Effects of piretanide on plasma fibrinolytic activity,platelet aggregation and platelet factor-4 release in man

Piretanide, 4-phenoxy-3-(pyrrolidinyl)-5-sulphamoyl benzoic acid, apart from being an efficient diuretic, enhances endogenous plasma fibrinolytic activity after a single dose of 6 mg administered by oral route. After ingestion of the drug, acceleration of fibrinolytic acitivity became manifest within 1 h, reached its peak in 3 h and was associated with a fall in fibrinogen and diminished urokinase excretion. Piretanide did not cause lysis of fibrinin vitro. Primary platelet aggregation, induced by adenosine-diphosphate, was inhibited by piretanide. Inin vitro experiments piretanide led to effective inhibition of adenosine-diphosphate-induced platelet aggregation with complete inhibition at 5 mM concentration. Piretanide led to a highly significant decrease of platelet factor-4 release.     

The influence of canavanine and related compounds on the water balance system of locusts

In vivo increase in haemolymph volume of canavanine-treated locusts substantiates our previous in vitro findings that canavanine inhibits fluid secretion by locust Malpighian tubules. Furthermore when diuretic hormone is applied in vivo after canavanine treatment haemolymph volume is drastically reduced below levels retained in locusts untreated with canavanine. Again this is in accord with canavanine potentiation of semi-isolated Malpighian tubules and enhanced fluid secretion in vitro. The response is specific to canavanine; compounds similar in structure (arginine, argininic acid, citrulline, canaline, ornithine and homoserine) have no effect on the rate of fluid secreted by Malpighian tubules. Only partial competition is obtained with uridine homoserine.     

Computational approaches from polymer physics to investigate chromatin folding

Microscopy and sequencing-based technologies are providing increasing insights into chromatin architecture. Nevertheless, a full comprehension of chromosome folding and its link with vital cell functions is far from accomplished at the molecular level. Recent theoretical and computational approaches are providing important support to experiments to dissect the three-dimensional structure of chromosomes and its organizational mechanisms. Here, we review, in particular, the String&Binders polymer model of chromatin that describes the textbook scenario where contacts between distal DNA sites are established by cognate binders. It has been shown to recapitulate key features of chromosome folding and to be able at predicting how phenotypes causing structural variants rewire the interactions between genes and regulators.     

Vegetation,herbivores and fires in savanna ecosystems: A network perspective

The dynamics of savanna ecosystems depends on the interplay between multiple factors such as grazing, browsing, fires, rainfall regime and interactions between grass and woody vegetation. In most modelling applications this interplay may not be fully understood because some of these drivers enter the models as dynamically independent factors. In this paper we consider such factors as dynamic variables. To analyze their interplay we focus on the structure of the interactive network of variables and exploit the properties of signed digraphs using the algorithm of Loop Analysis. Qualitative signed digraphs for the savanna ecosystem are developed and their predictions used to interpret patterns of abundance observed in case studies selected from the literature. The outcomes of this exercise unveil that: 1) the structure of the interactions is appropriate locus for the explanation of patterns observed in savannas; 2) signed digraph can help disentangling causative mechanisms by linking correlation patterns, source of change and network structure. This study highlights that central to the understanding of savanna dynamics is our ability to diagram the important relationships and understand how they interrelate with sources of variations to cause ecosystem change.     

Stiffness of the distal loop restricts the structural heterogeneity of the transition state ensemble in SH3 domains

