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W Machleidt

Publications and source records attributed to W Machleidt.

At least 19 recordsLinked to original sources
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↗
PubMed1991

A triazolodiazepine platelet activating factor receptor antagonist (WEB 2086) reduces pulmonary dysfunction during endotoxin shock in swine.

We wanted to determine the effects of WEB 2086, a platelet activating factor (PAF) antagonist, in lipopolysaccharide (LPS) shock in anesthetized pigs. In a randomized study, LPS from S. abortus equi, 2 micrograms/kg/h was given IV for six hours. Thirteen animals received LPS and WEB 2086, 10 mg/kg/h IV for 6.5 hours, beginning 30 minutes before LPS. Eleven septic controls received saline and LPS, three nonseptic controls received saline and WEB 2086, and three nonseptic controls received saline only. In six animals we investigated the effect of synthetic PAF in doses between 50 and 10,000 ng on arterial (AP) and pulmonary arterial (PAP) pressure before and during infusion of WEB 2086. The LPS-induced rise in PAP was reduced by WEB 2086 (p = 0.01) but not the decrease in AP. The LPS-induced leukopenia, hypoxia, increase in airway pressure, and release of plasminogen activator inhibitor were reduced by WEB 2086. Platelet activating factor produced an increase in PAP and a biphasic response in AP. All PAF dose response curves were shifted to the right by WEB 2086. Platelet activating factor was a pulmonary hypertensive agent and contributed to the LPS-induced respiratory alterations.

Animals↗
PubMed1991

Possible role of extracellularly released phagocyte proteinases in coagulation disorder during liver transplantation.

Orthotopic liver transplantation is frequently associated with a complex coagulation disorder, influencing the outcome of the procedure. In this respect, disseminated intravascular coagulation (DIC) had been suggested to be of causative importance for bleeding complications after reperfusion of the liver graft. In 10 consecutive patients undergoing orthotopic liver transplantations, we studied the occurrence of two phagocyte proteinases of different origin in the graft liver perfusate and in systemic blood during the operation, as well as their effects on hemostasis. As compared with plasma samples taken at the end of the anhepatic phase, highly significant increases of cathepsin B and thrombin-anti-thrombin III complexes (TAT), as well as highly significant decreases in antithrombin III, protein C, and C1-inhibitor were observed in graft liver perfusate. Von Willebrand factor and fibrinogen were slightly decreased, whereas the elastase-alpha 1 proteinase inhibitor complexes (EPI) were elevated. In plasma the activity of cathepsin B remained unchanged during the prereperfusion phases, but immediately after revascularization of the graft this cysteine proteinase increased. The EPI showed a gradual increase in plasma during the preanhepatic and anhepatic phases but a more pronounced increase in the reperfusion phase. In parallel with the rise in these two proteinases TAT increased and the activities of antithrombin III and C1-inhibitor in plasma decreased after reperfusion. At 12 hr after revascularization plasma levels of TAT, antithrombin III, and C1-inhibitor had returned to the prereperfusion ranges, whereas cathepsin B and EPI were significantly above the baseline levels. These observations are consistent with the hypothesis that extracellularly released lysosomal proteinases may play a role in the development of a DIC-like constellation, including thrombin formation after revascularization of the liver graft. For the first time we could prove the occurrence of phagocyte proteinases in graft liver perfusate and evaluate the importance of these proteinases for the understanding of the pathophysiology leading to bleeding complications in patients undergoing orthotopic liver transplantation.

Antithrombin III↗
PubMed1991

Proteolysis of defensive proteins in peritonitis exudate: pathobiochemical aspects and therapeutical approach.

Peritonitis exudate reveals strong proteolytic activity which is paralleled by deficient opsonic capacity and high concentrations of lysosomal proteinases (elastase and cathepsin B). Lysosomal serine and cysteine proteinases (elastase, cathepsins B, L) were shown to degrade immunoglobulin G(IgG) and seem to be at least partially responsible for the observed proteolytic inactivation of IgG in peritonitis exudates. Intraabdominal serum application seems to restore opsonic activity by substitution of opsonins and proteinase inhibitors.

Ascitic Fluid↗
PubMed1991

Molecular mechanism of inhibition of cysteine proteinases by their protein inhibitors: kinetic studies with natural and recombinant variants of cystatins and stefins.

Natural and recombinant variants of the cysteine proteinase inhibitors chicken cystatin and human stefin B were characterized by determination of their inhibition constants for papain, actinidin and human cathepsins B and H. The individual contributions of the three contact regions to the binding energy of the chicken cystatin-papain complex were calculated as 36% for the N-terminal segment, 51% for the first and 13% for the second hairpin loop. Removal of the N-terminal contact region of chicken cystatin resulted in a 10000-fold lower affinity for papain. In contrast, stefin B remained a tight-binding inhibitor of papain and actinidin without its N-terminal segment. Affinity of stefin B for papain was only slightly affected by exchange of the residue predicted to bind in the S2 subsite of papain. The essential contribution of the first hairpin loop to inhibitor binding was confirmed by the 240-fold lower affinity for papain of a Val48----Asp mutant of stefin B. Inhibition of cathepsin B by stefins A and B is slow-binding. Binding of stefin B, not of stefin A, follows a two-step mechanism involving a slow isomerisation of the enzyme-inhibitor complex.

