Kinetic analysis of a general model of activation of aspartic proteinase zymogens

Starting from a simple general reaction mechanism of activation of aspartic proteinase zymogens involving an uni- and a bimolecular simultaneous route, the time course equation of the concentration of the zymogen and of the activated enzyme have been derived. From these equations, an analysis quantifying the relative contribution to the global process of the two routes has been carried out for the first time. This analysis suggests a way to predict the time course of the relative contribution as well as the effect of the initial zymogen and activating enzyme concentrations, on the relative weight. An experimental design and kinetic data analysis is suggested to estimate the kinetic parameters involved in the reaction mechanism proposed. Finally, we apply some of our results to experimental data obtained by other authors in experimental studies of the activation of some aspartic proteinase zymogens.     

Contribution of the intra- and intermolecular routes in autocatalytic zymogen activation: application to pepsinogen activation

Taking as the starting point a recently suggested reaction scheme for zymogen activation involving intra- and intermolecular routes and the enzyme-zymogen complex, we carry out a complete analysis of the relative contribution of both routes in the process. This analysis suggests the definition of new dimensionless parameters allowing the elaboration, from the values of the rate constants and initial conditions, of the time course of the contribution of the two routes. The procedure mentioned above related to a concrete reaction scheme is extrapolated to any other model of autocatalytic zymogen activation involving intra- and intermolecular routes. Finally, we discuss the contribution of both of the activating routes in pepsinogen activation into pepsin using the values of the kinetic parameters given in the literature.     

Assembly and activation of a kinase ribozyme

RNA activities can be regulated by modulating the relative energies of all conformations in a folding landscape; however, it is often unknown precisely how peripheral elements perturb the overall landscape in the absence of discrete alternative folds (inactive ensemble). This work explores the effects of sequence and secondary structure in governing kinase ribozyme activity. Kin.46 catalyzes thiophosphoryl transfer from ATPγS onto the 5′ hydroxyl of polynucleotide substrates, and is regulated 10,000-fold by annealing an effector oligonucleotide to form activator helix P4. Transfer kinetics for an extensive series of ribozyme variants identified several dispensable internal single-stranded segments, in addition to a potential pseudoknot at the active site between segments J1/4 and J3/2 that is partially supported by compensatory rescue. Standard allosteric mechanisms were ruled out, such as formation of discrete repressive structures or docking P4 into the rest of the ribozyme via backbone 2′ hydroxyls. Instead, P4 serves both to complete an important structural element (100-fold contribution to the reaction relative to a P4-deleted variant) and to mitigate nonspecific, inhibitory effects of the single-stranded tail (an additional 100-fold contribution to the apparent rate constant, k_obs). Thermodynamic activation parameters ΔH^‡ and ΔS^‡, calculated from the temperature dependence of k_obs, varied with tail length and sequence. Inhibitory effects of the unpaired tail are largely enthalpic for short tails and are both enthalpic and entropic for longer tails. These results refine the structural view of this kinase ribozyme and highlight the importance of nonspecific ensemble effects in conformational regulation by peripheral elements.     

Cooperative mechanisms of thin filament activation and their contribution to the myocardial contractile function: Assessment in a mathematical model

A mathematical model was used for comparative analysis of the contribution to the myocardial mechanical activity of two potentially possible variants of the cooperative influence of myosin cross-bridges on calcium activation of sarcomere actin filaments. One of these variants implies that the cooperative action of the cross-bridge on the affinity of troponin C for calcium is localized within the functional group A₇TmTn (seven adjacent globular actin monomers, tropomyosin, and one troponin complex TnC + TnI + TnT) where this bridge is attached. The second variant is based on the assumption that cross-bridges may influence the troponin C affinity for calcium also in neighboring A₇TmTn groups (and the closer the group is positioned relative to the bridge, the stronger is the influence on the CaTnC complex affinity in this group). The contribution of each of these two variants to the active mechanical behavior of the cardiac muscle in the contraction-relaxation cycle was assessed. It turned out that adequate simulation of the muscle mechanical activity is provided only by the second variant. Thus, the results of modeling argue in favor of the existence of just this variant of cooperativity.     

