Regulation of cardiac cushion development by hyaluronan.

Hyaluronan is an extracellular matrix component implicated in expansion of the extracellular space, organization of supramolecular architecture, cell motility, proliferation, tumour metastases and wound healing. Hyaluronan is highly expressed in the developing heart but it is only a minor component of the mature heart. The loss of hyaluronan synthase-2 (Has2) results in embryonic lethality with a phenotype remarkably similar to that of the versican-deficient heart defect mouse. Has2-deficient embryos lack hyaluronan-containing cardiac jelly, and at embryonic day 9.5 show arrested development, with an apparent absence of the right ventricle and underdevelopment of the conustruncus segment, and pericardial effusion consistent with heart failure. Cardiac cushions are totally absent, and endocardial cell migration over collagen gels is not detectable in Has2-deficient atrioventricular (AV) canal explants. Endothelial to mesenchymal transformation is also defective in AV explants from Has2-null embryos. The normal phenotype is restored in AV canal explants from Has2-deficient embryos by co-culture with wild type AV canal explants, with conditioned media from wild type AV explants or with exogenous hyaluronan. These results provide evidence for a direct role for hyaluronan during endocardial cushion and AV canal morphogenesis.

Disruption of hyaluronan synthase-2 abrogates normal cardiac morphogenesis and hyaluronan-mediated transformation of epithelium to mesenchyme.

We identified hyaluronan synthase-2 (Has2) as a likely source of hyaluronan (HA) during embryonic development, and we used gene targeting to study its function in vivo. Has2(-/-) embryos lack HA, exhibit severe cardiac and vascular abnormalities, and die during midgestation (E9.5-10). Heart explants from Has2(-/-) embryos lack the characteristic transformation of cardiac endothelial cells into mesenchyme, an essential developmental event that depends on receptor-mediated intracellular signaling. This defect is reproduced by expression of a dominant-negative Ras in wild-type heart explants, and is reversed in Has2(-/-) explants by gene rescue, by administering exogenous HA, or by expressing activated Ras. Conversely, transformation in Has2(-/-) explants mediated by exogenous HA is inhibited by dominant-negative Ras. Collectively, our results demonstrate the importance of HA in mammalian embryogenesis and the pivotal role of Has2 during mammalian development. They also reveal a previously unrecognized pathway for cell migration and invasion that is HA-dependent and involves Ras activation.

Acid precipitation of mammalian cell fermentation broth.

The broth harvested from the "fermentation" of mammalian cells contains many contaminants produced by the cells, including nucleic acids, proteins, and complex polysaccharides, in addition to the product. These contaminants can foul filtration membranes, precipitate during storage or processing steps, and interfere with the performance of chromatographic separations. Acidification of hybridoma cell fermentation broth at the time of harvest from the fermenter has been investigated as a method of selectively precipitating the major contaminants from the soluble antibody product. Between pH values of 6.0 and 4.5, precipitation of the major contaminants is rapid and independent of the temperature (4-37 degrees C), with less than 10% of the antibody coprecipitating in 4 of 5 cases. Antibody activity and physical characteristics were found to be unaltered above a pH of 3.8. Recovery of antibody at the stage of concentrated (50 x), diafiltered bulk was improved from 63% to 84% by using the acid precipitation step. An additional benefit is that retrovirus is effectively inactivated by incubation at a pH below 4.2.