The transmembrane tyrosine kinase TIE-2, the receptor for the angiopoietins-1 and -2, has been shown to be involved in angiogenic processes. Investigating the regulation of TIE-2 expression on endothelial cells, we found that stimulators such as PMA induce a decrease of TIE-2 protein from the cell surface without affecting TIE-2 mRNA. In conditioned media of PMA stimulated endothelial cells, a soluble form of this receptor comprising parts of the extracellular domain can be detected. Using a sandwich ELISA, we were able to detect and quantify TIE-2 receptors in cell lysates (representing the whole transmembrane receptor) and in cell culture supernatants (representing a soluble form of this receptor, sTIE-2). Several factors influencing the shedding process e.g. basic FGF could be identified. Finally, the soluble form of TIE-2 could also be detected in human biological fluids such as sera and plasma from healthy controls.

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~In the sialic acid aldolase-catalysed addition of pyruvate with o-arabinose leading to a mixture of 3-deoxy-~-manno-oct-2-u~osonic acid (KDO) and 4-epi-KDO (both in pyranose and furanose form), a high concentration of the acceptor o-arabinose have been found to increase the percentage of KDO up to 83%. KDO synthesis scaled up in the enzyme membrane reactor is reported. N e w condensation products obtained from L-xylose, L-allose, D-altrose and o-ribose as substrates are described as well. Kinetic parameters for the cleavage of KDO and 4-epi-KDO and the addition reaction between pyruvate and xylose and arabinose are given. Some apparent relation between enzyme stereoselectivity and conformation and stereochemistry at carbon C-3 of the substrate is discussed.Sialic acid aldolase (EC 4.1.3.3), which catalyses the reversible aldol reaction of N-acetylmannosamine and pyruvate to give Nacetylneuraminic acid, has been extensively used in the synthesis of natural and unnatural sialic acids. The original procedure using immobilized enzyme has been scaled up by the use of the enzyme membrane reactor (EMR).2 Moreover, sialic acid aldolase-catalysed condensation of pyruvate with D-arabinose 2 has also been reported; this was the first example of a lack of stereoselectivity of an aldolase: two diastereoisomers present in equimolar amounts were characterized in their pyranose form, as 3-deoxy-~-manno-oct-2-ulosonic acid (KDO) and 3-deoxy-~-gluco-oct-2-ulosonic acid (4-epi-KDO). Enzymic synthesis of KDO, the universal component of lipopolysaccharides (LPS) in the outer membrane of Gram-negative bacteria, has been reported with KDO-8-phosphate synthetase (EC 4.1.2.16), from ~-arabinose-5-phosphate and phosphoen~lpyruvate.~ Moreover KDO aldolase (EC 4. l .2.23), using non-phosphorylated intermediates, has been identified in crude extracts of Gramnegative bacteria; this enzyme was very recently partially purified from Aureobacterium barkerei, a Gram-positive bacterium, and used in synthesk6We now report new findings in the sialic acid aldolasecatalysed condensation of pyruvate 1 with D-arabinose 2, allowing a practical preparation of KD0.7 Trying to find an interpretation for the occurrence of the inverted stereoselectivity with D-arabinose, we hypothesized that the inverted stereoselectivity could originate from the C4 major Darabinose conformer. This hypothesis was reinforced by the recent results of complete inversion of stereoselectivity obtained for this aldolase with various L sugars such as L-mannose, Lrhamnose, and L-talose, which are known to adopt exclusively the preferred C4 c o n f o r m a t i~n .~.~ Moreover, another case of lack of selectivity with the sialic acid aldolase was reported with 3-deoxymannose, allowing us to suppose that the hydroxy group on carbon C-3 is also involved in positioning of the substrate at the active site." Therefore, in order to gain more insight into the selectivity of this aldolase we wanted to clarify the question: is there any relationship between the enzyme stereoselectivity and eit...

The enzymatic synthesis of 2-keto-3-deoxy-D-glycero-D-galacto-nonopyranulosonic acid (KDN) starting from D-mannose and pyruvic acid using Neu5Ac-aldolase has been scaled up. A repetitive batch ultrafiltration bioreactor was used for the KDN synthesis on 100 g scale with a conversion of up to 85%. Furthermore, a 440 mL pilot-scale enzyme membrane reactor (EMR) was performed for the continuous production of KDN. Conversion of mannose was 75% at a space--time yield of 375 g/(L d). KDN was advanteageously isolated by crystallization with an overall yield of 75%.