Treatment of burn surfaces by proteinases: mathematical description of an enzyme distribution

The process of penetration of a proteolytic enzyme applied to the surface of burn wound into the depth of necrotic tissue was considered. The model approximation describes three factors by a series of mathematical equations: inward-directed enzyme diffusion, counter-flow filtration of interstitial fluid (exudates), and irreversible inactivation of the enzyme by specific inhibitors present in exudates. According to the model, a quasi-stationary distribution of enzymatic activity through the thickness of the necrotic layer is achieved within 3 h and persists as long as the enzyme concentration on the wound surface is constant. The enzyme activity diminishes linearly from the wound surface to the mid-part of the necrotic layer. No enzyme activity is retained in the inner mid-part of the necrotic layer completely protected by the prevalent inhibitor. The ratio of enzyme concentration on the wound surface to inhibitor concentration in the interstitial fluid is the same as the ratio of the depth of active enzyme area to the depth of the inhibitor-protected area through the necrotic layer. The dynamics of accumulation of the active enzyme in the necrotic zone and the rate of enzyme inactivation in the wound by inhibitors were described by formulas applicable for practical purposes.     

Determinants of intestinal mucosal uptake of short- and medium-chain fatty acids and alcohols

Uptake rates across the jejunal brush border have been measured for water-soluble fatty acids and alcohols and analyzed to determine the relative roles of the unstirred water layer and the lipid cell membrane as determinants of the intestinal absorptive process. Initial studies involving measurement of time courses of electrical transients developed across the intestine exposed to poorly permeant solute molecules showed no anomalous discrimination of probe molecules of different size or charge. This finding suggests that the diffusion barrier in the intestine can be considered as an unstirred water layer. Next, uptake rates of fatty acid were found to be linear with respect to concentration of the test solute, demonstrated no competitive inhibition or contralateral stimulation, had low temperature dependency, and were insensitive to metabolic inhibition, indicating that uptake proceeds by passive diffusion. Passive permeability coefficients, *P, varied from 22 +/- 1.4 to 395 +/- 9.2 nmoles.min(-1).100 mg(-1).mm(-1) for the saturated fatty acids 2:0 through 12:0 and from 119 +/- 3.3 to 581 +/- 45.2 for the saturated alcohols 6:0 through 10:0. Vigorous stirring of the bulk buffer solution enhanced *P values in direct proportion to chain length while the presence of bile acid micelles depressed apparent permeability coefficients in proportion to fatty acid chain length. These results demonstrate that uptake of short-chain fatty acid monomers is rate limited by the lipid cell membrane but diffusion through the unstirred water layer becomes increasingly rate limiting as the chain length increases. It is also possible to conclude from these data that diffusion through the unstirred water layer becomes totally rate limiting for uptake of long-chain fatty acid monomers of physiological importance.     

Protein kinase C penetration into lipid bilayers

Physical characteristics of the association and subsequent penetration of protein kinase C into defined lipid bilayers were analyzed using four different fluorescence probes. The enzyme demonstrated strong hydrophobic and electrostatic interactions with the bilayer as suggested by its ability to increase permeability of carboxyfluorescein-filled unilamellar vesicles. The intensity of interaction was dependent on the concentration of phosphatidylserine. The hydrophilic quencher, N-methylpicolinium perchlorate, was used to show that the tryptophan residues affected by ligand-induced conformational changes were in a hydrophobic region(s) of the enzyme. Using quenching of intrinsic tryptophan fluorescence, the enzyme was shown to penetrate the lipid bilayer to the C-16 position of labeled fatty acid probes. The association and subsequent penetration of the enzyme into the lipid bilayer was independent of divalent cations in these systems and had no significant effect on activator-independent substrate phosphorylation.     

