Tissue substitutes with improved angiogenic capabilities: an in vitro investigation with endothelial cells and endothelial progenitor cells.

The use of implantable biomaterials, such as artificial skin substitutes used for dermal defects, remains limited by the low angiogenic potential of these products. The rapid in vivo degradation of growth factors contributes to the limiting of angiogenesis in biomaterials. Here, we report on collagen sponges in which vascular endothelial growth factor (VEGF) was immobilized through physical binding to heparin, covalently incorporated in the matrix via cross-linking with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) and N-hydroxysuccinimide. The in vitro release of VEGF over time and endothelial cell proliferation were investigated in matrices modified at varying heparin to EDC ratios either nonloaded or loaded with VEGF. ELISA demonstrated a significantly slower in vitro release of VEGF over a period of 5 days from heparinized matrices as compared to their unmodified and cross-linked counterparts. The effects of these modifications on the proliferation of endothelial cells and endothelial progenitor cells were evaluated after 1, 3 and 5 days either according to the bromodeoxyuridine assay or total cell counting with a Neubauer chamber. The endothelial and endothelial progenitor cells cultured in contact with heparinized matrices loaded with VEGF revealed both the highest rate of DNA synthesis and the highest total cell count. Furthermore, these results show that the cross-linking of collagen matrices - both in the presence and absence of heparin - leads to increases of the proliferative activities. We can assume that these changes lead to matrices with increased angiogenic capabilities.

Biomimetic modification of the TiO(2)/glass composite Ecopore with heparinized collagen and the osteoinductive factor BMP-2.

The porous TiO(2)/glass composite Ecopore has potential applications in hard tissue replacement. We describe the modification of Ecopore with the growth factor bone morphogenetic protein-2 (BMP-2) to add osteoinductive properties. Ecopore covalently coated with BMP-2 caused a weak induction of alkaline phosphatase in murine embryonal fibroblasts. In a rabbit bone defect model, BMP-2-coated Ecopore had moderately higher bone apposition rates and ingrown bone quantities at 6 weeks after implantation. To overcome loss of function due to chemical surface coupling, we filled the pore system of Ecopore with heparinized collagen sponge and loaded this secondary matrix with BMP-2. Heparinization of collagen filling increased the BMP-2 loading capacity of the matrix approximately 1.28-fold. Within 96 h, 17.0+/-0.1 and 10.1+/-0.2% of the used BMP-2 was released from non-modified and heparinized Ecopore/collagen, respectively, indicating that the heparin modification retarded BMP-2 release. Revealed by energy-dispersive X-ray spectroscopy analysis of implant cross-sectional areas, BMP-2-loaded Ecopore/collagen had significantly higher bony ingrowth quantities in rabbits, with the heparinized modification yielding the highest value (16.09+/-3.51%, p<0.005) compared with the non-heparinized matrix (10.72+/-4.07%, p<0.05) and the BMP-2-free controls (5.60+/-1.47%). This suggested a beneficial effect of the biomimetic modification of Ecopore with heparinized collagen for bone healing and integration.

Enhancing angiogenesis in collagen matrices by covalent incorporation of VEGF.

Since the survival of ingrowing cells in biomaterials for regenerative processes largely depends on the supply of nutrients and oxygen, angiogenesis plays an important role in the development of new materials for tissue engineering. In this study we investigated the possibility of enhancing the angiogenic properties of collagen matrices by covalent incorporation of the vascular endothelial growth factor (VEGF). In a previous paper we already reported the use of homo- and heterobifunctional cross-linking agents for modifying collagen matrices [1]. In the present work the angiogenic growth factor was linked to the collagen with the homobifunctional cross-linker disuccinimidyldisuccinatepolyethyleneglycol (SS-PEG-SS) in a two step procedure. The efficiency of the first reaction step-the reaction of SS-PEG-SS with VEGF--was evaluated by western blot analysis. After 10 minutes virtually all of the dimeric molecules VEGF were on average modified by conjugation with 1 cross-linking molecule. The biological activity of the conjugate was investigated by exposing endothelial cells to non-modified VEGF and to VEGF conjugated to the cross-linker. The conjugation only had a limited effect on the mitogenic activity of VEGF. We therefore applied the cross-linking reaction to the VEGF-collagen system. In a first approach the changes were evaluated by the in vitro exposure of HUVECs to non-modified matrices, to matrices in which the VEGF was simply admixed and to matrices in which the VEGF was covalently incorporated. The angiogenic properties were evaluated in vivo with the chorioallantois membrane model. In this assay the chorioallantois membrane of the chicken embryo was exposed to the same set of matrices. The covalent incorporation of VEGF has a small but significant effect both on the formation of microvessels in the chorioallantois membrane and the tissue ingrowth into the implant. The covalent incorporation of angiogenic growth factors may thus be considered as a promising approach for enhancing the angiogenic capabilities of collagen matrices. Also the cross-linking with the homobifunctional cross-linking agent has a positive effect on the angiogenic potential of the collagen matrices.

