Effects of periodic input on the quasi-steady state assumptions for enzyme-catalysed reactions

In this paper we investigate the validity of a quasi-steady state assumption in approximating Michaelis-Menten type kinetics for enzyme-catalysed biochemical reactions that are subject to periodic substrate input.Acknowledgement This work was funded by the EPSRC Grant GR/R53098. JLL is supported by the Scottish Executive Environment and Rural Affairs Department.     

Input rate-dependent stereoselective pharmacokinetics: Effect of pulsatile oral input

Computer simulation was used to test the effects of pulsatile oral input on the stereoselectivity in the area under the blood concentration–time curves (AUCs) of the enantiomers of racemic drugs. The effects of input rate determinants, namely, dose, dosage interval, and formulation on the stereoselectivity were investigated under both steady-state and nonsteady-state conditions. Simulations were carried out for drugs undergoing Michaelis–Menten hepatic metabolism with different enantiomeric maximum velocity (V_max) or constant (K_m) values. With pulsatile input, the enantiomeric AUC ratios of both types of drugs were dependent on all the determinants of input rate. However, in most cases, the direction of input rate-dependent changes in the enantiomeric AUC ratios for drugs with different enantiomeric V_max was opposite of that for drugs with different enantiomeric K_m. The direction and magnitude of changes in the enantiomeric AUC ratios were also dependent on the selected dose, dosage interval, and formulation. Further, different conclusions could be reached based on the nonsteady-state and steady-state data. Additional simulations were then performed to test the effects of input rate-dependent stereoselective pharmacokinetics on the bioequivalence of chiral drugs with nonlinear metabolism. These simulations suggested that bioequivalence studies based on the racemic drug measurement may result in erroneous conclusions for the individual enantiomers. The results of this study may be used as a tool for the design of experiments to test the input rate dependence of stereoselective pharmacokinetics and bioequivalence of racemic drugs in animals and humans. © 1994 Wiley-Liss, Inc.     

Spline and General Regression of Polytomous Data

The paper shows a formulation for a general linear regression as well as a spline regression of multinomial responses on a quantitative input variables. Application of least squares and asymptotic theory yields the F-test for significance of coefficients and a t-test for structural discontinuity.     

Some system considerations of neuron pools with feedback

This paper describes an approach to the analysis of the inputoutput relationships present in a neuron pool that receives a number of inputs. These inputs consist of primary inputs to the neuron pool and inputs resulting from feedback of information from the neuron pool as well. Multiple input-output relationships are obtained in terms of the synaptic weightings of the inputs, the membrane response characteristics of the neurons and the conduction delays on the feedback pathways.y=(I+MHD) ^-1 ·MFx is the explicit representation of the cell pool behavior assuming quasi-linear conditions, wherey is the output vector of cell responses,I is the identity matrix,M is the response matrix of the cells,H is the feedback synaptic weighting matrix,D is the delay matrix,F is the input weighting matrix, andx is the input vector. It is shown that a solution to this formulation exists, is unique, is stable, and can be computed by specified algorithms. An insight gained from this formulation suggests that the output of each cell in the pool is related to virtually all of the inputs to the pool and the outputs of all cells in the pool.Supported by NSF Grant GK 38301 and US-PHS Grant NS 08470.Program in Bioengineering, Electrical and Computer Engineering Department, The University of Michigan.Computer Information and Control Program, and Department of Electrical and Computer Engineering, The University of Michigan.     

The lipases from Yarrowia lipolytica: genetics, production, regulation, biochemical characterization and biotechnological applications

