A kinetic study of lipase-catalyzed reversible kinetic resolution involving verification at miniplant-scale.

Lipase-catalyzed kinetic resolution of racemates is a popular method for synthesis of chiral synthons. Most of these resolutions are reversible equilibrium limited reactions. For the first time, an extensive kinetic model is proposed for kinetic resolution reactions, which takes into account the full reversibility of the reaction, substrate inhibition by an acyl donor and an acyl acceptor as well as alternative substrate inhibition by each enantiomer. For this purpose, the reversible enantioselective transesterification of (R/S)-1-methoxy-2-propanol with ethyl acetate catalyzed by Candida antarctica lipase B (CAL-B) is investigated. The detailed model presented here is valid for a wide range of substrate and product concentrations. Following model discrimination and the application of Haldane equations to reduce the degree of freedom in parameter estimation, the 11 free parameters are successfully identified. All parameters are fitted to the complete data set simultaneously. Six types of independent initial rate studies provide a solid data basis for the model. The effect of changes in substrate and product concentration on reaction kinetics is discussed. The developed model is used for simulations to study the behavior of reaction kinetics in a fixed bed reactor. The typical plot of enantiomeric excess versus conversion of substrate and product is evaluated at various initial substrate mixtures. The model is validated by comparison with experimental results obtained with a fixed bed reactor, which is part of a fully automated state-of-the-art miniplant.

Kinetic modeling of lipase catalyzed hydrolysis of (R/S)-1-methoxy-2-propyl-acetate as a model reaction for production of chiral secondary alcohols.

The Candida antarctica lipase B catalyzed kinetic resolution of (R/S)-1-methoxy-2-propyl-acetate was studied as a model system for the biocatalytic production of chiral secondary alcohols. For this purpose, a kinetic model is proposed involving both enantiomers of this reaction using model discrimination and parameter identification. Starting from a ping-pong bi-bi mechanism, a simplified model with sensitive parameters was derived for the R- and S-enantiomer, respectively. It was validated at pH 7.0, using time-course measurements at varying temperatures (30-60 degrees C) and initial substrate conditions (0.05-1.5 M). This model was then used for mechanistic interpretation of the kinetic resolution on a biochemical level. The effect of temperature on kinetic parameters and enantiomeric ratio was investigated and compared to findings from the field of molecular modeling to obtain a better understanding of the reaction system for process design. Values of 21.2 and 9.7 kJmol-1 were determined for the enthalpic (DeltaR-S DeltaH ++ degrees) and the entropic (-T x DeltaR-S DeltaS ++ degrees) contribution of the difference in transition state energy of both enantiomers at 30 degrees C. High enantiomeric ratio's (E of 47-110) especially at lower temperatures, in addition to enzyme activity at a wide pH range, indicate this biotransformation is a promising example for the industrial production of chiral secondary alcohols.

Modelling of the enzymatic kinetically controlled synthesis of cephalexin: influence of diffusion limitation.

In this study the influence of diffusion limitation on enzymatic kinetically controlled cephalexin synthesis from phenylglycine amide and 7-aminodeacetoxycephalosporinic acid (7-ADCA) was investigated systematically. It was found that if diffusion limitation occurred, both the synthesis/hydrolysis ratio (S/H ratio) and the yield decreased, resulting in lower product and higher by-product concentrations. The effect of pH, enzyme loading, and temperature was investigated, their influence on the course of the reaction was evaluated, and eventually diffusion limitation was minimised. It was found that at pH >or=7 the effect of diffusion limitation was eminent; the difference in S/H ratio and yield between free and immobilised enzyme was considerable. At lower pH, the influence of diffusion limitation was minimal. At low temperature, high yields and S/H ratios were found for all enzymes tested because the hydrolysis reactions were suppressed and the synthesis reaction was hardly influenced by temperature. The enzyme loading influenced the S/H ratio and yield, as expected for diffusion-limited particles. For Assemblase 3750 (the number refers to the degree of enzyme loading), it was proven that both cephalexin synthesis and hydrolysis were diffusion limited. For Assemblase 7500, which carries double the enzyme load of Assemblase 3750, these reactions were also proven to be diffusion limited, together with the binding-step of the substrate phenylglycine amide to the enzyme. For an actual process, the effects of diffusion limitation should preferably be minimised. This can be achieved at low temperature, low pH, and high substrate concentrations. An optimum in S/H ratio and yield was found at pH 7.5 and low temperature, where a relatively low reaction pH can be combined with a relatively high solubility of 7-ADCA. When comparing the different enzymes at these conditions, the free enzyme gave slightly better results than both immobilised biocatalysts, but the effect of diffusion limitation was minimal.

