Engineering aspects of beta-lactam biosynthesis.

The adaptation of an existing industrial production line to boost production capacity (a hardware analogue of strain improvement) or to enable the production of a derivative of the original product is used as an example to explore the similarities between process design and engineering and metabolic pathway engineering. In the two fields the same principles appear to apply: for most process engineering solutions metabolic pathway engineering analogues can be found. Analogues are illustrated by reference to literature (e.g. overproduction of amino acid precursors at the 'bottom' of the pathway, relocalisation of proteins and overexpression of genes from the secondary metabolism). Some possible future applications are defined (e.g. supervisory manipulations as regulation by gene transcription factors).

The role of scavenger receptor class A in the adhesion of cells is dependent on cell type and cellular activation state.

Scavenger receptor class A (SR-A) facilitates the development of atherosclerosis, which might be due to its role in the uptake of modified low-density lipoproteins. However, the receptor is also suggested to be important for cell adhesion, thereby potentially influencing the residence time of cells in vivo. Using SR-A-deficient mice, we investigated the role of SR-A in the adhesion of peritoneal macrophages (PM) and tissue macrophages (Kupffer cells). In resident PM no effect of the absence or presence of SR-A on cell adhesion was observed, either in the presence or in the absence of serum. However, in thioglycollate-induced PM, SR-A is important for adhesion both in the presence and in the absence of serum and more than 85% of the divalent-cation-independent adhesion in the presence of serum is mediated by SR-A. In unactivated Kupffer cells, like in resident PM, adhesion is not influenced by the absence or presence of SR-A. In vivo administration of phorbol 12-myristate 13-acetate leads to the activation of Kupffer cells, and it appears that under these conditions SR-A does contribute to adhesion, since both in the absence and in the presence of serum SR-A is responsible for about 35% of cell adhesion. It is concluded that SR-A is important for the divalent-cation-independent adhesion of activated PM and Kupffer cells, suggesting that SR-A may influence the residence time of cells at sites of cellular activation, e.g., in atherosclerotic plaques and during liver infection.

Characterization of a receptor for oxidized low-density lipoproteins on rat Kupffer cells: similarity to macrosialin.

Rat liver Kupffer cell membranes contain a protein that recognizes specifically oxidized low-density lipoproteins (oxLDL). Visualization after blotting under reducing conditions indicates that the receptor is a monomeric protein, with an estimated molecular mass of 115-120 kDa. N-Glycosidase F and endoglycosidase F treatment resulted in a fall in estimated molecular mass of 24 and 11 kDa respectively, whereas O-glycosidase was ineffective. No effect on the extent of interaction with oxLDL was noticed, suggesting that glycans are not essential for ligand recognition. Using a polyclonal antibody to mouse macrosialin, we visualized macrosialin on blot, and compared this glycoprotein with the oxLDL-binding protein. It appears that the two glycoproteins have a similar molecular mass and are comparably affected by treatment with the different glycosidases. Incubation with trypsin resulted in a reduction in the estimated molecular mass of about 25 kDa for both the oxLDL-binding protein and macrosialin. These results indicate that the oxLDL-binding protein and macrosialin are identical, suggesting a role for macrosialin in modified LDL catabolism.

LDL receptor-independent and -dependent uptake of lipoproteins.

The liver plays a decisive role in the regulation of the plasma levels of atherogenic lipoproteins. The primary liver interaction site for chylomicron-remnants and VLDL remnants (beta-VLDL) is still unidentified, while the subsequent cellular uptake is likely to be mediated in concert by the LDL receptor related protein (LRP) and the LDL receptor. The nature of the primary interaction site of remnants (remnant-receptor) might be a liver-specific proteoglycan or a liver-specific protein. Atherogenic modified LDL can be recognized by a family of scavenger-receptors. A newly identified 95-kDa protein forms the most likely candidate for mediating the in vivo uptake of oxidized LDL from the circulation and might thus protect the body against the presence of oxidized LDL in the blood compartment.

Epidermal growth factor induces rapid and transient association of phospholipase C-gamma 1 with EGF-receptor and filamentous actin at membrane ruffles of A431 cells.

Addition of epidermal growth factor to A431 cells results in dramatic changes in cell morphology. Initially the cells form membrane ruffles accompanied by increased actin polymerization, followed by cell rounding. Activation of the tyrosine kinase of the receptor by binding epidermal growth factor leads also to phosphorylation and activation of phospholipase C-gamma 1, a key enzyme in the phosphoinositide pathway. In this study we have investigated the localization of phospholipase C-gamma 1 during cell activation by epidermal growth factor. It is shown that addition of the growth factor to A431 cells leads to a translocation of phospholipase C-gamma 1 from the cytosol to the membrane fraction. Interestingly, this relocation is exclusively directed to the membrane ruffles. Most of the phospholipase C-gamma 1 associates to the membrane and a small fraction to the underlying skeleton. Immunocytochemical studies demonstrated that phospholipase C-gamma 1 co-localizes with the epidermal growth factor receptor and also filamentous actin at the membrane ruffles. Moreover, using anti-phosphotyrosine antibodies we found that the membrane ruffles are significantly enriched in phosphotyrosyl proteins. Between 5 and 10 minutes after stimulation the membrane ruffles disappear and also the co-localization of phospholipase C-gamma 1 with the epidermal growth factor receptor and filamentous actin. These results support the notion that activation of A431 cells by epidermal growth factor leads to the formation of a signalling complex of its receptor, phospholipase C-gamma 1 and filamentous actin which is primarily localized at membrane ruffles.