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ω-Transaminases (ω-TAs) catalyze the conversion of ketones to chiral amines, often with high enantioselectivity and specificity, which makes them attractive for industrial production of chiral amines. Tailoring ω-TAs to accept non-natural substrates is necessary because of their limited substrate range. We present a computational protocol for predicting the enantioselectivity and catalytic selectivity of an ω-TA from Vibrio fluvialiswith different substrates and benchmark it against 62 compounds gathered from the literature. Rosetta-generated complexes containing an external aldimine intermediate of the transamination reaction are used as starting conformations for multiple short independent molecular dynamics (MD) simulations. The combination of molecular docking and MD simulations ensures sufficient and accurate sampling of the relevant conformational space. Based on the frequency of near-attack conformations observed during the MD trajectories, enantioselectivities can be quantitatively predicted. The predicted enantioselectivities are in agreement with a benchmark dataset of experimentally determined ee% values. The substrate-range predictions can be based on the docking score of the external aldimine intermediate. The low computational cost required to run the presented framework makes it feasible for use in enzyme design to screen thousands of enzyme variants.

Xylose isomerase from Piromycessp. E2 (PirXI) can be used to equip Saccharomyces cerevisiaewith the capacity to ferment xylose to ethanol. The biochemical properties and structure of the enzyme have not been described even though its metal content, catalytic parameters, and expression level are critical for rapid xylose utilization. We have isolated the enzyme after high-level expression in Escherichia coli, analyzed the metal dependence of its catalytic properties, and determined 12 crystal structures in the presence of different metals, substrates, and substrate analogues. The activity assays revealed that various bivalent metals can activate PirXI for xylose isomerization. Among these metals, Mn2+is the most favorable for catalytic activity. Furthermore, the enzyme shows the highest affinity for Mn2+, which was established by measuring the activation constants (Kact) for different metals. Metal analysis of the purified enzyme showed that in vivothe enzyme binds a mixture of metals that is determined by metal availability as well as affinity, indicating that the native metal composition can influence activity. The crystal structures show the presence of an active site similar to that of other xylose isomerases, with a d-xylose binding site containing two tryptophans and a catalytic histidine, as well as two metal binding sites that are formed by carboxylate groups of conserved aspartates and glutamates. The binding positions and conformations of the metal-coordinating residues varied slightly for different metals, which is hypothesized to contribute to the observed metal dependence of the isomerase activity.

Deciphering the structural features that functionally separate ammonia lyases from aminomutases is of interest because it may allow for the engineering of more efficient aminomutases for the synthesis of unnatural amino acids (e.g., β-amino acids). However, this has proved to be a major challenge that involves understanding the factors that influence their activity and regioselectivity differences. Herein, we report evidence of a structural determinant that dictates the activity differences between a phenylalanine ammonia lyase (PAL) and aminomutase (PAM). An inner loop region that closes the active sites of both PAM and PAL was mutated within PAM (PAM residues 77–97) in a stepwise approach to study the effects when the equivalent residue(s) found in the PAL loop were introduced into the PAM loop. Almost all of the single loop mutations triggered a lyase phenotype in PAM. Experimental and computational evidence suggest that the induced lyase features result from inner loop mobility enhancements, which are possibly caused by a 310-helix cluster, flanking α-helices, and hydrophobic interactions. These findings pinpoint the inner loop as a structural determinant of the lyase and mutase activities of PAM.

Welcome to the Careers with Pharma Top 50 Companies: Focus on Diversity recruitment supplement. This 2003 edition features Pharmaceutical Executive's 'Top 50' companies and the opportunities available at the largest [...]

The purpose of this case report is to describe the use of mobilization and eccentric exercise training for a patient with ankle pain and a history of chronic ankle sprains and discuss the course of diagnostic decision making when the patient did not respond to care.

Turn to switch: A mutant of phenylalanine aminomutase was engineered that can catalyze the regioselective amination of cinnamate derivatives (see scheme, red) to, for example, β‐amino acids. This regioselectivity, along with the X‐ray crystal structures, suggests two distinct carboxylate binding modes differentiated by CβCipsobond rotation, which determines if β‐ (see scheme) or α‐addition takes place.