[Local anesthesia in children and mentally handicapped]. / Lokale anesthesie bij kinderen en verstandelijk gehandicapten.

Pain belief and pain thresholds vary greatly in children and mentally disabled persons. Whereas adults may tolerate discomfort and remain cooperative, children and mentally disabled persons become frightened when they are hurt and will not cooperate further. Effective pain control is a prerequisite for their successful treatment. There are several ways of producing dental anaesthesia, of which local anaesthesia remains the most widely used technique. The main issue is the preparation of the child and the parents for local anaesthesia. This article offers tools for this preparation, administering and control of local anaesthesia in children and mentally disabled persons.

Use of the excimer laser in high-flow bypass surgery of the brain.

The technique of laser-assisted anastomosis for high-flow bypass surgery using the excimer laser is described in 15 rabbits and in one patient. The left common carotid artery of the rabbits was excised and, with two anastomoses, connected to the right common carotid artery. An end-to-side anastomosis technique was used that obviated the temporary occlusion of the recipient artery. The end of the donor artery was connected for its full circumference with the exterior of the recipient artery and, with the aid of an excimer laser catheter (introduced via an artificial side branch of the donor artery), the wall of the recipient artery was evaporated. In two animals only, occlusion of the anastomosis sites occurred. In the remaining 13 animals both anastomosis sites were proven to be patent by inspection at different times, followed by scanning electron microscopy in six animals. In a patient with hypoperfusion of the brain caused by bilateral internal carotid artery occlusion, revascularization of the right hemisphere was obtained by placing a shunt between the proximal superficial temporal artery and the intracranial portion of the internal carotid artery, using a free transplant of the right inferior epigastric artery. The anastomosis with the internal carotid artery was created using the excimer laser-assisted technique without occlusion of the recipient artery.

End-to-side anastomosis of small vessels using an Nd:YAG laser with a hemispherical contact probe. Technical note.

A new technique is described which enables the surgeon to perform an end-to-side anastomosis between arteries with little (less than 2 minutes) or no occlusion of the recipient artery. The technique was developed in rabbits, but has recently been successfully used in a patient in whom an anastomosis between the superficial temporal artery and a proximal branch of the middle cerebral artery was created.

Fertilization and development of eggs of the South African clawed toad, Xenopus laevis, on sounding rockets in space.

Egg rotation and centrifugation experiments strongly suggest a role for gravity in the determination of the spatial structure of amphibian embryos. Decisive experiments can only be made in Space. Eggs of Xenopus laevis, the South African clawed toad, were the first vertebrate eggs which were successfully fertilized on Sounding Rockets in Space. Unfixed, newly fertilized eggs survived reentry, and a reasonable number showed a seemingly normal gastrulation but died between gastrulation and neurulation. Only a few reached the larval stage, but these developed abnormally. In the future, we intend to test whether this abnormal morphogenesis is due to reentry perturbations, or due to a real microgravity effect, through perturbation of the reinitiation of meiosis and other processes, or started by later sperm penetration.

Scanning electron microscopy of hair cells, stereocilia and cross-linkage systems in experimentally induced endolymphatic hydrops.

