Multiple mechanisms shape selectivity for FM sweep rate and direction in the pallid bat inferior colliculus and auditory cortex

The inferior colliculus and auditory cortex of the pallid bat contain a large percentage of neurons that are highly selective for the direction and rate of the downward frequency modulated (FM) sweep of the bat’s echolocation pulse. Approximately 25% of neurons tuned to the echolocation pulse respond exclusively to downward FM sweeps. This review focuses on the finding that this selectivity is generated by multiple mechanisms that may act alone or in concert. In the inferior colliculus, selectivity for sweep rate is shaped by at least three mechanisms: shortpass or bandpass tuning for signal duration, delayed high-frequency inhibition that prevents responses to slow sweep rates, and asymmetrical facilitation that occurs only when two tones are presented at appropriate delays. When acting alone, the three mechanisms can produce essentially identical rate selectivity. Direction selectivity can be produced by two mechanisms: an early low-frequency inhibition that prevents responses to upward sweeps, and the same asymmetrical two-tone inhibition that shapes rate tuning. All mechanisms except duration tuning are also present in the auditory cortex. Discussion centers on whether these mechanisms are redundant or complementary.     

5-Cyano-6-oxo-1,6-dihydro-pyrimidines as potent antagonists targeting exchange proteins directly activated by cAMP

Exchange proteins directly activated by cAMP (Epac) are a family of guanine nucleotide exchange factors that regulate a wide variety of intracellular processes in response to second messenger cAMP. To explore the structural determinants for Epac antagonist properties of high throughput screening (HTS) hit ESI-08, pyrimidine 1, a series of 5-cyano-6-oxo-1,6-dihydro-pyrimidine analogues have been synthesized and evaluated for their activities for Epac inhibition. Structure-activity relationship (SAR) analysis led to the identification of three more potent Epac antagonists (6b, 6g, and 6h). These inhibitors may serve as valuable pharmacological probes for further elucidation of the physiological functions and mechanisms of Epac regulation. Our SAR results and molecular docking studies have also revealed that further optimization of the moieties at the C-6 position of pyrimidine scaffold may allow us to discover more potent Epac-specific antagonists.     

Deriving the rate equations for product inhibition patterns in bisubstrate enzyme reactions

In this work, the full rate equations for 17 completely reversible bisubstrate enzyme kinetic mechanisms, with two substrates in the forward and two in the reverse direction, have been presented; among these are rapid equilibrium, steady-state, and mixed steady-state and rapid equilibrium mechanisms. From each rate equation eight product inhibition equations were derived, four for the forward and four for the reverse direction. All the corresponding product inhibition equations were derived in full; thus a total of 17 × 8 = 136 equations, were presented. From these equations a list of product inhibition patterns were constructed and presented in a tabular form, both for the primary plots (intercept effects) and the secondary plots (slope effects).

The purpose of this work is to help investigators in practical work, especially biologists working with enzymes, to choose quickly an appropriate product inhibition pattern for the identification of the kinetic mechanism. The practical application of above product inhibition analysis was illustrated with three examples of yeast alcohol dehydrogenase-catalyzed reactions.     

Deriving the rate equations for product inhibition patterns in bisubstrate enzyme reactions

In this work, the full rate equations for 17 completely reversible bisubstrate enzyme kinetic mechanisms, with two substrates in the forward and two in the reverse direction, have been presented; among these are rapid equilibrium, steady-state, and mixed steady-state and rapid equilibrium mechanisms. From each rate equation eight product inhibition equations were derived, four for the forward and four for the reverse direction. All the corresponding product inhibition equations were derived in full; thus a total of 17 x 8 = 136 equations, were presented. From these equations a list of product inhibition patterns were constructed and presented in a tabular form, both for the primary plots (intercept effects) and the secondary plots (slope effects). The purpose of this work is to help investigators in practical work, especially biologists working with enzymes, to choose quickly an appropriate product inhibition pattern for the identification of the kinetic mechanism. The practical application of above product inhibition analysis was illustrated with three examples of yeast alcohol dehydrogenase-catalyzed reactions.     

Constant-ratio alternative substrate approach for differentiation of enzyme mechanisms

A new kinetic approach using alternative substrates as a tool for studying enzyme mechanisms is described. In this method the substrate to alternative substrate ratio is maintained constant and the common product (or summation of product analogs) is measured. The double-reciprocal plots so obtained at several constant ratios generate different patterns for various mechanisms, thus permitting a choice of kinetic model. In some cases, secondary intercept plots are utilized as a diagnostic aid. Another feature of this approach is that most of the resultant plots are linear. The graphical patterns for four cases of two-substrate, two-product reactions are presented as examples. These patterns allow one to differentiate several mechanisms which are not distinguishable by conventional alternative substrate, competitive inhibitor, or product inhibition studies alone. When used in combination with other methods, various mechanisms involving isomerization and abortive complex formation can be differentiated even if only one alternative substrate is available.     