Simple and efficient: The combination of an enzymatic, enantioinverting reaction with simple follow‐up processes allows the transformation of readily available racemic compounds into versatile chiral α‐substituted amides (see picture; Ms=methanesulfonyl). These important building blocks are prepared in high overall yield and enantiomeric excess; the elimination of intermediate purification steps results in a time‐ and cost‐efficient process.

Haloalkane dehalogenases from five sources were heterologously expressed in Escherichia coli, isolated, and tested for their ability to achieve kinetic resolution of racemic α‐bromoamides, which are important intermediates used in the preparation of bioactive compounds. To explore the substrate scope, fourteen α‐bromoamides, with different Cα‐ and N‐substituents, were synthesized. Catalytic activity towards eight substrates was found, and for five of these compounds the conversion proceeded with a high enantioselectivity (E value >200). In all cases, the (R)‐α‐bromoamide is the preferred substrate. Conversions on a preparative scale with a catalytic amount of enzyme (enzyme:substrate ratio less 1:50 w/w) were all completed within 17–46 h and optically pure α‐bromoamides and α‐hydroxyamides were isolated with good yields (31–50%). Substrate docking followed by molecular dynamics simulations indicated that the high enantioselectivity results from differences in the percentage of the time in which the substrate enantiomers are bound favourably for catalysis. For the preferred (R)‐substrates, the angle between the attacking aspartate oxygen atom of the enzyme, the attacked carbon atom of the substrate, and the displaced halogen atom, is more often in the optimal range (>157°) for reactivity. This can explain the observed enantioselectivity of LinB dehalogenase in a kinetic resolution experiment.

The multistep catalytic process using designer cells, either added as freshly prepared suspensions or as stable lyophilized powder, and click reaction can be performed in one pot. The sequence of four reactions allows the production of both enantiomers of β‐hydroxytriazoles with high enantiomeric excess.

Gezielte Mutationender Oberflächenschleife von Halogenalkan‐Dehalogenasen haben Auswirkungen auf die Enantiodiskriminierung. Die Temperaturabhängigkeit der Enantioselektivität (siehe lnE‐1/T‐Kurve) von β‐Bromalkanen kehrt sich bei einer Deletion der Oberflächenschleife um (rote Datenpunkte), was bei Einführung einer zusätzlichen Einzelpunktmutation wieder rückgängig gemacht wird; bei α‐Bromestern findet keine Änderung statt.

The gene encoding aspartate ammonia lyase (aspB) from Bacillussp. YM55‐1 has been cloned and overexpressed, and the recombinant enzyme containing a C‐terminal His6tag has been purified to homogeneity and subjected to kinetic characterization. Kinetic studies have shown that the His6tag does not affect AspB activity. The enzyme processes L‐aspartic acid, but not D‐aspartic acid, with a Kmof ≈15 mMand a kcatof ≈40 s−1. By using this recombinant enzyme in the reverse reaction, a set of four N‐substituted aspartic acids were prepared by the Michael addition of hydroxylamine, hydrazine, methoxylamine, and methylamine to fumarate. Both hydroxylamine and hydrazine were found to be excellent substrates for AspB. The kcatvalues are comparable to those observed for the AspB‐catalyzed addition of ammonia to fumarate (≈90 s−1), whereas the Kmvalues are only slightly higher. The products of the enzyme‐catalyzed addition of hydrazine, methoxylamine, and methylamine to fumarate were isolated and characterized by NMR spectroscopy and HPLC analysis, which revealed that AspB catalyzes all the additions with excellent enantioselectivity (>97 % ee). Its broad nucleophile specificity and high catalytic activity make AspB an attractive enzyme for the enantioselective synthesis of N‐substituted aspartic acids, which are interesting building blocks for peptide and pharmaceutical synthesis as well as for peptidomimetics.