The impact of vacuum freeze-drying on collagen sponges after gas plasma sterilization.

The sterilization of porous collagen sponges remains a challenging procedure. Gamma irradiation denatures collagen, resulting in dramatic changes to its structure. Ethylene oxide leaves toxic residues requiring weeks to evaporate. This study investigated the impact on cell behavior of gas plasma treatment when combined with vacuum freeze-drying. The goal of this procedure is to eliminate the molecules of hydrogen peroxide remaining after the sterilization process, together with their decomposition products, from the scaffolds. These molecules hinder the immediate use of the porous designs. Collagen and EDC/NHS-heparinized collagen scaffolds were sterilized with gas plasma. H2O2 released by the collagen specimens was measured by peroxidase test both immediately and also 1 week after the plasma treatment. Further measurements were done 24, 36, 48 and 72 h after vacuum freeze-drying. The activity of these scaffolds was further evaluated in relation to the proliferation, migration and differentiation of human umbilical vein endothelial cells (HUVECs). Both immediately after exposure to gas plasma and also 1 week later, the collagen designs contained significantly higher concentrations of H2O2 than scaffolds having also undergone vacuum freeze-drying. This procedure achieved faster decontamination of the remaining H2O2. Following vacuum freeze-drying, sponges already allowed HUVEC proliferation after 48 h, but in non-lyophilized specimens after gas plasma treatment alone, cell death occurred as early as only 1 week later. These data highlight the advantages of carrying out vacuum freeze-drying following gas plasma sterilization. The results show the substantial impact of sterilization of porous materials made for tissue engineering.

Enhanced dermal regeneration using modified collagen scaffolds: experimental porcine study.

Ongoing research has achieved much progress towards the development of new artificial skin substitute products. However, effective implant material for correcting full-thickness defects (such as those arising from extensive burns, tumor resection, hereditary or congenital defects and chronic wounds) has not yet become available. Following split-thickness skin grafting, contraction and scar formation are unavoidable. These phenomena are believed to be due to poor dermis regeneration. Collagen dermis substitute has been developed for the treatment of deep, poorly vascularized tissue defects. However, their application is problematic, because scaffolds of this kind fail to adequately induce the neoangiogenesis needed for regeneration. To overcome these shortcomings a number of matrices have been chemically modified. Furthermore, these matrices first require implantation and follow-up by skin grafting 3 to 5 weeks later. In this article we describe new materials made of modified collagen which enhance dermal regeneration and neoangiogenesis. The procedure was applied in successfully dealing with full-thickness defects in pigs, with subsequent split-thickness skin grafting being performed immediately afterwards. Histological findings revealed that the neodermis obtained resembles a normal dermis structure. These scaffolds have the potential of serving as an off-the-shelf skin replacement in the reconstruction of deep wounds, thus supporting wound-healing processes.

Effects of modified collagen matrices on human umbilical vein endothelial cells.

The most commonly used biomaterials fail to ensure sufficient angiogenesis for fast in vivo incorporation. This results in central necrosis and consequent infection. One way of obtaining a high angiogenic response is the application of vascular endothelial growth factor (VEGF). To obtain a sustained release of these cytokines, heparin was incorporated into collagen matrices using 1-ethyl-3(3-dimethyl-aminopropyl) carbodiimide (EDC) and N-hydroxysuccinmide (NHS). The functionality of the heparinized collagen matrices was then enhanced by immobilization of VEGF via its heparin-binding domain. This procedure changed in vitro degradation behavior and water-binding capacity. Accelerated endothelial cell proliferation was also achieved. A range of different heparin and EDC/NHS concentrations in combination with VEGF induced variation in endothelial cell growth and tubulogenic formation. Polymerized collagen scaffolds presented biointeractive systems with integrated angiogenic activity. This may become a useful tool in the clinical therapy of disorders connected with wound healing.