Screening the Medicines for Malaria Venture "Malaria Box" against the Plasmodium falciparum Aminopeptidases,M1, M17 and M18

Malaria is a parasitic disease that remains a global health burden. The ability of the parasite to rapidly develop resistance to therapeutics drives an urgent need for the delivery of new drugs. The Medicines for Malaria Venture have compounds known for their antimalarial activity, but not necessarily the molecular targets. In this study, we assess the ability of the “MMV 400” compounds to inhibit the activity of three metalloaminopeptidases from Plasmodium falciparum, PfA-M1, PfA-M17 and PfM18 AAP. We have developed a multiplex assay system to allow rapid primary screening of compounds against all three metalloaminopeptidases, followed by detailed analysis of promising compounds. Our results show that there were no PfM18AAP inhibitors, whereas two moderate inhibitors of the neutral aminopeptidases PfA-M1 and PfA-M17 were identified. Further investigation through structure-activity relationship studies and molecular docking suggest that these compounds are competitive inhibitors with novel binding mechanisms, acting through either non-classical zinc coordination or independently of zinc binding altogether. Although it is unlikely that inhibition of PfA-M1 and/or PfA-M17 is the primary mechanism responsible for the antiplasmodial activity reported for these compounds, their detailed characterization, as presented in this work, pave the way for their further optimization as a novel class of dual PfA-M1/PfA-M17 inhibitors utilising non-classical zinc binding groups.     

Identifying main evolutionary mechanisms shaping genetic variation of Leuciscus cephalus L. 1758 (Cyprinidae) in western Greece: discordance between methods.

Numerous methods can be used in intraspecific phylogeographic studies to infer the evolutionary mechanisms that shaped observed genetic variation in populations. However, these methods are scarcely used jointly, and the evolutionary outcomes they could propose are not fully compared. In this study, using a chub (Leuciscus cephalus; Cyprinidae) mitochondrial DNA data set (13 populations in Western Greece, 14 haplotypes), we compare three distinct 'historical' methods that could possibly infer relative importance of basic evolutionary mechanisms (isolation vs migration) shaping genetic variation: the nested clade analysis, the psi-test and the 'mismatch distributions'. Taking together, interpretations of these analyses allow to draw a picture of the evolutionary history of chub in Western Greece based on isolation and genetic drift for higher clades. However, results issued each method can differ for low differentiated clades. We discuss such differences and suggest that methods should be used jointly in phylogeographic studies for a better evaluation of the evolutionary mechanisms that shaped genetic variation.     

Finding the right fold.

Using mutational analysis, three groups have compared the transition states for the folding of two pairs of homologous proteins. The results of these studies suggest that protein folding mechanisms are conserved and are defined primarily by the overall topology of the native structures, as opposed to specific details of the interactions stabilizing these structures.     

Ionic-strength-dependent substrate inhibition of the lysis of Micrococcus luteus by hen egg-white lysozyme

The kinetics of lysis of Micrococcus luteus by hen egg-white lysozyme in dilute buffer media is characterized by pronounced substrate inhibition. This effect occurs within the complete pH range where lysozyme activity is detectable. The electrostatic potential of the negatively charged cell-wall proteoglycan increases with decreasing ionic strength, resulting in an enhanced affinity between proteoglycan and lysozyme and probably favouring multipoint substrate attachment. For the lysozyme-catalyzed hydrolysis of cell-wall proteoglycan three plausible mechanisms of substrate inhibition can be postulated. Two out of the three models fit our experimental data, the simplest of the two providing the most rigorous information on the kinetic parameters Km, V and Ki. Three graphical methods consistent with the chosen model were applied for preliminary parameter estimation and the constants obtained were compared to those from nonlinear least-squares analysis. If substrate inhibition is neglected it is shown that serious bias is imposed upon the parameters.     