Zusammenfassung: Einleitung: Ein Element im Konzept des Qualitätsmanagements der Philipps-Universität Marburg ist die Implementierung evidenzbasierter Leitlinien über Behandlungspfade. Die Entwicklung von Behandlungspfaden wird für die Aspekte Suizidalität und Suizidversuch dargestellt.Methodik: Auf der Basis einer Leitlinien- und Literaturrecherche wurde die optimale Versorgungsqualität für den jeweiligen Aspekt als Soll-Zustand definiert. Über die Analyse des Behandlungsablaufs und die Auswertung einer Mitarbeiterbefragung wurde der Ist-Zustand beschrieben. Ein problemorientierter Ist-Soll-Vergleich bildete die Grundlage der Behandlungspfade. Im Konsens aller an der Versorgung Beteiligten und unter Berücksichtigung lokaler Ressourcen und Barrieren wurden die wichtigsten Entscheidungsebenen und Interventionen als Ablauf am einzelnen Patienten dargestellt.Ergebnisse: Das Fehlen konkreter Handlungsempfehlungen in den Leitlinien für den vollständigen Versorgungsablauf erforderte eine prozessspezifische, lokal adaptierte Ausarbeitung. Optimierungspotenziale wurden für die Bereiche Verlegungen und Konsiliaruntersuchungen, Information und Koordination im Team sowie Diagnostik der Suizidalität identifiziert und als Schwerpunkte der Behandlungspfade adressiert. Die Pfade beinhalten den fallbezogenen klinischen Algorithmus und Checklisten mit Zuweisung personeller Verantwortlichkeiten.Schlussfolgerungen: Die Entwicklung von Behandlungspfaden als Teil des Qualitätsmanagements und der transsektoral integrierten Versorgung in Psychiatrie und Psychotherapie ist machbar. Das vorgestellte Konzept eröffnet Potenziale im Hinblick auf eine Steigerung von Transparenz, Effektivität und Effizienz der Versorgung sowie Zufriedenheit der Beteiligten. Es bietet psychiatrischen Kliniken die Chance, eine optimale Versorgungsqualität bei zunehmendem Kostendruck und Leistungsverdichtung zu gewährleisten.

Contrast-enhanced MR angiography (CE-MRA) using a combined head and neck coil permits non-invasive imaging of the vasculature from the aortic arch through to the Circle of Willis in less than 2 minutes.

A semi-random mutagenesis approach was followed to increase the performance of penicillin acylase PAS2 in the kinetically controlled synthesis of ampicillin from 6-aminopenicillanic acid (6-APA) and activated d-phenylglycine derivatives. We directed changes in amino acid residues to positions close to the active site that are expected to affect the catalytic performance of penicillin acylase: αR160, αF161 and βF24. From the resulting triple mutant gene bank, six improved PAS2 mutants were recovered by screening only 700 active mutants with an HPLC-based screening method. A detailed kinetic analysis of the three most promising mutants, T23, TM33 and TM38, is presented. These mutants allowed the accumulation of ampicillin at 4–5 times higher concentrations than the wild-type enzyme, using d-phenylglycine methyl ester as the acyl donor. At the same time, the loss of activated acyl donor due to the competitive hydrolytic side reactions could be reduced to <20% with the mutant enzymes compared >80% wild-type PAS2. Although catalytic activity dropped by a factor of 5–10, the enhanced synthetic performance of the recovered penicillin acylase variants makes them interesting biocatalysts for the production of β-lactam antibiotics.

Penicillin acylase catalyses the condensation of Cα-substituted phenylacetic acids with β-lactam nucleophiles, producing semi-synthetic β-lactam antibiotics. For efficient synthesis a low affinity for phenylacetic acid and a high affinity for Cα-substituted phenylacetic acid derivatives is desirable. We made three active site mutants, αF146Y, βF24A and αF146Y/βF24A, which all had a 2- to 10-fold higher affinity for Cα-substituted compounds than wild-type enzyme. In addition, βF24A had a 20-fold reduced affinity for phenylacetic acid. The molecular basis of the improved properties was investigated by X-ray crystallography. These studies showed that the higher affinity of αF146Y for (R)-α-methylphenylacetic acid can be explained by van der Waals interactions between αY146:OH and the Cα-substituent. The βF24A mutation causes an opening of the phenylacetic acid binding site. Only (R)-α-methylphenylacetic acid, but not phenylacetic acid, induces a conformation with the ligand tightly bound, explaining the weak binding of phenylacetic acid. A comparison of the βF24A structure with other open conformations of penicillin acylase showed that βF24 has a fixed position, whereas αF146 acts as a flexible lid on the binding site and reorients its position to achieve optimal substrate binding.

