A thyroxine binding globulin (TBG)-like protein in the sera of developing and adult rats

We report evidence based on equilibrium binding, electrophoretic, autoradiographic studies, that the rat possesses a major high affinity thyroid hormone binding protein, with an electrophoretic mobility and binding properties similar to those of the human thyroxine binding globulin (TBG). We show that in the sera of postnatal developing animals, the thyroxine and the triiodothyronine binding activities increase up to 10 times over adult or foetal levels, due to a high transient post-natal surge of the rat TBG. In the adult serum, the TBG persists in decreased amounts: it then yields the predominant role as thyroxine carrier to the thyroid binding prealbumin, but retains the major role as binder of triiodothyronine i.e. of the biologically active thyroid hormone.     

Rheology of fibrin clots: III. Shear creep and creep recovery of fine ligated and coarse unligated clots

Creep and creep recovery of human fibrin clots in small shearing deformations have been investigated over a time scale from 24 to 10⁴ s. Coarse, unligated dots and fine dots ligated by fibrinoligase in the presence of calcium ions were studied to suppllement previous data on coarse ligated and fine unligated clots. Stress was found to be proportional to strain up to at least a maximum shear strain (in torsion geometry) of 2.6%. The initial modulus (25 s after imposition of stress) is proportional to approximately the 1.5 power of concentration for fine ligated and coarse unligated clots. For fine unligated clots, there is comparatively little creep subsequent to the initial deformation; ligation (in this case involving mostly the γ chains) reduces the creep to nearly zero. For coarse unligated dots, there is substantially more creep under constant stress, and creep recovery is not complete. legation (in this casa involving both γ and α chains) largely suppresses the creep and causes the recovery to be complete. If the structure is fully formed before creep begins, tests of creep recovery by the Boltzmann superposition principle show adherence to linear viscoelastic behavior for all four clot types. Otherwise, the Boltzmann test fails and the recovery is much less than calculated. For fine ligated clots, the observed recovery agrees well with that calculated on the basis of a dual structure model in which an additional independent structure is built up in the deformed state, so that the state of ease after removal of stress is a balance between two structures deformed in opposite senses, it is postulated that the coherence and elastic modulus of the fine ligated dot are largely due to steric blocking of long protofibrils with a high flexural stiffness. In the coarse clot, it is proposed that the structure involves extensive branching of thick bundles of protofibrils, which become permanently secured by the ligation of the α chains of the fibrin.     

Relations between fatty acids and oestrogen binding properties of pure rat alpha1-foetoprotein

A delipidation procedures based on treatment with charcoal at pH 3 has been applied to highly purified rat alpha₁-foetoprotein preparations. The oestrogen binding properties of the delipidated proteins have been studied with an equilibrium dialysis technique, and compared with the properties of the untreated foetal protein, as well as those of preparations reconstituted from the defatted α₁-foetoprotein and the removed lipids. An important increase has been evidenced for the binding levels of oestrone, oestradiol-17β and diethyl-stilboestrol by the delipidated α₁-foetoprotein. A reversal of this effect has been obtained by incubating the delipidated protein either with the lipids extracted from the purified α₁-foetoprotein or with a potent competitor of the rat α₁-foetoprotein-oestrogen interaction, designated as ‘L’, previously demonstrated and isolated from whole rat sera, and tentatively characterized as a mixture of fatty acids. Scatchard analysis of the oestrone and oestradiol-17β binding parameters show that the enhanced fixation of the hormones after defatting is primarily due to a two-fold increase of the apparent number of binding sites/ mol α₁-foetoprotein. The results are interpreted in terms of the probable, at least partial, identity between the lipids closely associated with the pure α₁-foetoprotein and the fatty acid mixture ‘L’ isolated from whole sera. The possible biological role of a complex interplay between oestrophilic α₁-foetoproteins, phenolsteroids and fatty acids in the control of eostrogen levels during development is discussed brieftly.     

Clinical improvement after treatment with VEGF165 in patients with severe chronic lower limb ischaemia

The present study focuses on the application of a therapeutic strategy in patients with chronic severe lower limb ischaemia using a plasmid vector encoding the vascular endothelial growth factor (phVEGF₁₆₅). It has been shown that VEGF promotes neo-vascularization and blood vessel network formation and thus might have the ability to improve blood-flow at the level of the affected limbs. However, little information is available regarding the necessary level of expression of VEGF and its possible related adverse effects. We have subcloned VEGF ₁₆₅ isoform into pCMV-Script expression vector (Stratagene) under the control of the CMV promoter. Three patients with chronic ischaemia of the lower limb, considered as not suitable for surgical re-vascularization, received intramuscular injection with 0.5 ml saline solution containing 10¹¹ copies of VEGF ₁₆₅ plasmid. The clinical evolution has been monitored by angiography and estimated by walking time on the rolling carpet (Gardner protocol). Two months after therapy, all three patients showed complete relief of rest pain, improvement of ischaemic ulcer lesions and increased walking distance on the rolling carpet most probably due to appearance of newly formed collateral vessels.     

