Urszula Piotrowska

Fibrinogen Fractions in the Third Trimester of Pregnancy and in Puerperium

The concentration of fibrinogen, its fractions, and the concentration of C-reactive protein were determined in 45 healthy pregnant women before and after vaginal (27) or caesarean section 18 delivery. The control group consisted of 33 blood donors. In pregnancy, increased concentration of total fibrinogen and its fractions and a normal concentration of C-reactive protein were noted. Three days after vaginal delivery the concentration of total and high molecular weight fibrinogen fraction decreased slightly and the ratio of high to low molecular weight fibrinogen increased. After caesarean section both total and high molecular weight fibrinogen and the ratio of high to low molecular weight fibrinogen increased. The C-reactive protein concentration increased after either type of delivery. The degree of augmentation, however, was ten times as strong after caesarean section. In all women at the end of puerperium the concentrations of the compounds studied returned to normal values. The results suggest that the mechanisms leading to the increase of fibrinogen during pregnancy and after delivery are different. The increase of all fibrinogen fractions in pregnancy may depend first of all on hormones, whereas the increased proportion of high molecular weight to low molecular weight fibrinogen after delivery depends on the acute phase reaction. The degree of this reaction depends on the type of delivery.

Distinct immunological and biochemical properties of thyroid peroxidase purified from human thyroid glands and recombinant protein produced in insect cells.

The biosynthesis of thyroid hormone from thyroglobulin is catalysed by thyroid peroxidase (TPO), an integral membrane protein. TPO is also a major autoantigen in autoimmune thyroid disease and autoantibodies to TPO are markers for disease activity. Large quantities of purified TPO are essential for elucidating its structure and understanding its role in disease activity. We describe the high yield purification of full-length recombinant human TPO from baculovirus infected insect cells and compare it to purified native TPO from human thyroid glands. In contrast to native human TPO, the human TPO produced in insect cells as a recombinant protein was insoluble and resistant to solubilisation in detergents. Reversible substitution of lysine residues with citraconic anhydride led to increased solubility of the recombinant TPO, allowing high-yield purification by monoclonal antibody chromatography. The purified enzyme preparation was shown to be TPO by its reactivity with monoclonal and polyclonal antibodies by enzyme linked immunosorbent assay and Western blotting. Both the human and recombinant purified TPO preparations also react with sera from patients with autoimmune thyroid disease, although the binding of conformational dependent autoantibodies was considerably lower to the recombinant TPO than to the native TPO. This suggests that the recombinant TPO may differ in some aspects of its tertiary structure. The purified recombinant TPO was devoid of enzyme activity, in contrast to the enzymatically active, purified human TPO preparations. Both preparations contained comparable amounts of haem (R(z)=0.269), but a shift in the Soret band of recombinant TPO (402 nm) from that of natural TPO (409 nm) indicates that the lack of enzymatic activity of the recombinant enzyme may be due to changes in the protein backbone surrounding the haem. Both the purified native and recombinant TPO, under non-denaturing conditions, show evidence of high molecular mass oligomers, although the latter preparation is prone to a greater degree of aggregation. In conclusion, our studies indicate that recombinant TPO generated in insect cells is conformationally distinct from the native TPO, is insoluble and enzymatically inactive, consistent with the difficulties associated with its purification and crystallisation.

Phosducin and monomeric β-actin have common epitope recognized by anti-phosducin antibodies.

Phosducin family proteins are regulators of cytoplasmic processes. The main function ascribed to phosducin is the binding and sequestration of the β subunit of heterotrimeric G proteins. Phosducin-like protein 1, longer than phosducin by 37 amino-acids, is involved in chaperoning of newly synthesized proteins. β-Actin, a component of the cytoskeleton, participates in cell movement. There is no apparent evolutionary relationship between phosducin and β-actin nor structure similarity. Nevertheless we obtained the polyclonal antibodies named ap33, originally directed against a phosducin-derived peptide (SQSLEEDFEGQATHTGPK), that also recognized β-actin. The epitope on the β-actin molecule was characterized. It is a conformational epitope grouping some of the L-D-F-E-Q-A-T-K amino-acids found in the peptide originally used to obtain the antibodies. The main part of the epitope is localized on the actin-actin interface of polymerized actin, so it is accessible only on monomeric actin. The existence of a common epitope on the molecules of phosducin and β-actin may reflect a topological similarity of a small region of their surfaces.