Lipases are serine hydrolases that catalyze in nature the hydrolysis of ester bonds of long chain triacylglycerol into fatty acid and glycerol. However, in favorable thermodynamic conditions, they are also able to catalyze reactions of synthesis such as esterification or amidation. The non-conventional yeast Yarrowia lipolytica possesses 16 paralogs of genes coding for lipase. However, little information on all those paralogs has been yet obtained and only three isoenzymes, namely Lip2p, Lip7p and Lip8p have been partly characterized so far. Microarray data suggest that only a few of them could be expressed and that lipase synthesis seems to be dependent on the fatty acid or oil used as carbon source confirming the high adaptation of Y. lipolytica to hydrophobic substrate utilization. This review focuses on the biochemical characterization of those enzymes with special emphasis on the Lip2p lipase which is the isoenzyme mainly synthesized by Y. lipolytica. Crystallographic data highlight that this latter is a lipase sensu stricto with a lid covering the active site of the enzyme in its closed conformation. Recent findings on enzyme conditioning in dehydrated or liquid formulation, in enzyme immobilization by entrapment in natural polymers from either organic or mineral origins are also discussed together with long-term storage strategies. The development of various biotechnological applications in different fields such as cheese ripening, waste treatment, drug synthesis or human therapeutics is also presented.     

Validity of anecdotal reports of suspected adverse drug reactions: the problem of false alarms

Suspected adverse drug reactions first reported in 1963 in the “British Medical Journal,” the “Lancet,” the “Journal of the American Medical Association,” and the “New England Journal of Medicine” were reviewed 18 years later to assess their initial validity and subsequent verification. Of 52 first reports, five were deliberate investigations into potential or predictable reactions, and in each case causality was reasonably established; the other 47 reports were essentially anecdotal. Of these 47 reports, 14 related to categories of adverse reaction where false-positive reports were unlikely: immediate reactions, local reactions, and known reactions caused by a different mode of administration or a brand previously thought or claimed to be safe. The problem of false alarms rose in the remaining types of reactions: general reactions that did not occur immediately after administration and arose for the first time with a new chemical entity. Of 33 reports of such suspected adverse reactions, validity was satisfactorily established in 14 cases on the basis of rechallenge, predictability from known pharmacology, or the unique nature of the reaction. Of the remaining 19 reports, further verification still has not been satisfactorily established in 12. Seven of these possible false alarms were haematological reactions.Although 35 of the 47 anecdotal reports were clearly correct, of the 19 reports that were not reasonably validated at the time of the report, only seven were subsequently verified. This suggests that agencies monitoring adverse drug reactions should adopt criteria for assessing the validity of first reports of suspected adverse reactions. Such criteria should include: reactions on rechallenge, a pharmacological basis for the adverse reaction, immediate acute reactions, local reactions at the site of administration, reactions with a new route of administration of a drug known to provoke such reactions by another route, and the repeated occurrence of very rare events.     

Nanoemulsions of sulfonamide carbonic anhydrase inhibitors strongly inhibit the growth of Trypanosoma cruzi

Sulfonamide carbonic anhydrase (CA, EC 4.2.1.1) inhibitors targeting the α-class enzyme from the protozoan pathogen Trypanosoma cruzi, responsible of Chagas disease, were recently reported. Although many such derivatives showed low nanomolar activity in vitro, they were inefficient anti-T. cruzi agents in vivo. Here, we show that by formulating such sulfonamides as nanoemulsions in clove (Eugenia caryophyllus) oil, highly efficient anti-protozoan effects are observed against two different strains of T. cruzi. These effects are probably due to an enhanced permeation of the enzyme inhibitor through the nanoemulsion formulation, interfering in this way with the life cycle of the pathogen either by inhibiting pH regulation or carboxylating reactions in which bicarbonate/CO₂ are involved. This type of formulation of sulfonamides with T. cruzi CA inhibitory effects may lead to novel therapeutic approaches against this orphan disease.     

On the validity of using semilogarithmic plots to determine initial velocities of enzyme-catalyzed reactions

It has been proposed (Johnston & Diven, 1969a) that it is valid to use semilogarithmic (first-order) plots of the extent of reaction versus time for graphic determination of initial velocities of enzyme-catalyzed reactions. This proposition suggests the assumption that the initial velocity error expected to be introduced by the proposed procedure is smaller than or comparable to the error introduced by the customary graphic procedure. The latter is based on the assumption that the progress curves of enzyme-catalyzed reactions have an initial linear segment of sufficient duration to permit accurate determination of slope. The validity of the procedure proposed by Johnston and Diven is examined in this report. It is concluded that the procedure is applicable to a very small class of enzyme-catalyzed reactions and only under certain experimental conditions.     