Coordinate assembly of lipids and enzyme proteins into epidermal lamellar bodies

Formation of the epidermal permeability barrier requires delivery of lamellar body (LB) contents to the stratum corneum interstices. LB are enriched in a mixture of polar lipids and a family of hydrolytic enzymes, required for the extracellular processing of the secreted polar lipids into the more hydrophobic products which mediate barrier function. Prior non-quantitative studies show that acute barrier disruption leads to immediate secretion of the contents of performed LB from the outermost layer of granular cells, followed by the synthesis and accelerated secretion of newly-formed (= nascent) organelles over 0.5-4 h. We asked here whether lipids and hydrolytic enzymes are packaged into nascent organelles separately, or in a parallel, linked process. We first quantified the rate of appearance of lipids (by the content of internal lamellae within LB) and enzyme content (by cytochemistry of neutral lipase and acid sphingomyelinase); both are concentrated in LB, and in nascent organelles. Immediately after barrier disruption, the density of LB in the cytosol of the outermost granular cell decreased by > 50% reduction at 30 min, returning to near-normal densities by 4 h. Nascent organelles budded off a trans-Golgi-like reticulum, in the outermost granular cells as early as 30 min. In quantitative studies, LB progressively accumulated lipid and enzyme contents in parallel. However, when lipid/lamellae generation was inhibited with lipid synthesis inhibitors, enzymes did not accumulate in organelles. Likewise, when exogenous physiologic lipids were delivered to sites of LB generation in the face of brefeldin A blockade of organellogenesis, or when lipids were delivered in conjunction with treatment with lipid synthesis inhibitors, enzymes accumulated only in those organelles that displayed lipid content. These studies demonstrate: (a) quantitative changes in the density of LB in the outermost granular cell at various time points after acute barrier disruption; (b) the origin of nascent organelles in a trans-Golgi-like reticulum; (c) co-ordinate packaging of lipid and enzyme contents into nascent organelles; (d) that lipid deposition in nascent organelles is required for enzyme accumulation; and (e) that enzymes can be delivered to nascent organelles, even if the source of lipid is of exogenous rather than endogenous origin.     

Enzymatic reaction in a vesicular microreactor: peptaibol-facilitated substrate transport

Catalytic reactions performed with enzymes localized in lipid vesicles or in whole cells represent a new, promising approach in biocatalysis. The delivery of different substrates into these micro- or nano-'reactors' requires a sufficient permeability of lipid membranes. To increase the permeability of lipid bilayers, one may use different membrane-active peptides, including peptaibols. In the present study, the trypsin-catalyzed hydrolysis of N(alpha)-benzoyl-L-arginine-para-nitroanilide (BAPA; 1) was studied in a phospholipid vesicular system made of phosphatidylcholine (POC), in the presence of the peptaibols alamethicin (ALM) or zervamicin IIB (ZER). Two different manners of compartmentalization of substrate and enzyme (enzyme- vs. substrate-containing vesicles) were used. The kinetics parameters of the reaction in homogeneous solution and in the vesicular systems were determined. The rate of the extra- or intravesicular enzymatic reaction was found to be controlled by substrate diffusion through the lipid bilayer. In comparison with untreated vesicular systems, an up to seven-fold increase in reaction rate was observed in the presence of either ALM or ZER.     

Determination of intrinsic parameters for immobilization reactions of catalase and amyloglucosidase in porous glass supports

Experiments have been carried out for immobilizing enzyme-catalase and amyloglucosidase in controlled-pore glass particles of two different pore sizes. The experimental results have been analyzed, initial-stage analysis for the rate parameters of immobilization reactions and long-time analysis for determining the evolution of the immobilization process. These investigations suggest that the overall process of immobilization is controlled by the restricted diffusion of enzymes into the pores of the support. As a result, immobilized enzyme (IME) can penetrate only up to a certain distance into the support. The penetration depth of IME for the support-enzyme system mentioned have been evaluated from the experimental bulk enzyme concentration data in a batch recirculation reactor.     

Preparation and characterization of a stratum corneum substitute for in vitro percutaneous penetration studies

The intercellular stratum corneum (SC) lipids form the main barrier for diffusion of substances through the skin. A porous substrate covered with synthetic SC lipids would be an attractive model to study percutaneous penetration, hereby replacing native human SC. Prerequisite is that this stratum corneum substitute (SCS) is prepared with a uniform lipid composition and layer thickness. Furthermore, the lipid organization and orientation should resemble that in SC. The objective of this study was to investigate the utility of an airbrush spraying device to prepare a SCS composed of cholesterol, ceramides and free fatty acids on a polycarbonate filter. The results demonstrate that a proper choice of solvent mixture and lipid concentration is crucial to achieve a uniform distribution of the applied lipids over the filter surface. A smooth and tightly packed lipid layer is only obtained when the equilibration conditions are appropriately chosen. The SCS possesses two crystalline lamellar phases with periodicities similar to those present in native SC. The orientation of these lamellae is mainly parallel to the surface of the polycarbonate filter, which resembles the orientation of the intercellular SC lipids. In conclusion, the airbrush technique enables generation of a homogeneous SCS, which ultimately may function as a predictive in vitro percutaneous penetration model.     

