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Biomedical subjects

H Fritz

Publications and source records attributed to H Fritz.

At least 19 recordsLinked to original sources
PubMed1995

Functional display and expression of chicken cystatin using a phagemid system.

A recombinant phage antibody system has been used to display functionally active chicken cystatin, a small protein-type inhibitor of papain-like cysteine proteinases. A synthetic gene, AEF-[S1M, M29I, M89L] chicken cystatin, was ligated into phagemid pCANTAB 5E, cloned, sequenced and displayed on the tips of filamentous phage as a fusion protein with protein III (gpIII). The gene was expressed also as soluble inhibitor in a non-suppressor E.coli strain. Recombinant phages were selected by binding to carboxymethylated-papain. The positive phages showed binding to papain in dose dependent manner. The chicken cystatin-gpIII fusion protein was detected with anti-chicken cystatin antibody and with anti-protein III antibody by western blot analysis. Unfused soluble inhibitor was identified in the periplasma and showed strong papain inhibitory activity. The results demonstrate that functionally active cystatin can be displayed on the phage surface. This technology can be employed further to select inhibitor variants which differ in their binding affinity to papain and papain-like enzymes.

Animals↗
PubMed1995

Temporary inhibition of papain by hairpin loop mutants of chicken cystatin. Distorted binding of the loops results in cleavage of the Gly(9)-Ala10 bond.

Temporary inhibition of the cysteine proteinases papain and cathepsin L was observed with several hairpin loop mutants of recombinant chicken cystatin at enzyme concentrations above nanomolar. Kinetic modelling of inhibition data, gel electrophoresis and amino acid sequencing revealed that reappearance of papain activity is due to selective cleavage of the Gly(9)-Ala10 bond in the N-terminal binding area of the chicken cystatin variants, resulting in truncated inhibitors of lower affinity. Cleavage of the same bond by contaminating papaya proteinase IV was ruled out by previous purification of papain and suitable control experiments. According to the proposed kinetic model, cleavage occurs within the enzyme-inhibitor complex with first order rate constants ktemp of 2.3 x 10(-3) up to 5 x 10(-1) s-1. A similar ktemp/Km ratio was found for all mutants (0.7 x 10(6)-2.1 x 10(6) s-1.M-1); it is almost identical with the kcat/Km ratio of the peptide substrate Z-Phe-Arg-NHMec. These results suggest that distorted contacts of one of the hairpin loops affect binding of the N-terminal contact area in a way that covalent interaction of the Gly(9)-Ala10 bond with the active-site Cys residue of papain can occur and the bond is cleaved in a substrate-like manner.

Alanine↗
PubMed1995

Hairpin loop mutations of chicken cystatin have different effects on the inhibition of cathepsin B, cathepsin L and papain.

Five recombinant hairpin loop variants of chicken cystatin (delta V55, delta V55-S56, delta P103-L105, delta I102-Q107, loop2-KD2) were constructed by cassette mutagenesis, expressed in E. coli, purified to homogeneity, characterized by protein-chemical means and by their inhibitory properties. The variant forms, modified in two of the three postulated cysteine proteinase binding regions, were inhibitorily active. However, the equilibrium dissociation constants of the complexes between papain as well as human cathepsin B or L and the cystatin variants show a weaker affinity for all three enzymes compared with recombinant chicken cystatin. These results prove the contribution of both hairpin loops to complex formation with the three enzymes. Furthermore, the kinetic constants indicate discrete differences in the molecular mechanism of interaction between chicken cystatin and papain, cathepsin B, and cathepsin L. Inhibition of cathepsin L was much less affected than inhibition of papain or cathepsin B by the modifications achieved in the five variants. Remarkably, at high enzyme concentration (above 0.5 nM) inhibition of papain by these variants was 'temporary', that means, active papain was released from the enzyme-inhibitor complex within minutes to hours (compare [1]).

Amino Acid Sequence↗
PubMed1995

Influence of recombinant human erythropoietin on hematological and hemostatic parameters with special reference to microhemolysis.