Protein engineering experiments and Phi(F)-value analysis of SH3 domains reveal that their transition state ensemble (TSE) is conformationally restricted, i.e. the fluctuations in the transition state (TS) structures are small. In the TS of src SH3 and alpha-spectrin SH3 the distal loop and the associated hairpin are fully structured, while the rest of the protein is relatively disordered. If native structure predominantly determines the folding mechanism, the findings for SH3 folds raise the question: What are the features of the native topology that determine the nature of the TSE? We propose that the presence of stiff loops in the native state that connect local structural elements (such as the distal hairpin in SH3 domains) conformationally restricts TSE. We validate this hypothesis using the simulations of a "control" system (16 residue beta-hairpin forming C-terminal fragment of the GBl protein) and its variants. In these fragments the role of bending rigidity in determining the nature of the TSE can be directly examined without complications arising from interactions with the rest of the protein. The TSE structures in the beta-hairpins are determined computationally using cluster analysis and limited Phi(F)-value analysis. Both techniques prove that the conformational heterogeneity decreases as the bending rigidity of the loop increases. To extend this finding to SH3 domains a measure of bending rigidity based on loop curvature, which utilizes native structures in the Protein Data Bank (PDB), is introduced. Using this measure we show that, with few exceptions, the ordering of stiffness of the distal, n-src, and RT loops in the 29 PDB structures of SH3 domains is conserved. Combining the simulation results for beta-hairpins and the analysis of PDB structures for SH3 domains, we propose that the stiff distal loop restricts the conformational fluctuations in the TSE. We also predict that constraining the distal loop to be preformed in the denatured ensemble should not alter the nature of TSE. On the other hand, if the amino and carboxy terminals are cross-linked to form a circular polypeptide chain, the pathways and TSs are altered. These contrasting scenarios are illustrated using simulations of cross-linked WT beta-hairpin fragments. Computations of bending rigidities for immunoglobulin-like domain proteins reveal no clear separation in the stiffness of their loops. In the beta-sandwich proteins, which have large fractions of non-local native contacts, the nature of the TSE cannot be apparently determined using purely local structural characteristics. Nevertheless, the measure of loop stiffness still provides qualitative predictions of the ordered regions in the TSE of Ig27 and TenFn3.     

Design and functional analysis of actomyosin motor domain chimera proteins

To gain more structural and functional information on the actomyosin complexes, we have engineered chimera proteins carrying the entire Dictyostelium actin in the loop 2 sequence of the motor domain of Dictyostelium myosin II. Although the chimera proteins were unable to polymerize by themselves, addition of skeletal actin promoted polymerization. Electron microscopic observation demonstrated that the chimera proteins were incorporated into actin filaments, when copolymerized with skeletal actin. Copolymerization with skeletal actin greatly enhanced the MgATPase, while the chimera proteins without added skeletal actin hydrolyzed ATP at a very low rate. These results indicate that the actin part and the motor domain part of the chimera proteins are correctly folded, but the chimera proteins are structurally stressed so that efficient polymerization is inhibited.     

Enzymatic function of loop movement in enolase: preparation and some properties of H159N,H159A,H159F,and N207A enolases

The hypothesis that His159 in yeast enolase moves on a polypeptide loop to protonate the phosphoryl of 2-phosphoglycerate to initiate its conversion to phosphoenolpyruvate was tested by preparing H159N, H159A, and H159F enolases. These have 0.07%–0.25% of the native activity under standard assay conditions and the pH dependence of maximum velocities of H159A and H159N mutants is markedly altered. Activation by Mg²⁺ is biphasic, with the smaller Mg²⁺ activation constant closer to that of the catalytic Mg²⁺ binding site of native enolase and the larger in the mM range in which native enolase is inhibited. A third Mg²⁺ may bind to the phosphoryl, functionally replacing proton donation by His159. N207A enolase lacks an intersubunit interaction that stabilizes the closed loop(s) conformation when 2-phosphoglycerate binds. It has 21% of the native activity, also exhibits biphasic Mg²⁺ activation, and its reaction with the aldehyde analogue of the substrate is more strongly inhibited than is its normal enzymatic reaction. Polypeptide loop(s) closure may keep a proton from His159 interacting with the substrate phosphoryl oxygen long enough to stabilize a carbanion intermediate.     

Citric acid production and morphology of Aspergillus niger as functions of the mixing intensity in a stirred tank and a tubular loop bioreactor

The relationship between Aspergillus niger morphology and citric acid production was investigated in two reactor systems with different configurations, a tubular loop and a stirred tank bioreactor, with operating volumes of 6 and 8 dm³, respectively. Morphology was quantified by image analysis. In each system, morphology, characterized by the parameter P (mean convex perimeter of clumps), and citric acid production, were agitation-dependent and closely linked. Increased agitation caused a reduction of clump sizes and results when both reactors demonstrate that the parameter P should not exceed a threshold value in order to achieve increased productivities. The results obtained from the two reactors were in agreement, both qualitatively and quantitatively. Reducing the fundamentally different mixing conditions of the two bioreactors to the order of the dimensionless mixing parameter relative mixing time (τ_m), results showed that the loop simulated the stirred tank. Also, relationships valid for one system accurately described the results obtained from the other system, demonstrating the validity of the relationship between morphology and productivity for the particular fermentation, regardless of the reactor type. Previous attempts to evaluate the use of loop configurations as scale-up tools and their performance as bioreactors, neglected the morphology of the producer micro-organisms. This study demonstrated the close link between morphology and productivity for citric acid production by A. niger, and identified a morphology parameter that was used successfully to characterize the process performance.