Preparation and characterization of a stratum corneum substitute for in vitro percutaneous penetration studies

The intercellular stratum corneum (SC) lipids form the main barrier for diffusion of substances through the skin. A porous substrate covered with synthetic SC lipids would be an attractive model to study percutaneous penetration, hereby replacing native human SC. Prerequisite is that this stratum corneum substitute (SCS) is prepared with a uniform lipid composition and layer thickness. Furthermore, the lipid organization and orientation should resemble that in SC. The objective of this study was to investigate the utility of an airbrush spraying device to prepare a SCS composed of cholesterol, ceramides and free fatty acids on a polycarbonate filter. The results demonstrate that a proper choice of solvent mixture and lipid concentration is crucial to achieve a uniform distribution of the applied lipids over the filter surface. A smooth and tightly packed lipid layer is only obtained when the equilibration conditions are appropriately chosen. The SCS possesses two crystalline lamellar phases with periodicities similar to those present in native SC. The orientation of these lamellae is mainly parallel to the surface of the polycarbonate filter, which resembles the orientation of the intercellular SC lipids. In conclusion, the airbrush technique enables generation of a homogeneous SCS, which ultimately may function as a predictive in vitro percutaneous penetration model.     

A modelling framework describing the enzyme regulation of membrane lipids underlying gradient perception in Dictyostelium cells II: input-output analysis

Spatial sensing in Dictyostelium involves localization of the phosphoinositide lipids PI(3,4,5)P3 and PI(3,4)P2 at the leading edge of the cell in response to an external gradient. We have previously proposed a modelling framework describing the regulation of these lipids by the enzymes PI3K and PTEN. In this paper we analyse this regulation from an input-output perspective. When the inputs are homogeneous, we obtain explicit analytical expressions for the lipid concentrations as a function of enzyme concentrations and model parameters. We also show that the system can be cast as an open-loop bilinear control system, and employ control engineering tools to show that a local three-dimensional region in the four-dimensional phase space can be accessed by temporally varying either or both enzyme concentrations. For spatially graded enzyme profiles, we show that diffusion limits the extent to which lipid profiles can be manipulated by enzymes. However, we also demonstrate that for certain ranges of network parameters, increasing lipid diffusion can lead to an increase in steady-state leading-edge concentrations of PI(3,4,5)P3 or PI(3,4)P2, even though all lipid diffusion coefficients are equal. Finally, in order to determine the extent to which lipid profiles can be regulated by the enzymes, we formulate and solve inverse problems, where we determine the enzyme profiles required to realize particular lipid profiles at steady state.     

Ultrastructure of the penetration of the crayfish integument by the fungal parasite, Aphanomyces astaci, Oomycetes

In the crayfish, Astacus astacus, susceptible to the crayfish plague fungus, penetration of the cuticle by the parasite occurred in the soft cuticle. The zoospore lysed the surface lipid layer, tore it away, and formed an infection peg (germ tube) that penetrated through the epicuticle. A septum was formed in the infection peg, and a hypha was formed below the inner epicuticular surface. In the endocuticle, hyphae grew preferrentially parallel to the surface, occassionally perpendicular to it. Growth direction in relation to cuticle architecture is discussed. Subsequently, some hyphae started to penetrate out through the epicuticle. This process was preceded by the swelling of the hyphal tip touching the inner side of the epicuticle. The hypha penetrating out through the epicuticle was much thicker than the infection peg. Histolytic activity, combined with mechanical penetration, seems to be evident in all stages and levels except in the outward penetration of the epicuticular lipid surface layer, where only mechanical rupture could be seen. Differences in the protoplasmic ultrastructure were found between the spore and the penetrant hyphae. Penetration of the cuticle of a resistant crayfish was essentially identical to that in susceptible ones. However, inward penetration of intact epicuticle was too scarce to allow for ultrastructural studies.     