In this study scanning electron microscopy was used to examine the stereocilia and their cross-linkages in guinea pig cochleas 1, 2, 4 and 8 months after endolymphatic sac obliteration. Initial changes were restricted to the stereocilia of the outer hair cells and consisted of a disarrangement and bulging of the stereocilia with an interruption of their cross-linkage systems. Subsequently, the stereocilia became fused and atrophied. Cross-linkages of inner hair cells remained intact. Loss of both outer and inner hair cells started in the apex and progressed towards the base of the cochlea. These findings indicate that early changes in the micro-architecture of the stereocilia may have a mechanical origin, with pressure fluctuations in the scala media possibly playing an important role.

Fertilization of frog eggs on a Sounding Rocket in space.

During the TEXUS-17 flight (April/May 1988) eggs of a higher organism, the anuran amphibian Xenopus laevis, have for the first time been successfully fertilized under microgravity on a Sounding Rocket. This result also implies that Life Sciences Experiments of Short Duration can be carried out on Sounding Rockets. The latter can therefore function as additional carriers for such experiments. Histological sections of the experimental material demonstrated the penetration of sperm into eggs, while SEM analysis revealed the differentiation of characteristic egg surface structures. Our TEXUS-17 experiment convincingly shows that the modified automatic experiment container, originally designed for experimental BR 52NL on the D1-mission, now functions flawlessly. Eight containers were flown in an airtight, well-isolated box (TEM 06-15), and a similar set was activated on Earth, two hours later. The analysis of the biological material is in progress.

New type of end-to-side anastomosis for small arteries: a technical and scanning electron microscopic study in rats.

A new type of end-to-side anastomosis between small arteries is described. The anastomosis is made for three-quarters of its circumference with a nonoccluded recipient artery. The recipient artery must be occluded for only 3 to 5 minutes to complete the anastomosis. The anastomosis site was studied with the scanning electron microscope at different times (1 hour to 3 months) after the operation in 20 rats. In every case, the anastomosis was widely patent and a gradual endothelialization of the anastomosis site was observed. After 7 to 10 days, the endothelialization was more or less complete and, during the following weeks, a further smoothing of the ridge protruding into the anastomosis was noted. As an addendum, the authors describe an improvement of this technique that was developed after the sapphire tip of the neodymium: YAG laser became available. Now the anastomosis is fully completed and the connection between the two vessels is made by the laser tip introduced via a side branch of the donor vessel. The recipient artery need not be occluded at all. The results are promising.

Abnormal transbilayer mobility of phosphatidylcholine in hereditary pyropoikilocytosis reflects the increased heat sensitivity of the membrane skeleton.

We determined whether the membrane defect in hereditary pyropoikilocytosis (HPP) is associated with thermally induced changes in the lipid bilayer, the stability of which was probed by the rate of translocation of phosphatidylcholine (PC) over the two leaflets. [14C]PC was incorporated into the outer leaflet of the lipid bilayer of the intact erythrocytes using a PC-specific phospholipid exchange protein. The transbilayer equilibration of this PC was determined by measuring the time-dependent changes in its accessibility to exogenous phospholipase A2. The rate of transbilayer equilibration of PC was increased in HPP cells at 37 degrees C when compared to normal erythrocytes (rate constants, 0.07 +/- 0.02 and 0.03 +/- 0.01 h-1, respectively). A further dramatic increase in PC transbilayer equilibration was noted in HPP cells incubated at 44 degrees C (rate constant, 0.15 +/- 0.02 h-1). A similar marked acceleration in transbilayer movement of PC was also seen in normal erythrocytes when incubated at 46 degrees C (rate constant, 0.13 +/- 0.03 h-1). Despite the enhanced transbilayer mobility of PC in HPP cells when compared to normal erythrocytes, no major alteration in the asymmetric distribution could be observed when probed with phospholipase A2. Since changes in transbilayer mobility of PC and cell morphology occur in HPP cells at lower temperature than in normal red cells, it may be concluded that the enhanced thermal sensitivity of spectrin is the major factor responsible for these changes. Our results therefore support the view that the structural integrity of the skeletal network is essential for stabilization of the lipid bilayer of the red cell membrane.

Shape changes in human erythrocytes induced by replacement of the native phosphatidylcholine with species containing various fatty acids.