Denitrification and its control

Denitrification in bacteria comprises a series of four reduction reactions; for nitrate, nitrite, nitric oxide and nitrous oxide. Nitrogen gas is the final product. The nature of the enzymes catalysing these reactions is described along with the the properties of the underlying electron transport systems. The factors influencing the expression of the reductases for the four reactions are reviewed along with the effect of oxygen on the activities of the enzymes of denitrification. The main emphasis is on observations made withParacoccus denitrificans andPseudomonas stutzeri.     

Expanding the boundary of biocatalysis: design and optimization of in vitro tandem catalytic reactions for biochemical production

Biocatalysts have been increasingly used in the synthesis of fine chemicals and medicinal compounds due to significant advances in enzyme discovery and engineering. To mimic the synergistic effects of cascade reactions catalyzed by multiple enzymes in nature, researchers have been developing artificial tandem enzymatic reactions in vivo by harnessing synthetic biology and metabolic engineering tools. There is also growing interest in the development of one-pot tandem enzymatic or chemo-enzymatic processes in vitro due to their neat and concise catalytic systems and product purification procedures. In this review, we will briefly summarize the strategies of designing and optimizing in vitro tandem catalytic reactions, highlight a few representative examples, and discuss the future trend in this field.     

Environmental Risk Management Decision-Making in a Societal Context

Recent studies point to the need for improved understanding of environmental management frameworks designed to combine qualitative public and quantitative technical inputs in decision-making processes. Flux in public perception and concern about risks imply frameworks must be iterative in nature and incorporate a variety of assessment triggers in the form of decision points. A conceptual model is proposed here to explain the de facto operation of standard risk analytic frameworks within the broader sociopolitical milieu of public policy. The model is presented as a decision flow diagram that emphasizes setting environmental management goals based on societal input and the formulation of decision criteria for selecting management actions to achieve those goals. Prospective and retrospective decision control points operate to select management options that, respectively, avoid or reduce actual or predicted effects. Feedback loops that modify risk management outcomes are identified. Technical and scientific inputs (i.e., risk analysis) are assigned an essential information role within the framework and are responsible for informing the management process with the results of appropriately conducted and reviewed investigations. The proposed model is intended primarily to indicate how environmental risk management decision-making and associated technical assessments may be influenced by social pressures. It is hoped this understanding will lead to analytical transparency and better public communication of the environmental implications of policy options.     

Theoretical Analysis of Time-to-Peak Responses in Biological Reaction Networks

Processing of information by signaling networks is characterized by properties of the induced kinetics of the activated pathway components. The maximal extent of pathway activation (maximum amplitude) and the time-to-peak-response (position) are key determinants of biological responses that have been linked to specific outcomes. We investigate how the maximum amplitude of pathway activation and its position depend on the input and wiring of a signaling network. For this purpose, we consider a simple reaction A→B that is regulated by a transient input and extended this to include back-reaction and additional partners. In particular, we show that a unique maximum of B(t) exists. Moreover, we prove that the position of the maximum is independent of the applied input but regulated by degradation reactions of B. Indeed, the time-to-peak-response decreases with increasing degradation rate, which we prove for small models and show in simulations for more complex ones. The identified dependencies provide insights into design principles that facilitate the realization dynamical characteristics like constant position of maximal pathway activation and thereby guide the characterization of unknown kinetics within larger protein networks.     

Nanoethosomal formulation for skin targeting of amphotericin B: an in vitro and in vivo assessment

Abstract

The present study is envisaged to develop nanoethosomal formulation for enhanced topical delivery of amphotericin B (AmB) for the treatment of cutaneous fungal infections. AmB encapsulated nanoethosomes were prepared using mechanical dispersion method in a strength of 0.1% w/w similar to the strength of marketed topical formulation. Vesicle size of nanoethosomal formulations was found to be in the range of 186?±?2 to 298?±?4?nm. The optimized nanoethosomal formulation was further incorporated in gel base to form AmB nanoethogel formulation. Rheological characterization study of nanoethogel demonstrated its viscoelastic nature with high elasticity and resistance to deformation at 37?°C. The yield stress value was found to be 108.05?±?2.4 and 52.15?±?0.9?Pa for nanoethogel and marketed gel formulation, respectively. The nanoethogel formulation exhibited 2.7- and 3.5-fold higher steady state transdermal flux and skin deposition of AmB, respectively, in comparison to marketed formulation. Confocal laser scanning microscopy (CLSM) study also revealed enhanced skin permeation and deposition with nanoethogel formulation. In vivo study showed that topical application of nanoethogel does not exhibit any skin irritation as tested by Draize test. The developed formulation, in comparison to the marketed gel, demonstrated a remarkable increase in the antifungal activity against Candida albicans. It is thus corroborated from the above results that nanoethosomal formulation represents an efficacious carrier for effective topical delivery of AmB.     

