Biochemical composition and heterogeneity of heparan sulfates isolated from AH-130 ascites hepatoma cells and fluid

The glycosaminoglycan composition of AH-130 ascites hepatoma cells and fluid were examined using enzymatic digestion, electrophoresis, and sequential partition fractionation. The cell-associated glycosaminoglycans were found to consist of 93% heparan sulfate, with the remainder consisting primarily of chondroitin sulfate. The glycosaminoglycans isolated from the ascitic fluid were found to consist of 58% heparan sulfate, 26% hyaluronic acid and 16% chondroitin sulfate. Dermatan sulfate was not detected in either cells or fluid. The heparan sulfate isolated from AH-130 cells is low-sulfate and highly heterogeneous with respect to biochemical composition. Fractions isolated by partition fractionation varied from 0.14 mol sulfate/mol uronic acid to 0.6 mol sulfate/mol uronic acid. Of the total sulfate 70–80% is N-sulfate in the former and 50% in the latter. Electrophoresis in 0.1 M HCl showed a highly heterogeneous material with mobility between that of hyaluronic acid and beef lung heparan sulfate. The heparan sulfate isolated from the fluid was similar to that isolated from the cells but was, however, somewhat more homogeneous with respect to charge.     

A dual rescaled Mitscherlich model of the simultaneous savings in phosphorus and zinc fertiliser from arbuscular mycorrhizal fungal colonisation of linseed (Linum usitatissimum L.)

Plant and Soil - Plant species with thin roots and high root-growth rate are thought to have greater root foraging precision than species with thick roots and low root-growth rate. However, whether...     

Development of a novel mammalian display system for selection of antibodies against membrane proteins

Reliable, specific polyclonal and monoclonal antibodies are important tools in research and medicine. However, the discovery of antibodies against their targets in their native forms is difficult. Here, we present a novel method for discovery of antibodies against membrane proteins in their native configuration in mammalian cells. The method involves the co-expression of an antibody library in a population of mammalian cells that express the target polypeptide within a natural membrane environment on the cell surface. Cells that secrete a single-chain fragment variable (scFv) that binds to the target membrane protein thereby become self-labeled, enabling enrichment and isolation by magnetic sorting and FRET-based flow sorting. Library sizes of up to 10⁹ variants can be screened, thus allowing campaigns of naïve scFv libraries to be selected against membrane protein antigens in a Chinese hamster ovary cell system. We validate this method by screening a synthetic naïve human scFv library against Chinese hamster ovary cells expressing the oncogenic target epithelial cell adhesion molecule and identify a panel of three novel binders to this membrane protein, one with a dissociation constant (K_D) as low as 0.8 nm. We further demonstrate that the identified antibodies have utility for killing epithelial cell adhesion molecule–positive cells when used as a targeting domain on chimeric antigen receptor T cells. Thus, we provide a new tool for identifying novel antibodies that act against membrane proteins, which could catalyze the discovery of new candidates for antibody-based therapies.     

Raising the sauropod neck: it costs more to get less

The long necks of gigantic sauropod dinosaurs are commonly assumed to have been used for high browsing to obtain enough food. However, this analysis questions whether such a posture was reasonable from the standpoint of energetics. The energy cost of circulating the blood can be estimated accurately from two physiological axioms that relate metabolic rate, blood flow rate and arterial blood pressure: (i) metabolic rate is proportional to blood flow rate and (ii) cardiac work rate is proportional to the product of blood flow rate and blood pressure. The analysis shows that it would have required the animal to expend approximately half of its energy intake just to circulate the blood, primarily because a vertical neck would have required a high systemic arterial blood pressure. It is therefore energetically more feasible to have used a more or less horizontal neck to enable wide browsing while keeping blood pressure low.