A new approach in the kinetics of biological transport the potential of reversible inhibition studies

Kinetic equations are derived for reversible inhibition of both active and facilitated transport systems for seven common experimental arrangements. It is shown that the unique features of transport kinetics may be exploited to give new kinds of information. It is also shown that the familiar rules of enzyme kinetics, though often applied to transport, can be seriously misleading. The analysis leads to the following general conclusions: (1) A competitive mechanism frequently gives rise to non-competitive kinetics, depending on the experimental design, but a non-competitive mechanism never produces competitive kinetics. (2) Inhibition studies on exchange diffusion at equilibrium in non-active systems or in the final steady state in active systems are the only unambiguous kinetic tests to distinguish competitive from non-competitive mechanisms. (3) Substrate analogs that are bound to the carrier and transported are readily distinguished by inhibition kinetics from those not transported, even though both may rapidly enter the cell by another route. (4) Even in non-active systems competitive inhibitors commonly have far different affinities for the substrate sites on the two membranes faces: where sufficient non-polarity allows their penetration into the cell, inhibition kinetics readily establish such sidedness in their action. (5) Inhibition kinetics of the mixed competitive and non-competitive type result from moderately asymmetrical binding of inhibitor at the substrate site. (6) Asymmetry is a necessary feature of active transport; hence studies of inhibition kinetics should provide important insights into its mechanism.     

Recent advances on the inhibition of human solute carriers: Therapeutic implications and mechanistic insights

Solute carriers (SLCs) are membrane transport proteins tasked with mediating passage of hydrophilic molecules across lipid bilayers. Despite the extensive roles played in all aspects of human biology, SLCs remain vastly under-explored as therapeutic targets. In this brief review, we first discuss a few successful cases of drugs that exert their mechanisms of action through inhibition of human SLCs, and introduce select examples of human SLCs that have untapped therapeutic potential. We then highlight two recent structural studies which uncovered detailed structural mechanisms of inhibition exhibited against two different human major facilitator superfamily (MFS) transporters of clinical relevance.     

Kinetic and in silico studies of hydroxy-based inhibitors of carbonic anhydrase isoforms I and II

A series of hydroxy and phenolic compounds have been assayed for the inhibition of two physiologically relevant carbonic anhydrase (CA, EC 4.2.1.1) isozymes, the cytosolic human isozymes I and II. The investigated molecules showed inhibition constants in the range of 1.07–4003 and 0.09–31.5?μM at the hCA I and hCA II enzymes, respectively. In order to investigate the binding mechanisms of these inhibitors, in silico studies were also applied. Molecular docking scores of the studied compounds are compared using three different scoring algorithms, namely Glide/SP, Glide/XP and Glide/IFD. In addition, different ADME (absorption, distribution, metabolism and excretion) analysis was performed. All the examined compounds were found within the acceptable range of pharmacokinetic profiles.     

Bisubstrate inhibition: Theory and application to N-acetyltransferases

Bisubstrate inhibitors represent a potentially powerful group of compounds that have found significant therapeutic utility. Although these compounds have been synthesized and tested against a number of enzymes that catalyze sequential bireactant reactions, the detailed theory for predicting the expected patterns of inhibition against the two substrates for various bireactant kinetic mechanisms has, heretofore, not been presented. We have derived the rate equations for all likely sequential bireactant mechanisms and provide two examples in which bisubstrate inhibitors allow the kinetic mechanism to be determined. Bisubstrate inhibitor kinetics is a powerful diagnostic for the determination of kinetic mechanisms.     

The kinetic mechanisms of the bifunctional enzyme aspartokinase-homoserine dehydrogenase I from Escherichia coli

The kinetic mechanisms of the reactions catalyzed by the two catalytic domains of aspartokinase-homoserine dehydrogenase I from Escherichia coli have been determined. Initial velocity, product inhibition, and dead-end inhibition studies of homoserine dehydrogenase are consistent with an ordered addition of NADPH and aspartate beta-semialdehyde followed by an ordered release of homoserine and NADP+. Aspartokinase I catalyzes the phosphorylation of a number of L-aspartic acid analogues and, moreover, can utilize MgdATP as a phosphoryl donor. Because of this broad substrate specificity, alternative substrate diagnostics was used to probe the kinetic mechanism of this enzyme. The kinetic patterns showed two sets of intersecting lines that are indicative of a random mechanism. Incorporation of these results with the data obtained from initial velocity, product inhibition, and dead-end inhibition studies at pH 8.0 are consistent with a random addition of L-aspartic acid and MgATP and an ordered release of MgADP and beta-aspartyl phosphate.     