Baeyer–Villiger monooxygenases (BVMOs) are flavoenzymes that catalyze a remarkably wide variety of oxidative reactions such as regio- and enantioselective Baeyer–Villiger oxidations and sulfoxidations. Several of these conversions are difficult to achieve using chemical approaches. Due to their selectivity and catalytic efficiency, BVMOs are highly valuable biocatalysts for the synthesis of a broad range of fine chemicals. For a long time, only one member of this class of flavin-containing biocatalysts had been cloned and overexpressed which has limited their application for synthetic processes. Recently a number of new genes that encode BVMOs have been sequenced and overexpressed. In this paper the biocatalytic properties of recently cloned BVMOs are reviewed. Furthermore, the potential for obtaining novel BVMOs from sequenced genomes will be discussed.

Optically pure epoxides are useful synthons for a variety of biologically active compounds. The epoxide hydrolase obtained from Agrobacterium radiobacter AD1 hydrolyses racemic aryl epoxides with moderate and aliphatic epoxides with low enantioselectivity. The three-dimensional structure of this enzyme indicates that two tyrosine residues interact with the epoxide oxygen. Mutating one of these, tyrosine 215, to a phenylalanine (Y215F) resulted in an enzyme with increased enantioselectivity towards aryl epoxides. The relatively strong decrease in activity towards the remaining enantiomers makes this enzyme a much better biocatalyst than the wild-type enzyme for the preparation of optically pure (S)-styrene oxide derivatives.

The alpha-amino acid ester hydrolase from Acetobacter turbidans ATCC 9325 is capable of hydrolyzing and synthesizing the side chain peptide bond in beta-lactam antibiotics. Data base searches revealed that the enzyme contains an active site serine consensus sequence Gly-X-Ser-Tyr-X-Gly that is also found in X-prolyl dipeptidyl aminopeptidase. The serine hydrolase inhibitor p-nitrophenyl-p'-guanidino-benzoate appeared to be an active site titrant and was used to label the alpha-amino acid ester hydrolase. Electrospray mass spectrometry and tandem mass spectrometry analysis of peptides from a CNBr digest of the labeled protein showed that Ser(205), situated in the consensus sequence, becomes covalently modified by reaction with the inhibitor. Extended sequence analysis showed alignment of this Ser(205) with the catalytic nucleophile of some alpha/beta-hydrolase fold enzymes, which posses a catalytic triad composed of a nucleophile, an acid, and a base. Based on the alignments, 10 amino acids were selected for site-directed mutagenesis (Arg(85), Asp(86), Tyr(143), Ser(156), Ser(205), Tyr(206), Asp(338), His(370), Asp(509), and His(610)). Mutation of Ser(205), Asp(338,) or His(370) to an alanine almost fully inactivated the enzyme, whereas mutation of the other residues did not seriously affect the enzyme activity. Circular dichroism measurements showed that the inactivation was not caused by drastic changes in the tertiary structure. Therefore, we conclude that the catalytic domain of the alpha-amino acid ester hydrolase has an alpha/beta-hydrolase fold structure with a catalytic triad of Ser(205), Asp(338), and His(370). This distinguishes the alpha-amino acid ester hydrolase from the Ntn-hydrolase family of beta-lactam antibiotic acylases.