Modulation of angiogenic potential of collagen matrices by covalent incorporation of heparin and loading with vascular endothelial growth factor.

One of the prominent shortcomings of matrices for tissue engineering is their poor ability to support angiogenesis. We report here on experiments to enhance the angiogenic properties of collagen matrices. Our aim is to achieve this goal by covalently incorporating heparin into collagen matrices and by physically immobilizing angiogenic vascular endothelial growth factor (VEGF) to the heparin. The immobilization of heparin was performed with 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC) and N-hydroxysuccinimide (NHS). Carboxyl groups on the heparin are activated to succinimidyl esters, which react with amino functions on the collagen to zero length cross-links. This modification leads--in addition to the incorporation of heparin--to gross changes in in vitro degradation behavior and water-binding capacity. As a first approach to testing angiogenic capabilities, endothelial cells were exposed to nonmodified and heparinized collagen matrices. This exposure leads to an increase in endothelial cell proliferation. The increase can be further enhanced by loading the (heparinized) collagen matrices with VEGF. Evaluation of the angiogenic potential of heparinized matrices was further investigated by exposing them to the chorioallantoic membrane of chicken embryos and to the subcutaneous tissue of rats. Both approaches show that heparinized matrices have substantially increased angiogenic potential. In particular, the loading of heparinized matrices with VEGF invokes a further increase in angiogenic potential. It is apparent that the physical binding of VEGF to heparin allows for a release that is beneficial to angiogenesis. By varying the heparin and EDC/NHS concentrations during the modification process and by varying the loading with VEGF, the angiogenic potential as well as the degradation behavior can be adapted to obtain matrices that fulfill specific angiogenic requirements in the field of tissue engineering.

High density binding of proteins and peptides to poly(D,L-lactide) grafted with polyacrylic acid.

The use of graft polymers for the functionalisation of biomaterial surfaces is already widespread. We investigated the adsorptive and covalent binding of a variety of proteins and peptides to poly(D,L-lactide) grafted with polyacrylic acid. Covalent attachment was achieved through coupling of amino groups of the protein/peptide to the carboxyl groups of the graft polymer by using a water-soluble carbodiimide and N-hydroxysuccinimide. Binding densities were determined by automated amino acid analysis after acid hydrolysis of both the poly(D,L-lactide) and the adsorbed and covalently bound proteins. Experiments in the absence and presence of the coupling reagents allow to discriminate between adsorptive and covalent binding. Although the adsorptivc binding is quite substantial in absolute terms, the amount of adsorbed protein is relatively low as compared to the total amount of bound protein. Total binding densities of 20-30 microg/cm2 can easily be achieved. Depending on the concentration and on the properties of the proteins and peptides, between 5% and 80% of the totally bound protein may be physically adsorbed. Densities expressed in molecules/10 nm2 vary from 0.5 molecule fibronectin to 2,000 laminin-peptide molecules: their binding densities clearly correlate with their respective molecular masses. Obviously, the binding densities are governed by their individual three-dimensional space requirements rather than the density of the available carboxyl groups. From the number of carboxyl groups/10 nm2 (18,000-30,000 COOH/10 nm2) the average length of the acrylic acid graft polymer molecules was estimated. Based on the assumption that about 10 copolymer chains can be accommodated on 10 nm2, the average length of the polymer chains, which corresponds to the thickness of the graft phase, is estimated to be 0.5-1 microm. The organisation of the proteins and peptides within the polyacrylic acid phase was further investigated by experiments in which a protein (BSA) and a peptide (Val-Lys) were allowed to react in either a singular, a consecutive or a simultaneous way. Together with XPS and IR-ATR surface characterisation experiments a three-dimensional picture of the arrangement of the immobilised proteins and peptides within the graft polymer phase emerges.