Twenty chronic hemodialysis patients with renal anemia (hematocrit < 25%) received recombinant human erythropoietin (40 IU/kg body weight 3 x weekly) intravenously after each dialysis. Prior to and at 4, 8 and 12 weeks after commencement of erythropoietin therapy, hematocrit together with hemostasis and microhemolysis parameters were determined. There were significant increases in hematocrit, platelet count and platelet retention, but a significant fall in the initial clearly prolonged bleeding time. Free plasma hemoglobin likewise increased. Conversely, lactate dehydrogenase, prothrombin time, fibrinogen, antithrombin III activity, protein C activity and protein S concentration were all unaltered. The positive effect on bleeding time and platelet retention is most probably caused by an increase in adenosine diphosphate due to the hematocrit-dependent rise in the blood shear stress via physiologic microhemolysis (raised free plasma hemoglobin).

Adult↗
PubMed1994

Dextran sulfate activates contact system and mediates arterial hypotension via B2 kinin receptors.

To define some of the mechanisms underlying dextran sulfate (DXS)-induced hypotension, we investigated the effects of either the plasma kallikrein inhibitor des-Pro2-[Arg15] aprotinin (BAY x 4620) or the specific bradykinin B2-receptor antagonist Hoe-140 on the hypotensive response to DXS. In the first study, anesthetized miniature pigs were given DXS alone, DXS plus BAY x 4620 in various doses, or saline. As expected, DXS alone produced a profound but transient systemic arterial hypotension with a concomitant reduction in kininogen. Circulating kinin levels, complement fragment des-Arg-C3a, and fibrin monomer were all increased. Treatment with BAY x 4620 produced a dose-dependent attenuation of these effects with complete blockade of the hypotension as well as the observed biochemical changes at the highest dose (360 mg). In a second study, two groups of pigs were given either DXS alone or DXS plus Hoe-140. DXS-induced hypotension was completely blocked by Hoe-140 pretreatment; however, kininogen was again depleted. We conclude, therefore, that DXS-induced hypotension is produced by activation of plasma kallikrein that results in the production of bradykinin and that liberation of bradykinin and its action on B2 receptors in the vasculature are both necessary and sufficient to produce the observed effects on circulatory pressure.

Animals↗
PubMed1993

Inhibition of plasma kallikrein with aprotinin in porcine endotoxin shock.

Activation of the contact phase of coagulation has been implicated in the pathogenesis of septic shock. We wanted to determine if inhibition of plasma kallikrein can prevent arterial hypotension and liberation of kinins from kininogen, induced by an infusion of bacterial lipopolysaccharide (LPS) in anesthetized, ventilated 20-kg pigs. The LPS was given IV in a dose of 5 micrograms/kg/h for 8 hours. The plasma kallikrein inhibitor aprotinin, 537 mumol, was given IV during 8 hours, resulting in plasma levels above 10 mumol/L. Ten animals (SA) received LPS and aprotinin and ten randomized controls (SC) received LPS and saline. Kinin-containing kininogen was determined on the basis of the amount of kinin releasable in plasma samples by incubation with trypsin. Kininogen decreased to 58% +/- 4% of the baseline value without any difference between groups. This may indicate participation of other processes than degradation by plasma kallikrein in the decrease of kininogen. Arterial blood pressure was higher at 7 hours in the SA animals than in the SC group (101% +/- 11% vs. 68% +/- 8%; mean +/- SEM; p = 0.026). Fibrin monomer and C3adesArg plasma levels were attenuated by aprotinin treatment. These findings underscore the important role of the contact system in LPS shock.

Animals↗
PubMed1992

Recombinant chicken egg white cystatin variants of the QLVSG region.