Amino Acid Sequence↗
PubMed1990

The complete amino-acid sequences of human ganglioside GM2 activator protein and cerebroside sulfate activator protein.

The complete amino-acid sequences of human ganglioside GM2 activator protein and cerebroside sulfate activator protein have been established by Edman degradation. The GM2 activator is composed of 162 amino acids, the first two serine residues being present in only 20% of the material. A single carbohydrate chain is N-glycosidically linked to Asn32. Three hydrophobic alpha-helices may contribute to its lipid-binding site. Three amino acids differ from those found by cDNA sequencing which may be due to a polymorphism. The cerebroside sulfate activator consists of 80 amino acids and carries one N-linked carbohydrate chain at Asn21. The C-terminal valine residue is lacking in about 80% of the material. In spite their similar functions, both activator proteins show no sequence or structural similarities.

Amino Acid Sequence↗
PubMed1990

[Coordination and planning in community psychiatry. 14-year-experience in Hannover between the recommendations of the Psychiatric Inquiry and the Expert Commission (1976-1989)].

Against the background of the model pattern conceived in West Germany in 1975 on the basis of the so-called "Psychiatrie-Enquête" and in 1988 on recommendations made by a committee of experts, the article describes and discusses the history, composition, manner of working, thematic scope and the results of the coordination round table concerning psychiatric care under community council auspices. This model has been realised in Hanover since 1976 as part of the segmentation of psychiatric care according to community districts. The study aims at stimulating suggestions for a general solution regarding coordination- and planning of communal psychiatric care and how to translate these suggestions into reality, both on a scientific basis and with due reference to the realities of the prevailing circumstances. This can help to avoid developments in a wrong direction and also to avoid delays that may arise in solving this core problem of sociopsychiatric reform.

Community Mental Health Services↗
PubMed1989

Mechanism of inhibition of papain by chicken egg white cystatin. Inhibition constants of N-terminally truncated forms and cyanogen bromide fragments of the inhibitor.

N-terminally truncated forms of chicken egg white cystatin and its cyanogen bromide fragments were isolated and assayed for inhibition of papain. Truncated forms beginning with Gly-9 and Ala-10 had a 5000-fold lower affinity for papain than the two isoelectric forms (pI = 6.5 and 5.6) of the full-length inhibitor (Ki = 6 pM and 7 pM) or a truncated form beginning with Leu-7 (Ki = 6 pM), indicating the outstanding importance of one or two residues preceding conserved Gly-9 for binding. A weak inhibition of papain (Ki = 900 nM) was exhibited by the intermediate cyanogen bromide fragment (residues 30-89) containing the chicken cystatin QLVSG variation of the QVVAG segment which is conserved in almost all members of the cystatin superfamily. The obtained affinity data provide independent evidence for the validity of the proposed docking model of a chicken cystatin-papain complex [(1988) EMBO J. 7, 2593-2599].

Amino Acid Sequence↗
PubMed1988

Proteinase-sensitive regions in the heavy chain of low molecular weight kininogen map to the inter-domain junctions.

Low molecular weight kininogen from human plasma was subjected to limited proteolysis with trypsin, chymotrypsin, elastase, and bromelain, and the resulting fragments of 20,000 or 40,000 Da were isolated. Amino-terminal sequence analysis of the fragments disclosed for the various proteinases eight independent cleavage sites distinct from the typical kallikrein cleavage sites flanking the kinin region. All the identified cleavage sites cluster in two stretches of 11-12 residues of the kininogen heavy chain. These short segments represent the primary attack sites for proteinases ("proteinase-sensitive regions") in the heavy chain portion of human low molecular weight kininogen. The amino acid sequences of the two proteinase-sensitive regions are mutually homologous; they are further characterized by the presence of a single copy each of the consensus tetrapeptide Cys-X-Gly-Cys known to form a narrow disulfide loop (Kellermann, J., Thelen, C., Lottspeich, F., Henschen, A., Vogel, R., and Müller-Esterl, W. (1987) Biochem. J. 247, 15-21). The proteinase-sensitive regions are located at the junctions of the three cystatin-like domains constituting the kininogen heavy chain. Proteolytic cleavage at the sensitive regions dissects the kininogen heavy chain and releases single domains of 20,000 Da and combined domains of 40,000 Da which can function as cysteine proteinase inhibitors. The presence of kininogen heavy chain domains in plasma samples under pathologic conditions suggests that cleavage of the proteinase-sensitive regions might also occur in vivo.

Amino Acid Sequence↗