The use of bifunctional polyethyleneglycol derivatives for coupling of proteins to and cross-linking of collagen matrices.

The realization of three-dimensional (3D) degradable matrices which slowly release bio-active components represents a major challenge in the field of tissue engineering. In this paper we report on the usage of commercially available bifunctional agents for both the covalent coupling of proteins to and the cross-linking of collagen matrices. Proteins - horse radish peroxidase (HRP) was used as a model protein - were cross-linked with either a homobifunctional (disuccinimidyldisuccinatepolyethylene-glycol) or a heterobifunctional (N-hydroxysuccinimidylvinylsulfonepolyethyleneglycol) agent. In the case of the heterobifunctional cross-linking agent the collagen matrices were previously modified with succinimidylacetylthioacetate in order to introduce sulfhydryl groups. As compared with control experiments a 10-fold and 50-fold increase of immobilized proteins were achieved with the homobifunctional and heterobifunctional cross-linker resp. The HRP-PEG conjugates demonstrated a better long-term stability as compared to the non-treated HRP. The effects of the cross-linking agents and the thiolation reagent succinimidylacetylthio acetate on the in vitro degradation of the collagen matrices by collagenase were also investigated. In particular the reaction with succinimidylacetylthio acetate appears to offer interesting opportunities both for coupling active proteins and modulating the degradation times of collagen matrices.

Mutations in the Ca2+ binding site of the Paracoccus denitrificans cytochrome c oxidase.

Recent structure determinations suggested a new binding site for a non-redox active metal ion in subunit I of cytochrome c oxidase both of mitochondrial and of bacterial origin. We analyzed the relevant metal composition of the bovine and the Paracoccus denitrificans enzyme and of bacterial site-directed mutants in several residues presumably liganding this ion. Unlike the mitochondrial enzyme where a low, substoichiometric content of Ca2+ was found, the bacterial wild-type (WT) oxidase showed a stoichiometry of one Ca per enzyme monomer. Mutants in Asp-477 (in immediate vicinity of this site) were clearly diminished in their Ca content and the isolated mutant enzyme revealed a spectral shift in the heme a visible absorption upon Ca addition, which was reversed by Na ions. This spectral behavior, largely comparable to that of the mitochondrial enzyme, was not observed for the bacterial WT oxidase. Further structure refinement revealed a tightly bound water molecule as an additional Ca2+ ligand.

A novel chimaeric derivative of saruplase, rscu-PA-40 kDA/Hir, binds to thrombin and exerts thrombus-specific fibrinolysis in arterial and venous thrombosis in dogs.

The chimaeric molecule rscu-PA-40 kDA/Hir (M23) comprises the kringle and protease domain of saruplase (rscu-PA) and a thrombin inhibitory domain fused to the C-terminus of the protease domain. The 27 amino cid long thrombin inhibitory domain contains a sequence directed to the active site of thrombin and a fragment from the C-terminal region of hirudin. 125I-radiolabelled M23 (0.03 microM) bound to thrombin that was immobilised onto CNBr-activated sepharose beads. Unlabelled M23 (0.01-10 microM) and hirudin (0.001-10 microM) concentration-dependently displaced 125I-M23 from its binding to thrombin. Saruplase (up to 10 microM) did not influence the thrombin binding of M23. The fibrinolytic properties of M23 and saruplase were compared in anaesthetized dogs with femoral artery and saphenous vein thrombosis. Under concomitant heparinization, the intravenous bolus injections of 1 mg/kg M23 or saruplase induced reperfusion of thrombotically occluded femoral arteries in 4 out of 5 treated animals in each case. There was one reocclusion in the M23-treated group. Time to reperfusion (23 +/- 4 vs 25 +/- 11 min) and maximal height of reperfusion blood flow (98 +/- 21 vs 108 +/- 15% of baseline flow) did not differ significantly between the treatment groups. The time course of the lysis of incorporated 125I-fibrin radioactivity in thrombosed saphenous-veins was similar after bolus injections of M23 and saruplase. The maximal dissolution of 125I-fibrin in the venous thrombosis model was 91 +/- 1% in M23- and 88 +/- 5% in saruplase-treated animals. Plasma levels of fibrinogen were not influenced and alpha 2-antiplasmin levels were slightly reduced (-27 +/- 3%) after bolus injection of M23. In contrast, bolus injection of saruplase was accompanied by a significant decrease of fibrinogen (-55 +/- 19%) and alpha 2-antiplasmin (-75 +/- 11%) plasma levels. Template bleeding times virtually did not differ before (2.8 +/- 0.3 min) and 60 min after bolus injection of M23 (3.1 +/- 0.3 min), whereas treatment with saruplase resulted in a significant prolongation of template bleeding time from 2.6 +/- 0.2 min to 28 +/- 13 min. It is concluded that the saruplase derivative M23, while inducing equieffective thrombolysis after intravenous bolus injection in dogs, causes much fewer haemostatic side effects than its parent molecule. The high thrombus-specific activity of M23 is tentatively attributed to its affinity to clot-bound thrombin.