Linear mixed irreversible inhibition of the autocatalytic activation of zymogens. Kinetic analysis checked by simulated progress curves

Autocatalytic zymogen activation is a phenomenon of great importance for understanding some fundamental physiological processes involved in the enzyme regulation of gastrointestinal-tract enzymes, blood coagulation, fibrinolysis and the complement system. Examples of such processes are the activation of prekallikrein, trypsinogen and pepsinogen, all of which are controlled by natural proteinase inhibitors. This work studies the kinetics of a general autocatalytic zymogen activation process overlapped by two two-step irreversible inhibitions, i.e. a linear mixed irreversible inhibition. The kinetic equations for the whole course of the reaction are derived for this mechanism. In addition, we determine the corresponding kinetics for a number of particular cases of the general model analyzed, i.e. for reversible and irreversible non-competitive, competitive and uncompetitive inhibition systems which are considered particular cases of the general mechanism studied. The kinetic behavior of the system is related to a parameter, a dimensionless quantity, which shows whether the inhibition or the activation route prevails, in a similar way to that which we have previously carried out for other mechanisms. Finally, based on the kinetic equations obtained, a procedure for discriminating between the different mechanisms considered is suggested. The results of this contribution can be directly applied to most physiological autocatalytic zymogen activations in the presence of an inhibitor, allowing their complete kinetic characterization and suggesting procedures for varying the relative weight of the catalytic and inhibition routes or for changing the predominant route.     

A four-year prospective study of the work of the practice nurse in the treatment room of a South Yorkshire practice.

During a four-year study period 43 985 patients were seen in the treatment room and 61 806 coded procedures carried out. Thirty per cent of these procedures were not part of usual nursing curricula and required initial supervision and assessment or training (or both). Nearly 15% of the patients seen were making a first visit and did not require referral to a doctor. A further 17% were also making a first visit but were referred to a doctor. The treatment room made an important contribution to the work of the practice, but this would not have been possible if the staff concerned had been attached nurses requiring area health authority authorisation for procedures carried out as opposed to practice nurses for whom procedures were authorised on a personal basis.     

GENESIS: a simulation model of phylogeny 2. A sensitivity analysis

In a former paper (Heijerman 1988) a simulation model of phylogeny, GENESIS, was presented. This paer describes the results of a sensitivity analysis of GENESIS. The analysis is performed by cganging the input parameter values and estimating the relative effects on the model's output, as summarized by several tree statistics. The results show that none of the statistics tested can be classified as an unambiguous estimator of accuracy of methods for estimating hylogenetic trees. The sensitivity analysis increases the insight into the behaviour and applicability of the model. This is a prerequisite for a correct interpretation of the results of the evaluation experiments that will be carried out using GENESIS.     

Lipid Rafts & Co.: an integrated model of membrane organization in T cell activation

The model of membrane compartmentalization by self-organizing functional lipid microdomains, named lipid rafts, has been a fruitful concept resulting in great progress in understanding T cell signal transduction. However, due to recent results it has become clear that lipid rafts describe only one out of several membrane organizing principles crucial for T cell activation besides fences and pickets and protein-protein interactions that take part in the formation of the immunological synapse as a highly organized structure at the T cell contact site to the antigen-presenting cell. This review describes the concepts of lipid rafts and other membrane organizing principles to evolve a novel integrated model on the functional role of microdomains in immunological synapse formation and T cell activation. Further research has to elucidate the relative contribution and interrelation of different modes of membrane organization in productive T cell activation.     

Light-induced activation of an inert surface for covalent immobilization of a protein ligand.

A simple and mild procedure is developed for the preparation of an activated polymer surface, used for immobilization of a protein ligand through a covalent linkage. Activation of the polymer surface is carried out by attaching an active functional group through 1-fluoro-2-nitro-4-azidobenzene (FNAB). UV irradiation of FNAB transforms its azido group into a highly reactive nitrene, which binds with the inert polymer surface, whereas the active fluoro group of FNAB, now part of the polymer, remains intact. Covalent linkage between the ligand and the inert surface is established through this active fluoro group in a thermochemical reaction. The photochemical step is carried out under dry conditions to exclude the possibility of undesirable reactions between the solvent and the highly reactive nitrene. The method can be used for activation of different inert polymer surfaces having carbon hydrogen bonds. The efficacy of our method is demonstrated by immobilizing horseradish peroxidase on an activated polystyrene surface. The enzyme, immobilized through the photolinker, is found to give a twofold increase in absorbance with the substrate as compared to the directly adsorbed enzyme. The method may have many applications in the preparation of bioreactors, biostrips, and biosensors, and in diagnostic tests involving the ELISA technique.     

Fourier transform infrared spectroscopy indicates a major conformational rearrangement in the activation of rhodopsin.

