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Biomedical subjects

W Machleidt

Publications and source records attributed to W Machleidt.

At least 37 records · Page 2Linked to original sources
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↗
PubMed1994

Production, inhibitory activity, folding and conformational analysis of an N-terminal and an internal deletion variant of chicken cystatin.

Two deletion variants of chicken cystatin were produced after cassette mutagenesis of the recombinant Arg-Glu-Phe-[Met1, Ile29, Leu89]-chicken egg white cystatin gene in Escherichia coli. The variant des-Ser1-Pro11-[Ala12, Glu13, Phe14, Met15, Ile29, Leu89]-chicken cystatin (N-del 2) and the variant Arg-Glu-Phe-[Met1, Ile29]-des-Cys71-Met89-chicken cystatin (del-helix II) were purified and characterized by inhibition kinetics, far-ultraviolet-CD and fluorescence spectroscopy, and their folding in guanidine hydrochloride (Gdn/HCl) was studied. The del-helix II variant, shortened by 19 amino acids, is a basic, stefin-like mini-cystatin with one disulfide bridge. Its inhibitory properties are identical to chicken cystatin and its stability against Gdn/HCl is similar. The folding of the del-helix II variant corresponds best to a single step process. In contrast to this, the reversible folding of natural and recombinant chicken cystatin is more complex when recorded by either tryptophan fluorescence or far-ultraviolet-CD. With increasing Gdn/HCl concentration, a stabilization of secondary-structural elements is initially observed, followed by unfolding with minor but distinct intermediate states. The N-del 2 variant has a neutral pI and shows folding behaviour very similar to natural and recombinant chicken cystatin. However its inhibition constants with papain, actinidin and cathepsin B and L are 1000-100,000-fold higher than those obtained with natural and recombinant chicken cystatin.

Amino Acid Sequence↗
PubMed1994

Delayed treatment with platelet activating factor receptor antagonist web 2086 attenuates pulmonary dysfunction in porcine endotoxin shock.

The triazolodiazepine WEB 2086, a specific platelet activating factor (PAF) receptor antagonist, has previously been shown to prevent pulmonary hypertension, hypoxia, and bronchoconstriction when given before bacterial lipopolysaccharide (LPS). The aim of the present study was to examine whether WEB 2086 reduced these changes even when given after the onset of LPS-induced shock. In a randomized trial LPS was given intravenously (i.v.) in a dose of 1 microgram/kg/h for 8 hours to anesthetized, ventilated pigs. Ten animals received LPS and WEB 2086, 10 mg/kg/h i.v. for 6.5 hours, beginning 1.5 hours after LPS. Ten control animals received LPS and saline. During treatment with WEB 2086, pulmonary hypertension was significantly attenuated compared with the findings in the control group. Gas exchange, airway pressure, extravascular lung water levels, intrapulmonary shunt, and cathepsin B levels in plasma showed a trend toward improvement but the group differences were not statistically significant. These data indicate that the PAF antagonist WEB 2086 can partially block pulmonary dysfunction and enzyme release from inflammatory cells when given during ongoing LPS shock in pigs, and that PAF may be an important mediator of the cardiopulmonary changes seen in septic shock.

Animals↗
PubMed1994

Mediators of leukocyte activation play a role in disseminated intravascular coagulation during orthotopic liver transplantation.

Leukocytes play an important role in the development of disseminated intravascular coagulation (DIC). In the reperfusion phase of OLT a DIC-like situation has been described and has been held responsible for the high blood loss during this phase. We investigated the role of leukocytes in the pathogenesis of DIC in OLT by measuring the leukocytic mediators released upon activation (cathepsin B, elastase, TNF, neopterin) and the levels of thrombin-antithrombin III (TAT) complexes, seen as markers of prothrombin activation. Arterial blood samples were taken at 10 different time points during and after OLT. Samples were also taken of the perfusate released from the liver graft vein during the flushing procedure before the reperfusion phase. Aprotinin was given as a continuous infusion (0.2-0.4 Mill. KIU/hr) and its plasma levels were determined. Significantly elevated levels of neopterin (15-fold; P < 0.01), cathepsin B (440-fold; P < 0.01) in the perfusate, as compared with the systemic circulation, as well as their significant increases in the early reperfusion phase suggested that they were released by the graft liver. This was paralleled by elevated levels of elastase (1.3-fold, P < 0.05), TNF (1.5-fold, P = NS), and TAT complexes (1.4-fold; P < 0.1) in the perfusate. Significant correlations could be identified between the parameters of leukocyte activation and TAT complexes, whereas no correlation was observed between any of the parameters investigated and the aprotinin levels. Our results strongly indicate a release of leukocytic mediators from the graft liver during its reperfusion which seems to be related to the parallely increased prothrombin activation. No correlation could be seen between levels of aprotinin and levels of leukocytic mediators.