A strong thrombin-inhibitory prourokinase derivative with sequence elements from hirudin and the human thrombin receptor.

In order to design plasminogen activators with improved thrombolytic properties, bifunctional proteins with both plasminogen-activating and anticoagulative activity were constructed by fusing a thrombin-inhibitory moiety itself comprises four elements: linker 1, a motif directed to thrombin's active site, linker 2 and a fragment of hirudin which binds to the fibrinogen-recognition site of thrombin. In order to improve further the anticoagulative activity, the thrombin-inhibitory domain was modified by substituting linker 2. Introduction of a linker (FLLRNP) from the human thrombin receptor conferred about a 10-fold increase in anticoagulative activity in protein M37 compared with the parent molecule M23 carrying an aliphatic linker. The increase in anticoagulative activity was also reflected in the shift of the Ki value from 159 +/- 20 nM for M23 to 2.0 +/- 0.5 nM for M37. The increased thrombin-inhibitory activity of M37 may be due to the presence of an arginine in the linker from the thrombin receptor which may interact with one of two glutamic acid residues located at the exit of the thrombin substrate binding pocket. This explanation is supported by the observation that another chimera (M35) carrying a linker sequence with two acidic residues has relatively weak thrombin-inhibitory activity. The thrombin-inhibitory activity of M37 may be strong enough to substitute anticoagulative co-medication during fibrinolytic treatment.

Antibacterial activity of antileukoprotease.

Antileukoprotease (ALP), or secretory leukocyte proteinase inhibitor, is an endogenous inhibitor of serine proteinases that is present in various external secretions. ALP, one of the major inhibitors of serine proteinases present in the human lung, is a potent reversible inhibitor of elastase and, to a lesser extent, of cathepsin G. In equine neutrophils, an antimicrobial polypeptide that has some of the characteristics of ALP has been identified (M. A. Couto, S. S. L. Harwig, J. S. Cullor, J. P. Hughes, and R. I. Lehrer, Infect. Immun. 60:5042-5047, 1992). This report, together with the cationic nature of ALP, led us to investigate the antimicrobial activity of ALP. ALP was shown to display marked in vitro antibacterial activity against Escherichia coli and Staphylococcus aureus. On a molar basis, the activity of ALP was lower than that of two other cationic antimicrobial polypeptides, lysozyme and defensin. ALP comprises two homologous domains: its proteinase-inhibitory activities are known to be located in the second COOH-terminal domain, and the function of its first NH2-terminal domain is largely unknown. Incubation of intact ALP or its isolated first domain with E. coli or S. aureus resulted in killing of these bacteria, whereas its second domain displayed very little antibacterial activity. Together these data suggest a putative antimicrobial role for the first domain of ALP and indicate that its antimicrobial activity may equip ALP to contribute to host defense against infection.

Thrombin inhibitory and clot-specific fibrinolytic activities of the urokinase variant, M23 (rscu-PA-40 kDa/Hir).