The study of the structural differences between rhodopsin and its active form (metarhodopsin II) has been carried out by means of deconvolution analysis of infrared spectra. Deconvolution techniques allow the direct identification of the spectral changes that have occurred, which results in a significantly different view of the conformational changes occurring after activation of the receptor as compared with previous difference spectroscopy analysis. Thus, a number of changes in the bands assigned to solvent-exposed domains of the receptor are detected, indicating significant decreases in extended (beta) sequences and in reverse turns, and increases in irregular/aperiodic sequences and in helices with a non-alpha geometry, whereas there is no decrease in alpha-helices. In addition to secondary structure conversions, qualitative alterations within a given secondary structure type are detected. These are seen to occur in both reverse turns and helices. The nature of this spectral change is of great importance, since a clear alteration in the helices bundle core is detected. All these changes indicate that the rhodopsin --> metarhodopsin II transition involves not a minor but a major conformational rearrangement, reconciling the infrared data with the energetics of the activation process.     

Functional analysis of a Brassica oleracea SLR1 gene promoter.

The Brassica oleracea S-locus-related gene 1 (SLR1) is expressed in the papillar cells of Brassica stigmas from a few days before anthesis. We have previously shown that a 1500-bp fragment of the SLR1 gene promoter is sufficient to direct high-level, temporally regulated expression of the beta-glucuronidase reporter gene in the pistils of transgenic tobacco. We have carried out a deletion analysis of the SLR1 promoter and found that elements required for pistil expression are located between -258 and -327 bp (relative to the translation start site). Furthermore, specific binding of pistil nuclear factors to sequences within this region was demonstrated by gel retardation analysis. Sequences between -1350 and -1500 were found to be required for high-level expression.     

Structure-activity relationships in a new class of non-substrate-like covalent inhibitors of the bacterial glycosyltransferase LgtC

Lipooligosaccharide (LOS) structures in the outer core of Gram-negative mucosal pathogens such as Neisseria meningitidis and Haemophilus influenzae contain characteristic glycoepitopes that contribute significantly to bacterial virulence. An important example is the digalactoside epitope generated by the retaining α-1,4-galactosyltransferase LgtC. These digalactosides camouflage the pathogen from the host immune system and increase its serum resistance. Small molecular inhibitors of LgtC are therefore sought after as chemical tools to study bacterial virulence, and as potential candidates for anti-virulence drug discovery. We have recently discovered a new class of non-substrate-like inhibitors of LgtC. The new inhibitors act via a covalent mode of action, targeting a non-catalytic cysteine residue in the LgtC active site. Here, we describe, for the first time, structure-activity relationships for this new class of glycosyltransferase inhibitors. We have carried out a detailed analysis of the inhibition kinetics to establish the relative contribution of the non-covalent binding and the covalent inactivation steps for overall inhibitory activity. Selected inhibitors were also evaluated against a serum-resistant strain of Haemophilus influenzae, but did not enhance the killing effect of human serum.     

An economic evaluation of a genetic screening program for Tay-Sachs disease.

The resolution of policy questions relating to medical genetic screening programs will not be without considerable difficulty. Examples include such issues as the optimal degree of screening program expansion, the relative values of screening for different genetic diseases, the appropriate sources of program funding (public vs. private), and the relative value of funding expanded genetic screening programs vs. research directed toward elimination of genetic traits themselves. Information on the net impact of the relevant alternatives is greatly needed, and this need will increase if the National Genetics Act receives funding approval. We have provided what is hopefully a contribution toward this end. While our analysis pertains to a specific disease and a specific screening program for that disease, the methodology is readily generalizable to other genetic diseases, as well as programs of any size or structure. Hopefully, this will serve to stimulate further research efforts that we believe are needed for the objective consideration of resource allocation alternatives.     

An analysis of the work and tasks of the microbiology laboratory at a multidisciplinary research institute

The comparative analysis of the work of 3 groups of microbiologists at a multiple-discipline clinical research institute has permitted the development of the method for the evaluation of the work of researchers in the microbiological laboratory. The volume of investigations carried out in the laboratory, the relative significance of individual samples of clinical material, the degree of contamination of the samples under study, the level of the identification of microbes and the analysis of the data obtained in the course of investigations have been used as evaluation criteria. The method may be used for the objective quantitative evaluation of the work and the level of professional training of individual researchers, as well as for the calculation of expenditures in materials and time, necessary for performing investigations. According to the data of the analysis carried out by the authors with the use of the proposed method, the work of scientific-practical groups at the institutions of this category holds the greatest promise.     