The active site of penicillin acylase of Escherichia coli contains two conserved arginine residues. The function of these arginines, αArg145 and βArg263, was studied by site-directed mutagenesis and kinetic analysis of the mutant enzymes. The mutants αArg145→Leu (αArg145Leu), αArg145Cys and αArg145Lys were normally processed and exported to the periplasm, whereas expression of the mutants βArg263Leu, βArg263Asn and βArg263Lys yielded large amounts of precursor protein in the periplasm, indicating that βArg263 is crucial for efficient processing of the enzyme. Either modification of both arginine residues by 2,3-butanedione or replacement by site-directed mutagenesis yielded enzymes with a decreased specificity (kcat/Km) for 2-nitro-5-[(phenylacetyl)amino]benzoic acid, indicating that both residues are important in catalysis. Compared with the wild type, the αArg145 mutants exhibited a 3–6-fold-increased preference for 6-aminopenicillanic acid as the deacylating nucleophile compared with water. Analysis of the steady-state parameters of these mutants for the hydrolysis of penicillin G and phenylacetamide indicated that destabilization of the Michaelis—Menten complex accounts for the improved activity with β-lactam substrates. Analysis of pH—activity profiles of wild-type enzyme and the βArg263Lys mutant showed that βArg263 has to be positively charged for catalysis, but is not involved in substrate binding. The results provide an insight into the catalytic mechanism of penicillin acylase, in which αArg145 is involved in binding of β-lactam substrates and βArg263 is important both for stabilizing the transition state in the reaction and for correct processing of the precursor protein.

Haloalkane dehalogenase (DhlA) was used as a model protein to explore the possibility to use molecular dynamics (MD) simulations as a tool to identify flexible regions in proteins that can serve as a target for stability enhancement by introduction of a disulfide bond. DhlA consists of two domains: an α/β-hydrolase fold main domain and a cap domain composed of five α-helices. MD simulations of DhlA showed high mobility in a helix–loop–helix region in the cap domain, involving residues 184–211. A disulfide cross-link was engineered between residue 201 of this flexible region and residue 16 of the main domain. The mutant enzyme showed substantial changes in both thermal and urea denaturation. The oxidized form of the mutant enzyme showed an increase of the apparent transition temperature from 47.5 to 52.5°C, whereas the Tm,app of the reduced mutant decreased by more than 8°C compared to the wild-type enzyme. Urea denaturation results showed a similar trend. Measurement of the kinetic stability showed that the introduction of the disulfide bond caused a decrease in activation free energy of unfolding of 0.43 kcal mol–1 compared to the wild-type enzyme and also indicated that the helix–loop–helix region was involved early in the unfolding process. The results show that MD simulations are capable of identifying mobile protein domains that can successfully be used as a target for stability enhancement by the introduction of a disulfide cross-link.

Optically active epoxides can be obtained by kinetic resolution of racemic mixtures using enantioselective epoxide hydrolases. To increase the productivity of the conversion of sparingly aqueous soluble epoxides, we investigated the use of a two-phase aqueous/organic system. A kinetic model which takes into account interphase mass transfer, enzymatic reaction, and enzyme inactivation was developed to describe epoxide conversion in the system by the epoxide hydrolase from Agrobacterium radiobacter. A Lewis cell was used to determine model parameters and results from resolutions carried out in the Lewis cell were compared to model predictions to validate the model. It was found that n-octane is a biocompatible immiscible solvent suitable for use as the organic phase. Good agreement between the model predictions and experimental data was found when the enzyme inactivation rate was fitted. Simulations showed that mass transfer limitations have to be avoided in order to maximize the yield of enantiomerically pure epoxide. Resolution of a 39 g/L solution of racemic styrene oxide in octane was successfully carried out in an emulsion batch reactor to obtain (S)-styrene oxide in high enantiomeric excess (>95% e.e.) with a yield of 30%. © 2001 John Wiley & Sons, Inc. Biotechnol Bioeng 73:44–54, 2001.