The recombinant bifunctional urokinase variant, M23 (rscu-PA-40 kDA/Hir), comprising the kringle and protease domain of single-chain urokinase-type plasminogen activator and a C-terminal fragment of hirudin in one single-chain molecule, was evaluated for its thrombin-inhibitory and fibrinolytic properties in vitro and in vivo. M23 inhibited thrombin-activated coagulation of human blood and thrombin-induced aggregation of human platelet rich plasma in a concentration-dependent manner. The ADP-induced aggregation of human platelet rich plasma was not influenced by M23. In contrast, recombinant single-chain urokinase-type plasminogen activator (saruplase) inhibited neither blood coagulation nor platelet rich plasma aggregation. M23 and saruplase both lysed radiolabelled human thrombi immersed in human plasma (Chandler Loop system) with equal potency. However, there was a significantly lower systemic generation of plasmin (measured as consumption of alpha 2-antiplasmin) by M23 compared to saruplase. In anaesthetized non-heparinized rabbits, experimental femoral artery thrombosis was treated with intravenous bolus injections of M23 or saruplase (6 mg/kg, each). Thrombolytic restoration of arterial blood perfusion was significantly higher in M23- than in saruplase-treated rabbits. Plasma fibrinogen concentrations were decreased markedly in saruplase-treated animals, but remained at significantly higher levels in M23-treated rabbits. In conclusion, the bifunctional molecule, M23, showed thrombin inhibitory and fibrinolytic properties in human in vitro systems and exerted superior thrombolytic effects to saruplase in rabbit femoral artery thrombosis. In vitro and in vivo data indicate that the fibrinolytic activity of M23 is highly clot-specific.

Construction and structure-activity relationships of chimeric prourokinase derivatives with intrinsic thrombin-inhibitory potential.

The blood clotting enzyme thrombin plays a central role in the aetiology of occlusive disorders such as stroke and acute myocardial infarction. During fibrinolytic therapy with plasminogen activators, thrombin is neutralized by anticoagulative drugs. In order to combine plasminogen-activating and thrombin-inhibitory activities we constructed chimeric derivatives of recombinant single-chain, urokinase-type plasminogen activator (rscu-PA) which comprise the kringle and protease domain of rscu-PA fused via a linker sequence to a thrombin-inhibitory domain. The inhibitory domain contains a sequence element directed to the active site of thrombin and a sequence taken from either hirudin or the human thrombin receptor both binding to the fibrinogen recognition site of thrombin. Analysing different sets of point mutants showed that the linker between the protease domain and the active site-directed sequence is contributing significantly to the thrombin-inhibitory potential. Kinetic analysis of thrombin inhibition revealed that most of the chimeras tested competitively inhibit the thrombin-mediated cleavage of a peptide substrate in a concentration-dependent manner; however, in two examples the insertion of one glycine residue into the active site directed-sequence abolished the blockade of the active site. This supports the conclusion that the chimeras with high thrombin-inhibitory potential interact with the active site and the fibrinogen recognition site of thrombin.

Perturbation of the CuA site in cytochrome-c oxidase of Paracoccus denitrificans by replacement of Met227 with isoleucine.

Subunit II of cytochrome-c oxidase contains a redox centre, CuA, with unusual spectroscopic properties; this site consists of two copper atoms and acts as the entry point for electrons from cytochrome c. We have constructed a site-directed mutant of cytochrome-c oxidase from Paracoccus denitrificans in which the CuA site has been disturbed by replacement of Met227 with isoleucine. The purified, fully assembled enzyme complex has been investigated with various techniques including metal analysis, EPR and visible spectroscopies, steady-state and fast kinetics. The stoichiometry of the metals in the enzyme remains unchanged but a clear perturbation of the CuA site can be observed in the EPR and near-infrared optical spectra. It is concluded that in the mutant CuA is still binuclear but that the two nuclei are no longer equivalent, converting the delocalized [Cu(1.5)....Cu(1.5)] centre of the wild type into a localized [Cu(I)....Cu(II)] system. Changes in the overall kinetics of the mutant are correlated with a diminished electron transfer rate between CuA and heme alpha.

Functional properties of a recombinant chimeric protein with combined thrombin inhibitory and plasminogen-activating potential.