Assessment of the Influence Factors on Nasal Spray Droplet Velocity Using Phase-Doppler Anemometry (PDA)

Droplet velocity is an important parameter that can be used to characterize nasal spray products. In this study, a phase-Doppler anemometry (PDA) system was used to measure the droplet velocities of nasal sprays. A survey of seven commercial nasal spray products showed a range of droplet velocities from 6.7 to 19.2 m/s, all significantly different from each other. A three-level, four-factor Box–Behnken design of experiments (DOE) methodology were applied to investigate the influences of actuation parameters and formulation properties on nasal spray droplet velocity using a set of placebo formulations. The DOE study shows that all four input factors (stroke length, actuation velocity, concentration of the gelling agent, and concentration of the surfactant) have significant influence on droplet velocity. An optimized quadratic model generated from the DOE results describes the inherent relationships between the input factors and droplet velocity thus providing a better understanding of the input factor influences. Overall, PDA provides a new in vitro characterization method for the evaluation of inhalation drugs through assessment of spray velocity and may assist in product development to meet drug delivery equivalency requirements.     

Presteady-state kinetics and carrier-mediated transport: A theoretical analysis

Summary Kinetic studies of cotransport mechanisms have so far been limited to the conventional steady-state approach which does not allow in general to resolve either isomerization or ratelimiting steps and to determine the values of the individual rate constants for the elementary reactions involved along a given transport pathway. Such questions can only be answered using presteady-state or relaxation experiments which, for technical reasons, have not yet been introduced into the field of cotransport kinetics. However, since two recent reports seem compatible with the observation of such transient kinetics, it would appear that theoretical studies are needed to evaluate the validity of such claims and to critically evaluate the expectations from a presteady-state approach. We thus report such a study which was performed on a simple four-state mechanism of carrier-mediated transport. The time-dependent equation for zero-trans substrate uptake was thus derived and then extended to models withp intermediary steps. It is concluded that (p-1) exponential terms will describe the approach to the steady state but that such equations have low analytical value since the parameters of the flux equation cannot be expressed in terms of the individual rate constants of the elementary reactions for models withp>5. We thus propose realistic simplifications based on the time-scale separation hypothesis which allows replacement of the rate constants of the rapid steps by their equilibrium constants, thereby reducing the complexity of the kinetic system. Assuming that only one relaxation can be observed, this treatment generates approximate models for which analytical expressions can easily be derived and simulated through computer modeling. When performed on the four-state mechanism of carrier-mediated transport, the simulations demonstrate the validity of the approximate solutions derived according to this hypothesis. Moreover, our approach clearly shows that presteady-state kinetics, should they become applicable to (co)transport kinetics, could be invaluable in determining more precise transport mechanisms.     

A scaled-down model for the translation of bacteriophage culture to manufacturing scale