Using recombinant DNA methods, seven cystatin variants were produced by cassette mutagenesis of a chicken egg white cystatin variant which already contains the mutations Ala3, Glu2, Phe1, Ser1-->Met, Met29-->and Met 89-->Leu. When characterized by structural and functional studies, they were all found to harbour mutations in the first hairpin loop, the so-called 'QXVXG' region, which is highly conserved within the cystatin superfamily and thought to be important for its inhibitory activity towards cysteine proteinases. They were purified to more than 90% homogeneity and analysed by SDS/PAGE, HPLC, tryptic peptide mapping, N-terminal amino acid sequencing and ELISA. Structural model building of the variants and their complexes with papain was performed using computer graphics based on the crystallographic coordinates of chicken egg white cystatin and the papain-stefin complex. Only minor conformational changes were required for modelling the mutants or complexes. Equilibrium dissociation constants and rate constants of complex formation of the variants with papain, actinidin as well as cathepsin B and L were determined by kinetic measurements using fluorogenic substrates. The single exchanges Gln53-->Glu, Gln53-->Asn, Val44-->Asp, Gly57-->Ala and the double exchanges Arg52-->Leu, Gln53-->Glu, Gln53-->Asn, Ser56-->Ala, Leu54-->Met, Gly57-->Ala reduced the inhibition of papain, actinidin and cathespin B significantly by 10-1000-fold. With the exception of the Val55-->Asp variant, the differences in the Ki values are mainly due to larger k off values, whereas the kon values seem to be more or less unaffected by the selected mutations. The effect on the inhibition of papain is generally smaller than the effects on actinidin and cathepsin B inhibition. Cathepsin L inhibition is strikingly insensitive to all mutations. These distinct effects of the inhibitor variants indicate differences in proteinase-inhibitor-protein interactions between closely related cysteine proteinases. In addition, the results verify the prediction, made earlier from sequence alignment studies and from a docking model of the chicken cystatin-papain complex, that the first hairpin loop of cystatins is essential for effective inhibition.

Amino Acid Sequence↗
PubMed1992

Inflammatory mediators, infection, sepsis, and multiple organ failure after severe trauma.

The relation of (multiple) organ failure (OF) to the release of inflammatory mediators and the incidence of infection and sepsis was studied prospectively in 100 patients with multiple trauma (injury severity score = 37). Sixteen patients died of OF, 47 patients survived OF, and 37 patients had no OF. Fifteen (24%) of the patients with OF showed no signs of infection. In patients with early onset of OF (n=45), infection followed with a lag of 2 or more days. In 16 (44%) of these patients, infection led to a deterioration in organ function. With late onset of OF (n=18), infection preceded OF in nine patients. Polymorphonuclear leukocyte-elastase, neopterin, C-reactive protein, lactate, antithrombin III, and phospholipase A discriminated significantly among the three outcome groups. Of all factors, only polymorphonuclear leukocyte-elastase showed a difference between patients with and without infection or sepsis, respectively. These data indicate that infection might not play a crucial role in the pathogenesis of posttraumatic OF in a substantial portion of patients with trauma. Early OF, especially, seems to be mainly influenced by the direct sequelae of tissue damage and shock (eg, the release of inflammatory mediators). Since infection and sepsis did not lead to an augmented release of mediators in patients with trauma, the role of both entities remains unclear.

Adult↗
PubMed1992

Effect of the elastase inhibitor eglin C in porcine endotoxin shock.

We wanted to demonstrate that the neutrophil elastase inhibitor eglin C reduces the loss of intravascular protein in endotoxin (LPS) shock and that effective concentrations are present in lymph. We used 45 anesthetized miniature pigs in a randomized, controlled trial. LPS 10 micrograms/kg/hr was given for 6 hr. Recombinant eglin C was given before LPS (250 nmol/kg) and continuously at 250 nmol/kg/hr (n = 18); septic controls received saline (n = 18); nine nonseptic controls were used. Eglin C significantly reduced the loss of intravascular protein (plasma volume times plasma protein concentration) from -0.79 g/kg in septic controls to 0.42 g/kg in eglin C-treated animals and reduced plasma concentration of neutrophil elastase, bound to the leukocyte neutral protease inhibitor (LNPI), but systemic hypotension was unchanged. Six additional pigs were anesthetized, prepared with a thoracic lymph fistula, and received eglin C at 475 nmol/kg/hr; three received saline; three received LPS. Eglin C concentrations in lymph were comparable to those in plasma. We conclude that eglin C reduced the loss of intravascular protein during LPS shock and attained effective concentrations in lymph.