A chimeric protein (rscu-PA-40-kDa/Hir), consisting of the C-terminal amino acids 53-65 of hirudin (Hir), fused via a 14-amino-acid linker sequence to the C-terminal of a 40-kDa fragment (Ser47-Leu411) of recombinant (r) single-chain (sc) urokinase-type plasminogen activator (rscu-PA), was produced by expression of the corresponding chimeric cDNA in Escherichia coli cells. The thrombin inhibitory potential of purified rscu-PA-40-kDa/Hir was confirmed by complete inhibition of the coagulant activity of thrombin at 20-30-fold molar excess of the chimera, and by the resistance of rscu-PA-40-kDa/Hir to proteolytic cleavage by thrombin, rscu-PA-40-kDa/Hir prolonged the thrombin time of normal human plasma in a dose-dependent way (reduction of the apparent thrombin concentration to 50% with 95 nM chimeric protein as compared to 4.7 nM hirudin), and inhibited thrombin-mediated platelet aggregation (reduction of the apparent thrombin concentration to 50% with 40 nM chimeric protein). The chimera had a specific activity on fibrin films of 57,000 IU/mg as compared to 95,000 IU/mg for rscu-PA. The urokinase-like amidolytic activity of the single-chain protein was only 220 IU/mg but increased to 169,000 IU/mg after treatment with plasmin, which resulted in quantitative conversion to a two-chain (tc) derivative (rtcu-PA-40-kDa/Hir). Corresponding values for rscu-PA were 270 and 226,000 IU/mg. The catalytic efficiencies for plasmin-mediated conversion to two-chain molecules were comparable for rscu-PA-40-kDa/Hir and rscu-PA (0.63 and 0.65 microM-1.s-1, respectively). The plasminogen-activating potential of the single-chain chimera was comparable to that of rscu-PA; the catalytic efficiencies for plasminogen activation by their two-chain counterparts were also similar (0.55 and 0.73 microM-1.s-1, respectively). In 2 h, 50% lysis of 125I-fibrin-labeled clots prepared from platelet-poor human plasma and immersed in normal plasma was obtained with 1.3 micrograms/ml rscu-PA-40-kDa/Hir and with 0.67 micrograms/ml rscu-PA, with corresponding residual fibrinogen levels of 74% and 87%, respectively. In the absence of fibrin, 50% fibrinogenolysis in 2 h in normal human plasma required 2.1 micrograms/ml rscu-PA, but 7.9 micrograms/ml rscu-PA-40-kDa/Hir. Thus, the chimera rscu-PA-40-kDa/Hir has maintained the specific fibrinolytic and plasminogen activating activity of rscu-PA as well as its fibrinolytic potency in plasma, whereas it displayed a similar or somewhat better fibrin specificity.(ABSTRACT TRUNCATED AT 250 WORDS)

Segmental differentiation processes in embryonic muscle development of the grasshopper.

To analyse segmental differentiation processes in muscle development, we studied the embryogenesis of the ventral body wall muscles in thoracic and abdominal segments of the grasshopper Schistocerca gregaria at the identified cell level. We visualized differentiating muscle pioneer and muscle precursor cells by staining with a muscle-specific monoclonal antibody and with rhodamine-coupled phalloidin. Our results show that a similar pattern of serially reiterated early muscle pioneers is initially established in all segments. Subsequently, two major segmental differentiation processes occur. First, segment-specific sets of additional, later differentiating muscle pioneers are generated de novo. Second, segment-specific sets of existing early muscle precursors are eliminated through atrophy and eventual loss. These events have consequences for matching homonomy of muscles and their innervating motoneurons. Taken together, these processes in the embryo, in concert with postembryonic differentiation events, play critical roles in shaping the highly specialized muscular structures of the mature animal.

Stoichiometry and redox behaviour of metals in cytochrome-c oxidase.

The early observation of extra copper in preparations of cytochrome-c oxidase has recently lead to a renewed interest in its stoichiometry and possible redox function. In various, pure preparations (heme A contents close to the theoretical value of 9.79 nmol/mg protein for the 13-subunit bovine enzyme) protein-related metal stoichiometries of 3 Cu, 2 Fe, 1 Zn, 1 Mg/monomer with M(r) 204266 were determined. Despite the presence of five potential redox metal ions, reductive and reoxidative titrations indicate the presence of only four one-electron-accepting/donating species in the ligand-free enzyme. Participation of two copper ions in a binuclear copper site acting as one-electron acceptor may explain both the observed copper stoichiometry and the redox behaviour. The homology of the C-terminal sequence of subunit II with one of the copper-binding sites in nitrous-oxide reductases provides possible ligands for complexing two copper ions in a binuclear center.