Purpose Allopurinol has beneficial effects in the systemic treatment of lens-induced uveitis and experimental autoimmune uveitis (EAU). This is believed to be due to a reduction of oxidative tissue damage through its dose-dependent free radical scavenging ability, and to an immunomodulating effect. The purpose of this study was to demonstrate the immunological effects on the IgG-antibody repertoire in EAU after topical and systemic allopurinol and steroids.Methods We assigned 43 male Lewis rats to 6 different groups: healthy rats (BASE, n=3), EAU without therapy (CTRL, n=9); systemic treatment with allopurinol (ALSYS, n=9, 50 mg/kg body wt.i.v., given every three days for two weeks), topical allopurinol (ALLOC, n=6, 8 times/day), systemic methylprednisolone (STSYS, n=10, 7.5 mg/kg body wt. i.v. (Hoechst, Frankfurt, Germany)), and topical treatment with prednisolone acetate 1% (Inflanefran Forte®) (STLOC, n=6). Sera were tested against Western blots (WB) of SDS-PAGE (sodium-dodecyl-sulfate polyacrylamide-gelelectorphoresis) of retinal proteins. Based on digital image analysis, discriminance was analysed.Results The analysis of discriminance indicated that all therapy groups were significantly different from untreated controls (Wilks' lambda = 0.174; p<0.01). Comparing only the number of peaks and the intensities, the WB of allopurinol treated animals showed a much stronger, significant, immunomodulating effect than those treated with steroids (p>0.05), even after topical application (p<0.01).Conlcusions Allopurinol had an immunomodulating effect in EAU. Surprisingly, the topical application had more effect than systemically applied allopurinol. Allopurinol had stronger effects than systemic or topical steroids. Allopurinol appears to offer promise in the treatment of uveitis. Topical application of the drug helps to reduce possible complications such as the allopurinol hypersensitivity syndrome.

Lactobacilli have been shown to be important in the maintenance of the healthy urogenital flora. One strain, Lactobacillus fermentumRC‐14, releases surface‐active components which can inhibit adhesion of uropathogenic bacteria. Using a quantitative method for determining inhibition of adhesion, a protein with high anti‐adhesive properties against Enterococcus faecalis1131 was purified. The N‐terminal sequence of the 29‐kDa protein was identical to that of a collagen‐binding protein from Lactobacillus reuteriNCIB 11951, and exhibited close homology with a basic surface protein from L. fermentumBR11. The results suggest that this anti‐adhesive cell surface protein of Lactobacilluscould protect against uropathogens by preventing their adhesion.

Two different molecular dynamics-based models are compared with respect to their ability to predict the number and the distribution of trapping sites of a molecule in a rare-gas matrix. The two approaches are applied to the same problem:  anthracene molecules trapped in an argon matrix. Both methods give a small number of trapping sites with similar structures, but the distributions of sites in each model are different. In all stable sites, the molecule was found to lie on either the {001} or the {111} plane of the crystalline argon. We propose a structure for the most stable site in which anthracene lies in the 6 substitutional site in the {001} plane.

A metabolic model describing growth of Methylosinus trichosporiumOB3b and cometabolic contaminant conversion is used to optimize trichloroethene (TCE) conversion in a bioreactor system. Different process configurations are compared: a growing culture and a nongrowing culture to which TCE is added at both constant and pulsed levels. The growth part of the model, presented in the preceding article, gives a detailed description of the NADH regeneration required for continued TCE conversion. It is based on the metabolic pathways, includes Michaelis‐Menten type enzyme kinetics, and uses NADH as an integrating and controlling factor. Here the model is extended to include TCE transformation, incorporating the kinetics of contaminant conversion, the related NADH consumption, toxic effects, and competitive inhibition between TCE and methane. The model realistically describes the experimentally observed negative effects of the TCE conversion products, both on soluble methane monooxygenase through the explicit incorporation of the activity of this enzyme and on cell viability through the distinction between dividing and nondividing cells. In growth‐based systems, the toxicity of the TCE conversion products causes rapid cell death, which leads to wash‐out of suspended cultures at low TCE loads (below μM inlet concentrations). Enzyme activity, which is less sensitive, is hardly affected by the toxicity of the TCE conversion products and ensures high conversions (>95%) up to the point of wash‐out. Pulsed addition of TCE (0.014–0.048 mM) leads to a complete loss of viability. However, the remaining enzyme activity can still almost completely convert the subsequently added large TCE pulses (0.33–0.64 mM). This emphasizes the inefficient use of enzyme activity in growth‐based systems. A comparison of growth‐based and similar non‐growth‐based systems reveals that the highest TCE conversions per amount of cells grown can be obtained in the latter. Using small amounts of methane (negligible compared to the amount needed to grow the cells), NADH limitation in the second step of this two‐step system can be eliminated. This results in complete utilization of enzyme activity and thus in a very effective treatment system.