Structural and functional organization of a developing brain and formation of cognitive functions in child ontogeny

Results of multidisciplinary studies, including neuromorphological, neurophysiological, neuropsychological, and psychphysiological studies, are reviewed. They allow the brain mechanisms of cognition formation and development during maturation to be identified. The role of regulatory (modulatory) brain systems in forming the cognitive function in the child is demonstrated. Data on considerable changes in the brain systems responsible for the development of cognitive functions in children between the ages of five to six and seven to eight years are presented. At this age, the morphological and functional maturations of the frontal cortical areas and their descending connections with other cerebral structures increase the efficiencies of arbitrary selective attention, learning the activity program, inhibition of spontaneous responses, and regulation and organization of activity, i.e., the functions that are important for successful schooling.     

Inhibition of human-type 1 3beta-hydroxysteroid deshydrogenase/Delta(5)-Delta(4)-isomerase expression using siRNA

Specific inhibition of type 1 3beta-HSD is of particular interest since it will allow us to control the formation of androgens and estrogens in peripheral target tissues without affecting type 2 3beta-HSD, which is responsible for the biosynthesis of glucocorticoids and mineralocorticoids in the adrenals. The high homology between types 1 and 2 3beta-HSD is a major difficulty in the development of specific inhibitors through classical chemical synthesis. In this report, we describe the use of small interference RNA (siRNA) to specifically inhibit human type 1 3beta-HSD. We have constructed three DNA vector-based RNAi vectors that allow us to produce three RNA duplexes of 21 nucleotides targeting three different coding regions of human type 1 3beta-HSD mRNA. The resulting constructs were co-transfected into HEK-293 cells with a vector expressing type 1 3beta-HSD. The results indicate that while the two duplexes that target sequences in the 5'-region do not have a strong inhibitory effect, the duplex that targets the 3'-region efficiently inhibits 3beta-HSD activity. Up to 98% inhibition has been observed. To our knowledge, this is the first report showing successful inhibition of steroidogenic enzymes using siRNA technology.     

Mathematical Models for Sleep-Wake Dynamics: Comparison of the Two-Process Model and a Mutual Inhibition Neuronal Model

Sleep is essential for the maintenance of the brain and the body, yet many features of sleep are poorly understood and mathematical models are an important tool for probing proposed biological mechanisms. The most well-known mathematical model of sleep regulation, the two-process model, models the sleep-wake cycle by two oscillators: a circadian oscillator and a homeostatic oscillator. An alternative, more recent, model considers the mutual inhibition of sleep promoting neurons and the ascending arousal system regulated by homeostatic and circadian processes. Here we show there are fundamental similarities between these two models. The implications are illustrated with two important sleep-wake phenomena. Firstly, we show that in the two-process model, transitions between different numbers of daily sleep episodes can be classified as grazing bifurcations. This provides the theoretical underpinning for numerical results showing that the sleep patterns of many mammals can be explained by the mutual inhibition model. Secondly, we show that when sleep deprivation disrupts the sleep-wake cycle, ostensibly different measures of sleepiness in the two models are closely related. The demonstration of the mathematical similarities of the two models is valuable because not only does it allow some features of the two-process model to be interpreted physiologically but it also means that knowledge gained from study of the two-process model can be used to inform understanding of the behaviour of the mutual inhibition model. This is important because the mutual inhibition model and its extensions are increasingly being used as a tool to understand a diverse range of sleep-wake phenomena such as the design of optimal shift-patterns, yet the values it uses for parameters associated with the circadian and homeostatic processes are very different from those that have been experimentally measured in the context of the two-process model.     

Studies on the regulation of plasma cholesterol levels in squirrel monkeys of two genotypes

Certain individual squirrel monkeys ("hypo-responders") are able to remain normocholesterolemic when fed diets containing cholesterol (0.5 mg/kcal). Other squirrel monkeys ("hyperresponders") when fed the same diet become hypercholesterolemic. The purpose of these studies was to identify the mechanisms which allow hyporesponders to compensate for dietary cholesterol. Using formula diets and sterol balance techniques, we have compared cholesterol absorption, synthesis, excretion, and turnover in hypo- and hyperresponding monkeys. Cholesterol absorption was essentially identical in the two groups (about 55 mg/day). Cholesterol synthesis was likewise similar in the two groups (about 35 mg/day) and there was no evidence of feedback inhibition at the level of cholesterol fed. Hyporesponders had faster turnover rates and smaller body cholesterol pools than did hyperresponders. Excretion of neutral steroids was similar for hypo- and hyperresponders and did not change with cholesterol feeding. In contrast, hyporesponders increased bile acid excretion shortly after cholesterol feeding was begun. Hyperresponders responded more slowly and to a lesser degree. It is concluded that, in this species, the mechanism of control of plasma cholesterol levels is related to the rate of conversion of cholesterol to bile acids.