Animals↗
PubMed1992

[Biochemical factors as objective parameters for assessing the prognosis in polytrauma].

One hundred patients with multiple injuries (mean ISS 37 patients) were prospectively evaluated over a period of 14 days following trauma. Significant differences in the blood levels of PMN elastase, cathepsin B, lactate, neopterin, C-reactive protein (CRP) and antithrombin III (ATIII) were found in non-survivors and in survivors with and without organ failure. On admission, a prediction of organ failure was possible with an accuracy of 63% to 69% (PMN elastase, cathepsin B, ATIII). Death was predictable with an 80% to 90% accuracy within the first 4 days (PMN elastase, lactate, CRP, neopterin). The prognostic value of these factors was comparable to trauma scores regarding organ failure and better with respect to death. Biochemical parameters may be helpful in estimating the severity of the injury and prognosis and in monitoring the ICU course of such patients.

Acute-Phase Proteins↗
PubMed1992

Reproduction toxicity studies with pamidronate.

Peroral Segment I, II and III reproduction toxicity studies in rats and peroral Segment II studies in rabbits were conducted with pamidronate (disodium 3-amino-1-hydroxypropylidene-1,1-bisphosphonate pentahydrate, CGP 23339 A, CAS 57248-88-1). In addition, intravenous Segment II studies were carried out in rats and rabbits. Only about 2% of a peroral dose of pamidronate is absorbed from the gastro-intestinal tract. However, with both modes of administration, it was evident that after equivalent dosing peroral and parenteral studies yielded similar results. The most relevant consistent findings in the rat were failure of the dams to complete and/or survive a protracted parturition and a reduced number of viable pups. There was evidence that the distressed state of the dams shortly before parturition was associated with acutely reduced serum calcium concentrations. In both species, at daily doses about ten times higher than the recommended human therapeutic dose, maternal toxicity, embryolethality or severe general underdevelopment and a marked skeletal retardation of the fetuses were observed. There was no evidence that pamidronate has a teratogenic potential, nor did it affect reproductive performance. Nevertheless, in view of the effects of pamidronate on parturition and the fact that it crosses the placenta and binds to fetal bone, use of the drug in pregnancy should be avoided unless there is a life-saving medical need.

Administration, Oral↗
PubMed1991

Differential patterns of PMN-elastase and type III procollagen peptide in knee joint effusions due to acute and chronic sports injuries.

In 38 traumatic knee joint effusions the proteolytic enzyme PMN-elastase (PMN-E) and the repair marker procollagen III aminoterminal peptide (PIIINP) were determined. According to the period between trauma and first aspiration of the effusion, the patients were divided into 3 groups. Group I (17 patients; period between trauma and first aspiration not longer than 72 hours) showed high concentrations of PMN-E (up to 5400 ng/ml) and low concentrations of PIIINP (less than 13 U/ml). Group II (11 patients; aspiration within 4 to 14 days) had mean PMN-E and PIIINP concentrations of 125.6 ng/ml and 52.1 U/ml, respectively. In group III (10 patients, aspiration after 14 days) mean PMN-E concentration was 123.8 ng/ml and mean PIIINP concentration was 63.4 U/ml. Graphic depiction of PMN-E and PIIINP levels in each individual sample as a function of time between trauma and fluid collection revealed highly increasing PMN-E levels during the first 24 posttraumatic hours, followed by rapidly decreasing levels within 72 hours post trauma, and no change after the 4th posttraumatic day. In contrast, PIIINP increased continuously up to the first posttraumatic week and stayed at high levels up to 90 days (end of the observation period). The differential patterns of PMN-E and PIIINP concentration in knee joint effusions may be useful in estimating the period between trauma and first treatment (aspiration of effusion) and should, therefore, be helpful in detecting degenerative lesions, which seem to be characterized by low PMN-E concomitantly with high PIIINP levels.