A biochemical model is presented that describes growth of Methylosinus trichosporiumOB3b on methane. The model, which was developed to compare strategies to alleviate NADH limitation resulting from cometabolic contaminant conversion, includes (1) catabolism of methane via methanol, formaldehyde, and formate to carbon dioxide; (2) growth as formaldehyde assimilation; and (3) storage material (poly‐β‐hydroxybutyric acid, PHB) metabolism. To integrate the three processes, the cofactor NADH is used as central intermediate and controlling factor­instead of the commonly applied energy carrier ATP. This way a stable and well‐regulated growth model is obtained that gives a realistic description of a variety of steady‐state and transient‐state experimental data. An analysis of the cells' physiological properties is given to illustrate the applicability of the model. Steady‐state model calculations showed that in strain OB3b flux control is located primarily at the first enzyme of the metabolic pathway. Since no adaptation in VMAXvalues is necessary to describe growth at different dilution rates, the organism seems to have a “rigid enzyme system”, the activity of which is not regulated in response to continued growth at low rates. During transient periods of excess carbon and energy source availability, PHB is found to accumulate, serving as a sink for transiently available excess reducing power.

Zusammenfassung: Der Antimetabolit Mitomycin C wird bei Trabekulektomien mit erhöhtem Vernarbungsrisiko intraoperativ appliziert. Als Risikofaktoren für eine Sickerkissenvernarbung dürfen ein Patientenalter unter 40 Jahren, vorangegangene Entzündungen, eine durch Voroperationen oder durch langjährige antiglaukomatöse Therapie beeinträchtigte Bindehaut und eine starke Pigmentierung angesehen werden. Es wurde der postoperative Verlauf von 54 konsekutiv mittels Trabekulektomie mit Mitomycin C (MMC) operierten Augen von 50 Patienten mit erhöhtem Vernarbungsrisiko über durchschnittlich 49,6 Wochen (24 bis 185 Wochen) erfasst und ausgewertet. Das durchschnittliche Alter der Patienten betrug 52,1 Jahre (2 bis 93 Jahre). Vor der Trabekulektomie waren im Mittel 1,6 Operationen und 0,8 Argonlaser-Trabekuloplastiken vorgenommen worden. Präoperativ wurden im Mittel 2,9 Antiglaukomatosa (Wirkstoffgruppen) verwendet. Der präoperative Augeninnendruck (IOD) betrug 29,3 ± 8,2 mmHg mit Therapie und 36,0 ± 10,0 mmHg ohne Therapie. Bei jedem Auge wurde eine Trabekulektomie mit limbusbasalem Bindehautlappen und 5-minütiger Applikation von 0,1 ml MMC der Konzentration 0,2 mg/ml vorgenommen. Die Nachkontrollen erfolgten nach 1, 3, 6, 12 und 24 Wochen sowie 12 und 18 Monate postoperativ. Der mittlere IOD nach 24 Wochen betrug 14,3 ± 5,6 mmHg. Bei 39% der Augen kam es zu passageren Hypotonien mit Ausbildung einer serösen Aderhautschwellung, in einem Fall zu einer chorioidalen Hämorrhagie. Längere Hypotoniephasen von mehr als 6 Monate wurden bei zwei Augen beobachtet. Hornhautkomplikationen waren selten und bildeten sich in allen Fällen nach entsprechender Therapie zurück. Bei 34 Augen waren postoperativ weitere Eingriffe wie z. B. Fadendurchtrennung, Needling, Revisions-Trabekulektomie oder Sickerkissenübernähung erforderlich. 