Therapeutic bacteriophages are emerging as a potential alternative to antibiotics and synergistic treatment of antimicrobial-resistant infections. This is reflected by their use in an increasing number of recent clinical trials. Many more therapeutic bacteriophage is being investigated in preclinical research and due to the bespoke nature of these products with respect to their limited infection spectrum, translation to the clinic requires combined understanding of the biology underpinning the bioprocess and how this can be optimized and streamlined for efficient methods of scalable manufacture. Bacteriophage research is currently limited to laboratory scale studies ranging from 1–20 ml, emerging therapies include bacteriophage cocktails to increase the spectrum of infectivity and require multiple large-scale bioreactors (up to 50 L) containing different bacteriophage–bacterial host reactions. Scaling bioprocesses from the milliliter scale to multi-liter large-scale bioreactors is challenging in itself, but performing this for individual phage-host bioprocesses to facilitate reliable and robust manufacture of phage cocktails increases the complexity. This study used a full factorial design of experiments approach to explore key process input variables (temperature, time of infection, multiplicity of infection, agitation) for their influence on key process outputs (bacteriophage yield, infection kinetics) for two bacteriophage–bacterial host bioprocesses (T4 – Escherichia coli; Phage K – Staphylococcus aureus). The research aimed to determine common input variables that positively influence output yield and found that the temperature at the point of infection had the greatest influence on bacteriophage yield for both bioprocesses. The study also aimed to develop a scaled down shake-flask model to enable rapid optimization of bacteriophage batch bioprocessing and translate the bioprocess into a scale-up model with a 3 L working volume in stirred tank bioreactors. The optimization performed in the shake flask model achieved a 550-fold increase in bacteriophage yield and these improvements successfully translated to the large-scale cultures.     

Synapse-like contacts between axons of the pineal tract and the subcommissural organ in Rana perezi (Anra) and their absence in Carassius auratus (Teleostei): ultrastructural tracer studies

The present study was designed to investigate the controversial subject of the existence of a neural input from the pineal organ via the pineal tract to the subcommissural organ (SCO) in teleosts and anurans. Horseradish peroxidase was injected into the pineal organ and pineal tract of Carassius auratus and Rana perezi. Within the pinealofugal fibers the tracer was visualized at the light-and electron-microscopic levels either by immunocytochemistry using an anti-peroxidase serum, or by revealing the enzymatic activity of peroxidase. In both species, labeled myelinated and unmyelinated fibers of the pineal tract were readily traced by means of electron microscopy. In R. perezi, numerous terminals contacting the SCO cells in a synapse-like (synaptoid, hemisynaptic) manner bore the label, whereas a different population of endings was devoid of the tracer, indicating that in this species the SCO receives a dual neural input, one of pineal origin, the other of unknown source and nature. In the SCO of C. auratus, neither labeled nor unlabeled synapse-like contacts were found. Thus, in this latter species, a direct neural input to the SCO is missing. It is concluded that the secretory activity of the SCO can be controlled by different mechanisms in different species, and that more than one neural input mechanism may operate in the same species.     

Influence of fractal kinetics on molecular recognition

Molecular recognition is a central issue for nearly every biological mechanism. The analysis of molecular recognition to has been conducted within the framework of classical chemical kinetics, in which the kinetic orders of a reaction have positive integer values. However, recent theoretical and experimental advances have shown that the assumption inherent in this classical framework are invalid under a variety of conditions in shown that the assumptions inherent in this classical framework are invalid under a variety of condition in which the reaction environment may be considered nonideal. A good example is provided by reactions that are spatially constrainal and diffusion limited. Bio molecular reactions confined within two-dimensional membranes, one-dimensional channels or fractal surfaces in general exhibit kinetic orders that are noninteger. An appropriate framework for the study of these nonideal phenomena is provided by the Power-Law formalism, which includes as special cases the Mass-Action formalism of chemical kinetics and the Michaelis–Menten formalism of enzyme kinetics. The Power-Law formalism is an appropriate representation not only for fractal kinetics per se, but also for other nonideal kinetic phenomena, provided the range of variation in concentration is not too large. After defining some elementary concepts of molecular recognition, and showing how these are manifested in classical kinetic terms, this paper contrasts the implications of classical and fractal kinetics in a few simple cases. The principal distinction lies in the ability of fractal kinetics to nonlinearly transform, rather than proportionally transmit, the input S/N ratio. As a consequence, fractal kinetics create a threshold for the input signal below which no recognition occurs and above which amplified recognition takes place. Thus, fractal kinetics implies an intimate relationship between design of the physiological mechanisms regulating the environment of the process and design of the molecular process itself. These results also suggest that recognition in the presence of a favorable input ration would emphasize rapid reactions, while recognition in the presence of an unfavorable input ratio would emphasize slow reactions.