Phosphatidylcholine-specific transfer protein from beef liver has been used to replace native phosphatidylcholine (PC) molecules from intact human erythrocytes by a variety of PC species differing in fatty acid composition. These replacements changed neither the total phospholipid content of the membrane, nor the composition of this fraction in terms of the various phospholipid classes. The morphology of the erythrocyte was not modified when native PC was replaced by 1-palmitoyl,2-oleoyl PC, 1-palmitoyl,2-linoleoyl PC, egg PC, or PC isolated from rat liver microsomes. Replacement with the disaturated species 1,2-dimyristoyl PC, 1,2-dipalmitoyl PC, and 1,2-distearoyl PC resulted in the formation of echinocytes and, at higher levels of replacement, in spheroechinocytes. Echinocyte-like erythrocytes were also observed after replacement with 1-palmitoyl,2-arachidonoyl PC, whereas stomatocytes were formed upon replacement with PC species containing two unsaturated fatty acids, e.g., 1,2-dioleoyl PC and 1,2-dilinoleoyl PC. The observations show that the erythrocyte membrane structure and the overall discoid cell shape of the human erythrocyte are optimally stabilized by PC species that contain one saturated and one mono- or diunsaturated fatty acid, and that the cell tolerates only limited variations in the species composition of its PC.

Comparative studies of heat sensitivity of several rat hepatoma cell lines and hepatocytes in primary culture.

Cell survival, cell morphology, rate of protein synthesis, and recovery of protein synthesis have been investigated in two continuously growing rat hepatoma cell lines, namely, Reuber H35 and HTC, and rat hepatocytes in primary culture after exposure at temperatures between 37 and 44 degrees. H35 and HTC cells heated under identical culture conditions and at similar cell densities showed a remarkable difference in thermal sensitivity. For the continuously growing cell lines, the thermosensitivity of survival is reflected by thermally induced morphological alterations and the rate of recovery of protein synthesis but not by the thermally induced initial changes in protein synthesis. With the use of time-lapse cinematography, application of morphological criteria, and determination of the recovery of protein synthesis, the relative heat sensitivity of hepatocytes was evaluated as less than that of H35 cells but comparable to that of HTC cells. Therefore, it appears that hepatoma cell lines originating from the same tissue possess different heat sensitivities.

The morphology of cultured melanophores from tadpoles of Xenopus laevis: scanning electron microscopical observations.

Tail-fin melanophores of tadpoles of Xenopus laevis (Daudin) in primary culture were examined scanning electron microscopically in the aggregated and in the dispersed state. After isolation, the melanophores are spherical, but within 24 h they develop thin filopodia for attachment to the substratum. Subsequently, cylinder-like as well as flat sheet-like processes are formed, which adhere to the substratum with terminal pseudopodia and filopodia. The processes of adjacent melanophores contact each other, thus forming an interconnecting network between the melanophores. In the aggregated state the central part of the melanophore is spherical and voluminous. Both the central part and the processes bear microvilli. In melanophores with dispersed melanosomes the central part is much flatter; the distal parts have a thickness that equals a monolayer of melanosomes. The surface of the cell bears only scarce microvilli. These features indicate that melanophores do not have a fixed shape and that pigment migration is accompanied by reciprocal volume transformation between the cell body and its processes.

Re-endothelialization of microvascular carotid end-to-side anastomosis in the rat.

A carotid end-to-side anastomosis was performed on 25 male and female Wistar rats (mean weight 197.8 gm). The animals were sacrificed at time intervals varying from 0 to 21 days after the operation. The anastomosis was exposed, the aorta cannulated, and the animals perfused with a 2.5% buffered glutaraldehyde solution at a constant pressure of 80 mm Hg. The anastomoses were removed for scanning electron microscopic (SEM) and light microscopic studies. The SEM results indicate that after the acute platelet-fibrin reaction in the first 48 hours, the suture line itself becomes re-endothelialized after 4 days. On the stitches, however, a cellular population consisting of leukocytes transforming into flattened cells was seen after 2 days. The morphology of these cells and their role in the regeneration of endothelium is discussed. This study presents evidence supporting a blood-borne genesis of endothelial cells in vivo.