Adult↗
PubMed1994

Local serum application: restoration of sufficient host defense in human peritonitis.

Intra-abdominal host defense in human peritonitis is hampered by a severe dysfunction of phagocytosis due to an almost complete breakdown of bacteria opsonization. This defect relates to some opsonin consumption, but mainly to proteolytic and oxidative opsonin destruction. To restore and protect intact opsonins we have developed a clinical approach of intra-operative peritoneal serum application. In a prospective, controlled, and randomized study of 30 patients with generalized peritonitis we have investigated the impact of this adjuvant therapy on biochemical parameters and clinical features. Serum application induced a rise in opsonin concentration and, even more pronounced, opsonin function (P < 0.01) of several hours' duration, leading to a distinct improvement of bacteria elimination. In addition, alpha 1-proteinase inhibitor (alpha 1-P)I levels were significantly increased after 1 h (P < 0.05) in the treatment group. The follow-up by APACHE II scoring indicated an improvement in the therapy group over the whole observation period of 14 days. Lethality in the therapy group was 33% compared to 53% in controls. These results indicate that the intra-operative restoration of physiologic intra-abdominal milieu can improve bacteria opsonization and elimination, thus contributing to a favourable clinical course in abdominal sepsis.

Adult↗
PubMed1994

The role of phagocyte proteinases and proteinase inhibitors in multiple organ failure.

Although numerous other inflammatory mediators are important, the following review of our research and that of other authors reveals a prominent role for the phagocyte proteinases, polymorphonuclear (PMN) elastase and cathepsin B, in the development of multiple organ failure. The release of these enzymes in relation to the severity of trauma- and/or infection-induced inflammation was clearly verified in a variety of clinical studies. The amounts of the extracellularly discharged phagocyte proteinases were highly predictive of forthcoming organ failure and ultimate patient outcome. Moreover, the consumption of important proteinase inhibitors (e.g., alpha 1-proteinase inhibitor, antithrombin III) and other plasma proteins (e.g., fibrinogen), which are highly susceptible to proteolytic degradation, coincided with the occurrence of proteolytic activity, especially that of PMN elastase. Therefore, the therapeutic use of specific PMN elastase and/or thrombin inhibitors should prevent multiple organ failure or at least reduce severe signs of inflammation.

Animals↗
PubMed1994

[Not Available].

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Animal Population Groups↗
PubMed1993

[Operative injury in spinal surgery in the management of polytrauma patients].

The release of indicators and mediators of the posttraumatic inflammatory response following spine surgery was measured in patients with multiple injuries. Eight of a group of 113 patients (mean injury severity score 36) who underwent delayed operative stabilization of vertebral fractures (> 24 h after trauma) were studied. The following significant postoperative changes of blood levels (median values, Wilcoxon signed-rank test) compared with the preoperative starting point were found: polymorphonuclear granulocyte elastase rose from 220 to 337 ng/ml, cathepsin B from 84.5 to 135.5 mU/l, C-reactive protein from 9.1 to 11.6 mg/dl, lactate from 9.6 to 15.2 mg/dl and neopterin from 6.9 to 15.2 nmol/l, while antithrombin III fell from 107.5% to 84%, platelet count from 102 to 88 x 10(9)/l and pO2/FiO2-ratio from 361 to 260. The alterations in the blood levels of these parameters following spine surgery showed a pattern similar, albeit of lesser magnitude, to that which can be observed after severe accidental trauma. We conclude that the additional activation of the inflammatory response following surgery for vertebral lesions should be taken into account when planning these operations in patients with multiple injuries.

Acute-Phase Proteins↗
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

The role of the kininogens as cysteine proteinase inhibitors in local and systemic inflammation.

The contribution of the kininogens and cystatin C to the functional inhibitory capacity for cysteine proteinases of blood plasma and inflammatory secretions was estimated from ex vivo experiments. 98.5% of the inhibitory capacity of blood plasma for cathepsin L (4-5 microM) is provided by the kininogens ensuring a complete control of this enzyme even at a lowered kininogen concentration. Control of cathepsin B activity by the kininogens is incomplete and depends critically on the active concentration of cystatin C (70 nM in normal plasma), which is reduced in blood plasma of polytraumatized and septic patients and very low in epithelial lining fluid of the shock lung.