Thermodynamics and kinetics of joint action of antiviral agent tilorone and DMSO on model lipid membranes

Individual and joint action of two water-soluble drugs, DMSO and tilorone, on model l-α-dipalmitoylphosphatidylcholine (DPPC) membranes were studied in equilibrium and kinetic regimes by differential scanning calorimetry (DSC). For equilibrium experiments, the drugs were introduced during preparation of the model membrane. In kinetic studies, one of the drugs was added to the DPPC membrane already containing the other drug, and the effects of drug-membrane interactions were monitored in real-time regime. It was found that tilorone and DMSO had opposite effects on the membrane melting temperature, which were non-additive under joint introduction of these drugs. Analysis of kinetics of DSC profiles under drugs introduction allowed us to discriminate two processes in drug-membrane interactions with different characteristic times, i.e., drug sorption onto the membrane (minutes) and drug diffusion through stacks of lipid bilayers (hours). It was established that 0.1?mol% DMSO effectively enhanced membrane penetration for tilorone with the rate of tilorone diffusion being dependent upon the scheme of drugs administration. A model was proposed describing how sorption of a dopant onto lipid membrane could affect the membrane permeability for other dopants. Conditions were determined for enhancement of membrane permeability, as it was observed for DPPC/DMSO/tilorone system.     

The Structure of the Mitochondrial Membrane: Inferences from Permeability Properties

Mitochondria possess a semipermeable membrane with properties similar to the cell membrane. Despite the presence of a limiting membrane, mitochondria swell approximately 4 to 5 times their original volume without lysis or loss of internal solute. For this reason, it has been argued that the membrane might be convoluted. The present kinetic study of the permeability of isolated mitochondria was undertaken to clarify this question. A photometric method described previously was used. In the case of highly lipid soluble penetrants, the results suggest that neither the permeability nor the surface area available for penetration varies significantly during considerable swelling. These results may be interpreted to mean that the mitochondrial membrane is convoluted. For highly polar compounds, the permeability of the membrane also remains unchanged during swelling, but the surface area available to penetration increases. These results may be interpreted to mean that in this latter case, the surface of the convolutions becomes available only after they are unfolded by swelling. The simplest model that can explain the permeability properties of this membrane consists of a bimolecular lipid layer where the inner monomolecular layer is convoluted.     

The effect of crosslink density on permeability in biosensors: An unsteady-state approach

Summary The influence of cross-linker density (2%, 5% and 10% of glutaraldehyde) on the permeability, P, of the enzyme layer (267.7, 203.5 and 146.0 m/s respectively for ¹²⁵I-cortisol-histamine conjugate, CHC (MW: 651) as tracer) in Clark enzyme electrode is reported. A diffusion chamber technique based on unsteady-state analysis for the estimation of permeabilities of radiolabelled molecules through enzyme layer is performed.     

On the large error introduced in the estimate of the density of membrane pores from permeability measurements when diffusion in "unstirred layer" within the cells is disregarded

After the development of the "black lipid membrane" techniques, studies of the permeability of labeled water and nonelectrolytes across these artificial membranes have yielded permeability constants comparable in magnitude to those obtained from tracer studies of living cell membranes. This general agreement has affirmed the belief that the living cell membranes are indeed closely similar to these bilayer phospholipid membranes. In this report, we draw attention to a hidden assumption behind such comparisons made: the assumption that labeled material passing through the cell membrane barriers instantly reaches diffusion equilibrium inside the cell. The permeability constants to labeled water (and nonelectrolytes) across lipid layers were obtained using setups in which the lipid membrane was sandwiched between aqueous compartments both of which were vigorously stirred. In studies of permeability of living cell membranes only the outside solution was stirred, the intracellular water remained stationary. Yet the calculations of permeability constants of the cell membrane were made with the tacit assumption, that once the labeled materials pass through the cell membrane, they were instantly mixed with the entire cell contents as if a stirrer operating at infinite speed had been present inside the cells. Ignoring this unstirred condition of the intracellular water, in fact, lumped all the real-life delay due to diffusion in the cytoplasm and added it to the resistance to diffusion of the membrane barrier. The result is an estimated membrane permeability to labeled water (and nonelectrolytes) many times slower than it actually is. The present report begins with a detailed analysis of a specific case: tritiated water diffusion from giant barnacle muscle fibers and two non-living models, one real, one imagined.(ABSTRACT TRUNCATED AT 250 WORDS)     

Lipid vesicles penetrate into intact skin owing to the transdermal osmotic gradients and hydration force.

Gradients across the outer skin layers may result in fields enforcing a lipid flow into or through the intact skin surface provided that lipids are applied in the form of special vesicles. The osmotic gradient, for example, which is created by the difference in the total water concentrations between the skin surface and the skin interior, provides one possible source of such driving force. It is sufficiently strong to push at least 0.5 mg of lipids per hour and cm2 through the skin permeability barrier in the region of stratum corneum. The lipid concentration gradient, on the contrary, does not contribute much to the lipid penetration into dermis. Occlusion, therefore, is detrimental for the vesicle penetration into intact skin.     