Determinants of beta diversity of spiders in coastal dunes along a gradient of mediterraneity

Aim The Mediterranean Basin is recognized for its high levels of species richness, rarity and endemicity. Our main aim was to evaluate the relative effects of environmental and spatial variables and their scale‐specific importance on beta diversity patterns along a gradient of mediterraneity, using spiders as a model group. Location This study was carried out in 18 coastal dune sites along the Portuguese Atlantic coast. This area encompasses 445 km and comprises two distinct biogeographic regions, Eurosiberian (northern coast) and Mediterranean (centre and south). Methods A forward selection procedure was carried out to select environmental and spatial variables responsible for determining beta diversity patterns. Variation partitioning and principal coordinates of neighbour matrices (PCNM) were used to estimate the contribution of pure environmental and pure spatial effects and their shared influence on beta diversity patterns and to estimate the relative importance of environmental structured variation and pure spatial variation at multiple spatial scales. Results Climate, ground vegetation dune cover and area were selected by a forward selection procedure. The same procedure identified three PCNM variables, all corresponding to large and medium spatial scales. Variation partitioning revealed that 46.1% of the variation of beta diversity patterns was explained by a combination of environmental and PCNM variables. Most of this variation (42.5%) corresponded to spatial variation (environmental spatially structured and pure spatial). Climate and vegetation structure influences were predominant at the PCNM1 and PCNM3 scales, while area was more important at the intermediate PCNM2 scale. Main conclusions Our study revealed that beta diversity of spiders was primarily controlled by a broad‐scale gradient of mediterraneity. The relative importance of environmental variables on the spider assemblage composition varied with spatial scale. This study highlights the need of considering the scale‐specific influence of niche and neutral processes on beta diversity patterns.     

The effects of precision demands during a low intensity pinching task on muscle activation and load sharing of the fingers.

High precision demands in manual tasks can be expected to cause more selective use of a part of the muscular synergy involved. To test this expectation, load sharing of the index finger and middle finger was investigated during a pinching task. Myoelectric activation of lower arm and neck-shoulder muscles was measured to see if overall level of effort was affected by precision demands. Ten healthy female subjects performed pinching tasks with three levels of force and three levels of precision demands. The force level did not significantly affect the relative contribution of the index and middle finger to the force. Higher precision demands, however, led to higher contribution of the index finger to the pinch force. Consequently, a more selective load of the forearm and hand occurs during tasks with high precision demands. The variability of the force contribution of the fingers increased during the task. No effects of precision demand on the activation of forearm and neck-shoulder muscles were found. Force level did affect the EMG parameters of several muscles. The effects were most apparent in the muscles responsible for the pinch force, the forearm muscles. Activation of these muscles was higher at higher force levels. In the trapezius muscle at the dominant side EMG amplitudes were lower at the high pinch force compared to the low force and median force conditions.     

Yeast mitochondrial deoxyribonuclease stimulated by ethidium bromide. 2. Mechanism of enzyme activation.

An endonuclease (EtdBr DNase), which is more active in the presence of EtdBr, has been purified from yeast mitochondrial membrane (Jacquemin-Sablon, H., et al. (1979) Biochemistry 18 (preceding paper in this issue)). This paper deals with the analysis of the mechanism of this activation. Determination of the enzyme activity in the presence of intercalating and nonintercalating agents showed that the enzyme does not recognize the DNA structure modifiction provoked by drug intercalation. Studies carried out with a series of phenanthridinium derivatives led to the following model. The EtdBr DNase activation would result from the formation of a ternary complex, DNA--drug--Triton X-100. The activation capacity of a drug depends on its ability to bind simultaneoulsy to the DNA (not necessarily by intercalation) and the detergent. When this complex is formed, the DNA molecule is surrounded with Triton X-100 molecules which constitute an hydrophobic environment and make the substrate more prone to interaction with the enzyme. The implications of this model are discussed.     

Detection and functional characterization of a large genomic deletion resulting in decreased pathogenicity in Ralstonia solanacearum race 3 biovar 2 strains

Bacterial wilt (brown rot) disease of potato caused by Ralstonia solanacearum is one of the most important bacterial diseases and a major constraint on potato production worldwide. Through a comparative genomic analysis between R.?solanacearum'race 3 biovar 2' (R3bv2) strains, we identified a 77 kb region in strain UW551 which is specifically absent in the hypoaggressive strain IPO1609. We proved that IPO1609 indeed carries a 77 kb genomic deletion and provide genetic evidence that occurrence of this deletion is responsible for almost complete loss of pathogenicity of this strain. We carried out a functional analysis of this 77 kb region in strain UW551 using a combination of gene deletion and functional complementation approaches which identified the methionine biosynthesis genes metER as having a major contribution to IPO1609 pathogenesis. Deletion of the metER genes significantly impacts pathogenicity of R3bv2 strains but does not lead to methionine auxotrophy nor reduced ability to multiply in planta. In addition, this study indicated that three type III secretion system effectors or a type VI secretion system present within the 77 kb region have no or very minor contribution to pathogenicity.