Cathepsin B↗
PubMed1992

Basic emotions reflected in EEG-coherences.

The differentiation of basic emotions by means of EEG power spectra has been discussed extensively by Machleidt et al. (1989). The present contribution concentrates on the interhemispheric coupling of different EEG-signals as depending on the modulation of these emotions. In the first part the estimation and interpretation of squared coherence spectra is outlined. Results from this technique are presented in the following section. Intention, Aggression and Joy are mainly characterized by an increase of alpha-coherence, whereas a decrease is seen for Anxiety and Sorrow. These effects showed a widespread distribution.

Adult↗
PubMed1992

Membrane permeable fluorogenic rhodamine substrates for selective determination of cathepsin L.

The dipeptidyl rhodamine diamide substrates (Z-Phe-Arg)2-R110 and (Z-Arg-Arg)2-R110 are 820- and 360-fold more selective for cathepsin L than for cathepsin B allowing a sensitive determination of cathepsin L activity in the presence of high activity of cathepsin B. The results obtained with cell lysates suggest that the cysteine proteinase activity of vital macrophages detected by flow cytometry with these substrates is mainly due to cathepsin L.

Animals↗
PubMed1992

Flow cytometric analysis of protease activities in vital cells.

The analysis of lysosomal proteases in cell lysates is complicated by pH-dependent and oxidative changes of their activity and complex formation with cytosolic inhibitors. Therefore, new flow cytometric methods were developed for the intracellular measurement of protease activities in viable cells. Intracellular cleavage of substrates such as Z-Arg-Arg-4-trifluoromethylcoumarinyl-7-amide to green fluorescent 7-amino-4-trifluoromethylcoumarin (AFC) in viable neutrophils and monocytes was only detected following phagocytosis of Escherichia coli. A measurement of the cysteine or serine proteinase activities in resting human leukocytes was, however, not possible with AFC derivatives as the overlapping blue fluorescence of the substrates reduces sensitivity. Nonfluorescent bis-substituted peptide derivatives of rhodamine 110 (R110), which are intracellularly cleaved to green fluorescent mono-substituted R110 and free R110 proved to be more sensitive substrates. The activity of the lysosomal cysteine proteinases of human monocytes or rat macrophages, i.e. cathepsin B and L, was specifically measured with (Z-Arg-Arg)2-R110, (Z-Phe-Arg)2-R110, or (Z-Ala-Arg-Arg)2-R110. Fluorescence generation was completely inhibited by Z-Phe-Ala-diazomethane or E-64. The serine proteinases of human neutrophils were analyzed with Elastase-substrates such as (Z-Ala-Ala)2-R110 or (Z-Ala-Ala-Ala)2-R110. Specificity was shown by inhibition with diisopropylfluorophosphate.

Amino Acid Sequence↗
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↗
PubMed1991

[The dynamics of emotional states in patients with epilepsy. A phenomenologic EEG analysis study].

The history of the main ideas of psychogenetic epilepsy-theory is outlined. Using the antithesis of omnipotency and impotency further implications of this theoretical approach are explained. Epileptic patients suffer from a deficient structure of their self and do not overcome infantile obsolutistic thought, behavioral and emotional patterns. Their behavior and symptoms exhibit the characteristics of the "all or nothing schema". In particular conflict solving strategies of epileptics consist either in an exaggerated spirit of toleration or in violent outbursts of aggression. Destructive fits of rage, epileptic fits and psychotic derangements can be understood as manifestations of aggression on ontogenetically earlier developmental stages. From this point of view we studied the influence of the emotional states of epileptics on EEG-background activity and discharges: Conflict-centered interviews were recorded simultaneously in ten epileptic patients with EEG- and video-techniques. FFT-spectralanalysed EEG-data and discharges from visually evaluated EEG-records were correlated with hermeneutic analyses and the ratings of the emotional states. Results show significant correlations between spectral EEG-parameters and aggression, anxiety, sorrow and joy. These results were validated by further studies. Normal aggressive episodes were correlated with an increase of power in all frequency-bands. Epileptic discharges occurred in over or latent aggressive impulses. In the theory of the "Kontinuitätsparadigma" a parallelism of two transitional stages is suggested: ranging from normal to pathological aggression with all its symptoms and from EEG-dynamics in normal aggression to typical discharges in epileptic aggression. The latter represents the extreme pathological end of the stage while the former is its starting-point.

Adult↗