Transglutaminases

Summary This paper is intended as a background to the topic of transglutaminases, while focusing on current ideas regarding the biological roles of these enzymes. Specifically, the following topics are discussed: geometry of forming -glutamyl--lysine cross-linked structures; energetic considerations; the -glutamyl--lysine cross-link; amine incorporation assays; artefactual incorporation of amines in cells and tissue homogenates; synthetic substrate systems; regulation of transglutaminase activities; strategies for probing transglutaminase-mediated events in biological systems; the blood clotting paradigm; transglutaminase and cell aging: the Ca²⁺-enriched human erythrocyte; transglutaminase and cell activation: the thrombin-stimulated human platelet and the fertilized sea urchin egg.     

Biotinylated peptides containing a factor XIIIa or a tissue transglutaminase-reactive glutaminyl residue that block protein cross-linking phenomena by becoming incorporated into amine donor sites.

Biotinylated peptides Biot-Gln-Gln-Ile-Val and Biot-epsilon-Aca-Gln-Gln-Ile-Val were shown to act as acceptor substrates for amines in reactions catalyzed by both tissue transglutaminase and coagulation factor XIIIa. Moreover, the peptides could be employed for specifically blocking the potential amine donor sites of protein substrates participating in biological cross-linking with these enzymes. The presence of the biotin label allowed for ready detectability of the marked donor substrates during the cross-linking of crystallins in lens homogenate by the intrinsic transglutaminase and that of the alpha chains of human fibrin by factor XIIIa.     

Visualization of purified fibronectin-transglutaminase complexes.

It has been reported previously (Turner, P.M., and Lorand, L. (1989) Biochemistry 28, 628-635) that human erythrocyte transglutaminase forms a noncovalent complex with human plasma fibronectin near its collagen-binding domain. In the present study, we show by nondenaturing electrophoresis that guinea pig liver transglutaminase, similarly to the erythrocyte enzyme, forms a complex with human fibronectin. Studies of anisotropic shifts of fluorescein-labeled liver and erythrocyte transglutaminases, upon addition of fibronectin, indicated that both transglutaminases bind to fibronectin with a stoichiometry of about 2:1. Polymerization of fibrinogen by human erythrocyte transglutaminase was inhibited after complex formation with fibronectin. Complexes of fibronectin with either erythrocyte or liver transglutaminase were isolated by glycerol gradient zone sedimentation and examined by rotary shadowing electron microscopy. The globular transglutaminase could be readily identified binding to the thin fibronectin strand. The binding site for transglutaminase was within 5-10 nm of the N terminus of fibronectin, consistent with its proximity to the collagen-binding domain. Under some experimental conditions, the complex of fibronectin with erythrocyte transglutaminase appeared as a ring-shaped structure in which two transglutaminase molecules had probably dimerized. The molecular weight of the erythrocyte transglutaminase was determined by sedimentation equilibrium to be 71,440 +/- 830.     

A trypsin-like proteinase localized in cortical granules isolated from unfertilized sea urchin eggs by zonal centrifugation. Role of the enzyme in fertilization

A trypsin-like proteinase was localized within a single subcellular compartment of unfertilized Strongylocentrotus purpuratus eggs, the cortical granules. Homogenates of eggs were fractionated by rate-zonal centrifugation. Enzymatic markers were used to determine the distribution of mitochondria (cytochrome oxidase), yolk platelets (acid nitrophenyl phosphatase), and cortical granules (β-1, 3-glucanase) in the sucrose density gradient. A bimodal distribution pattern was obtained for aryl esterase activity (substrate: β-naphthyl acetate), with one peak in the microsomal and the other in the cortical granule fractions. The cortical granule enzyme was characterized as a trypsin-like proteinase, since it also hydrolyzed another typical tryptic substrate α-N-benzoyl-l-arginine ethyl ester and was completely inactivated by soybean trypsin inhibitor (SBTI). The aryl esterase activity in the microsomal fractions was not inhibited by SBTI, while 50% of the total aryl esterase activity in the original egg homogenate was inactivated by SBTI. The identity of the enzyme(s) responsible for the aryl esterase activity associated with the microsomal particles is unknown at present.The cortical granule proteinase functions in the elevation of the fertilization membrane and establishment of the block to polyspermy at fertilization. Arbacia punctulata eggs inseminated in the presence of trypsin inhibitors, SBTI or tosyl lysine chloromethyl ketone (TLCK), failed to elevate normal fertilization membranes and became heavily polyspermic.On the basis of these results and observations made by other investigators with a wide variety of biological systems, it is proposed that trypsin-like proteinases function in the discharge of secretory granules from all types of cells.