The inhibition of cell growth is probably due to a direct inhibition of cell proliferation.     

A high field NMR study of the products ensuing from konjak glucomannan C(6)-oxidation followed by enzymatic C(5)-epimerization

Konjak glucomannan (KGM) is a water-soluble linear copolymer of (1-->4) linked beta-D-mannopyranosyl and beta-D-glucopyranosyl units. It has been selectively C6-oxidized by a 2,2,6,6-tetramethylpiperidin-1-oxy mediated reaction to obtain the corresponding uronan. Oxidized KGM has been treated with three different C-5 epimerases, AlgE4, AlgE6, and AlgE1, to obtain uronans with a various content of alpha-L-gulopyranuronate residues, namely, KGME4, KGME6, and KGME1. By use of 1D selective and 2D NMR techniques, a full assignment of the high field (600 MHz) NMR spectra of the purified native KGM and of the oxidized and epimerized derivatives has been obtained. Since in the anomeric region of the (1)H NMR spectrum of native KGM, diads sensitivity is present, the glucose-glucose, glucose-mannose, mannose-mannose, and mannose-glucose distribution has been obtained. In the (13)C spectrum of oxidized KGM, due to the presence of triad sensitivity on the C-4 resonance of glucuronic and mannuronic units, a better sequential investigation has been possible. As a result the average length of mannuronic blocks, N(M) is obtained. When AlgE4, AlgE6, and AlgE1 enzymes are used for the epimerization of oxidized KGM, the reaction products differ significantly both in the proportion and in the distribution of the mannuronic and guluronic residues. In epimerized KGM derivatives, a careful deconvolution of (1)H spectra allows the measurement of the degree of epimerization. In the case of KGME1 and KGME6, the average blocks length, N(G), of the guluronic blocks introduced in the polysaccharidic chain with the epimerization has also been calculated. Due to the shortness of mannuronic blocks in the oxidized KGM before the epimerization, N(G) in the epimerized compounds is also very low.