Adolescent↗
PubMed1991

Purification and characterization of a chicken egg white cystatin variant expressed in an Escherichia coli pIN-III-ompA system.

A synthetic gene coding for a chicken egg white cystatin variant was cloned and expressed using the pIN-III-ompA Escherichia coli expression system. After osmotic shock of the E. coli cells, the cysteine proteinase inhibitor was isolated from periplasm and purified by S-carboxymethylpapain affinity chromatography. The resulting inhibitory material was characterized by SDS/PAGE, reversed-phase HPLC, peptide mapping and amino acid sequencing. The recombinant variant chicken AEF-[S1----M, M29----I, M89----L]cystatin shows strong inhibitory activity and displays Ki values in the complex with papain, actinidin and cathepsin B similar to those found for natural chicken cystatin. The purified variant showed a native-chicken-cystatin-like conformational state, as determined by NMR spectroscopy, if the NMR data of 15N-labelled recombinant inhibitor were compared with those of the natural inhibitor.

Amino Acid Sequence↗
PubMed1991

Studies for revealing a possible sensitization to hirudin after repeated intravenous injections in baboons.

The immunogenic potential of natural and recombinant hirudin was investigated using baboons. Animals received 4 intravenous hirudin injections of 1,000 antithrombin units/kg at certain times (day 1, 3, 8 and 42) and were checked for an immune response. None of the tests performed (in vitro histamine-release, ELISA for detection of humoral hirudin-specific IgG and IgM antibodies by indirect ELISA, direct skin test) revealed any kind of sensitization to hirudin. Urinary excretion of hirudin was not diminished by successive injections, if diuresis was within normal ranges. In summary, these results confirm previous observations indicating the weak antigenic potential of hirudin. Therefore, immune reactions to hirudin in man during therapeutic measures requiring several exposures to the inhibitor should not to be expected under normal circumstances.

Animals↗
PubMed1991

Hirudin, a new therapeutic tool?

Hirudin is the most potent natural inhibitor of thrombin known to date. It is gaining popularity as an anticoagulant now that recombinant and synthesized forms are available. It is a monospecific and co-factor-independent thrombin inhibitor with otherwise inert pharmacological properties. Being a surprisingly weak immunogen, its administration has exhibited no side effects, particularly on platelets. Bleeding complications are not to be expected at therapeutic doses. Effective anticoagulatory doses can be easily predicted and laboratory control is no problem. Application of hirudin or derivatives thereof may be indicated for: prophylaxis and treatment of postoperative venous thrombosis and diffuse microthrombosis; prevention of arterial thrombosis, especially in cardiac surgery; enhancement of fibrinolytic therapy and/or angioplasty to prevent reocclusion; extracorporeal circulation; and plastic surgery. Hirudin may be a particularly useful alternative anticoagulatory agent in patients sensitized to heparin or in patients with hereditary or acquired antithrombin III deficiency. However, whether hirudin is really an effective therapeutic tool and whether it can replace heparin in certain clinical indications can be judged only after extended clinical experience has been accumulated.

Animals↗
PubMed1991

Determination of thrombin-hirudin complex in plasma with an enzyme-linked immunosorbent assay.

An enzyme-linked immunosorbent assay (ELISA) was developed for the determination of the thrombin-hirudin complex (TH) in plasma. The test is based on the sandwich principle and uses appropriate antibodies which selectively bind the corresponding moieties of the complex. The assay was calibrated by adding performed TH to normal human citrated plasma. The detection limit of the assay was 1.2 ng/ml. Mean coefficients of variation of 6.0% (intra-assay) and 6.4% (inter-assay) were found. The presence of TH was demonstrated in normal human plasma that was spiked with hirudin and subsequently activated in vitro by calcium-thromboplastin to generate thrombin. This complex was also found in plasma samples from pigs which had been treated with hirudin during experimentally induced septicaemia.

Animals↗