Ultrastructural aspects of and observations on the permeability of the rat mesentery to colloidal iron

In works already published, it was made clear that many researches were interested in the absorption phenomena, permeability and structure of the visceral mesothelial tissue. Attention was concentrated on the mesentery and observations were made using the application of lanthanum nitrate and osmium-amine. The penetration of lanthanum nitrate is impeded by the basement membrane situated between the connective and mesothelial tissues. The heavy salt moves through and not between the mesothelial cells by passive diffusion. No reaction was observed in general with osmium-amine, with the exception of a few cases. In those instances, the osmium-amine reacted not only in the outer surface of the mesentery, but also penetrated with no visible reaction all the way to the connective tissue where it was detected in the elastic layer. In this paper, the colloidal iron was employed using different techniques, and depositions were detected in the surface of the mesentery, in the mesothelial cells and also in the connective tissue. A final conclusion that the permeability of different layers of tissues is of great variety and has a definite capacity for selectivity is suggested.     

The effect of pH and organic ester penetration enhancers on skin permeation kinetics of terbutaline sulfate from pseudolatex-type transdermal delivery systems through mouse and human cadaver skins

The purpose of this research was to prepare a pseudolatex transdermal delivery system for terbutaline sulfate and to evaluate the effect of pH and organic ester penetration enhancers on permeation kinetics of terbutaline sulfate through mice abdominal skin and human cadaver skin. An increase in the permeation flux by increasing pH was observed. The distribution coefficient of terbutaline sulfate between 1-octanol and buffers of different pH values was also pH-dependent. Furthermore, the change of the permeability coefficient with pH correlated well with the distribution coefficient by a 2-degree polynomial equation. The permeation profile and related kinetic parameters of terbutaline sulfate was determined in presence of 3 estertype permeation enhancers incorporated in the films, viz methyl laureate, isopropyl lanolate, and isopropyl myristate. Among the 3, the more pronounced enhancing effect was obtained with isopropyl myristate, regarding the permeatin flux, permeability coefficient, and diffusion coefficient. This was attributed to solubility parameter of isopropyl myristate being closer to the solubility parameter of human skin, and such a pronounced enhancing effect was probably caused by its passage across the skin barrier through the lipid pathway. Published: September 30, 2005     

Transport of protons and hydrochloric acid through lipid bilayer membranes

Transport of protons and hydrochloric acid through lipid bilayer membranes was studied by a combination of electrical conductance and pH electrode techniques. In the presence of large pH gradients, proton transport occurs primarily by diffusion of molecular HCl. The permeability of egg phosphatidylcholine/decane bilayers to HCl is about 3 cm . s-1, seven to nine order of magnitude higher than the permeability to H+, OH- or Cl-. The HCl permeability of phosphatidylserine or egg phosphatidylcholine/cholesterol (1 : 1) bilayers is about 50% lower than the permeability of egg phosphatidylcholine bilayers. Diffusion of molecular HCl may be an important process in tissues exposed to high HCl concentrations, e.g., gastric mucosa. However, at neutral pH the diffusion of molecular HCl is too slow to contribute significantly to net movements of H+ or Cl-.     

Leakage of periplasmic enzymes from envA1 strains of Escherichia coli

Previous work ascribed antibiotic hypersensitivity of the envA1 mutant to lowered lipopolysaccharide levels and exposure of the lipid bilayer. In the detailed characterization of the EnvA permeability phenotype presented here, the envA1 mutation was shown to confer leakage of the periplasmic enzymes beta-lactamase and RNase I. Leakage was observed in three different genetic backgrounds, including the original envA1 strain and its parent. In contrast, no detectable leakage of the cytoplasmic enzyme beta-galactosidase was observed. Sensitivity of envA1 strains to a range of antibiotics not previously reported was tested, and lipophilicity (partition coefficient) of a number of antibiotics was determined. On the basis of observations of periplasmic leakage and sensitivity to large hydrophilic antibiotics and lysozyme, part of the permeability phenotype of the envA1 mutant is proposed to be due to transient rupture and resealing of the EDTA-sensitive outer membrane layer. In this regard, the EnvA permeability phenotype falls into a general class of permeability/leaky mutants of both Escherichia coli and Salmonella typhimurium.