16 Augen bedurften einer medikamentösen IOD-Senkung. Nur bei 10 Augen (19%) war der IOD innerhalb der ersten sechs Monate postoperativ ohne weitere operative oder medikamentöse Maßnahmen reguliert. Schlussfolgerung: Die Trabekulektomie mit intraoperativer Gabe von MMC führte in unserem Kollektiv von Augen mit hohen Vernarbungsrisiko zu einer guten Senkung des Augeninnendrucks. Allerdings erwiesen sich regelmäßige postoperative Kontrollen als erforderlich, um den Behandlungserfolg zu sichern. Lokale toxische Komplikationen durch Mitomycin C traten selten auf. Summary: The antifibrotic agent mitomycin C is used as an adjunct in trabeculectomies with poor surgical prognosis. Risk factors for scrarring of the filtering bleb are age under 40 years, previous inflammations, distinct pigmentation and conjunctival scarring due to previous operations or prolonged antiglaucomatous therapy. 54 eyes of 50 patients were examined for a mean follow-up period of 49.6 weeks (24 to 185 weeks). Mean age of the patients was 52.1 years (range 2–93 years). Trabeculectomy was preceded by 1.6 operations and 0.8 argon laser trabeculoplasties. The patients had a mean intake of 2.9 antiglaucomatous drugs. Before surgery, the intraocular pressure with and without medication was 29.3 ± 8.2 mmHg and 36.0 ± 10.0 mmHg, respectively. Trabeculectomies were performed using a limbus-based conjunctival flap. A volume of 0.1 ml mitomycin C (concentration: 0.2 mg/ml) was applied for 5 minutes prior to entering the eye. Postoperatively, the course was examined after 1, 3, 6, 12, 24 weeks and 12 as well as 18 months. Mean intraocular pressure after 24 weeks was 14.3 ± 5.6 mmHg. In 39% of the eyes a transient hypotony with choroidal detachment occurred, in one eye a hemorrhagic choroidal detachment was observed. Prolonged hypotonies of more than 6 months were seen in two eyes. Corneal epithelial defects were rare. In 34 eyes further surgical treatment was required, including suture lysis, needling, revision of trabeculectomy, surgical revision after leakage or cataract extraction. In 16 eyes, antiglaumatous drugs had to be applied. In only 10 eyes (19%) no further treatment, neither medical nor surgical, was necessary. In our group of glaucoma patients with poor surgical prognosis trabeculectomy with intraoperative application of mitomycin C induced a good lowering of the IOP. Nevertheless, regular postoperative controls were necessary. Complications due to localized tissue toxicity were rare.

In this study, 133 patients with incapacitating low back pain underwent temporary external transpedicular fixation of the lumbosacral spine in a prospective trial. Of these patients, 67 had undergone one or more spinal procedures in the past. On the basis of temporary external transpedicular fixation, 55 of 133 patients were selected for spinal fusion and the other 78 patients were treated conservatively. With an average followup period of 37 months, the clinical results were analyzed.

Determination of kinetic parameters of penicillin acylases for phenylacetylated compounds is complicated due to the low Kmvalues for these substrates, the lack of a spectroscopic signal, and the strong product inhibition by phenylacetic acid. To overcome these difficulties, a spectrophotometric method was developed, with which kinetic parameters could be determined by measuring the effects on the hydrolysis of the chromogenic reference substrate 2-nitro-5-[(phenylacetyl)amino]benzoic acid (NIPAB). To that end, spectrophotometric progress curves with NIPAB in the absence and presence of the phenylacetylated substrates and their products were measured and analyzed by numerical fitting to the appropriate equations for competing substrates with product inhibition. This analysis yielded kinetic constants for phenylacetylated substrates such as penicillin G, which are in close agreement with those obtained in independent initial velocity experiments. Using NIPAB analogs with lower kcat/Kmvalues, kinetic parameters for the hydrolysis of cephalexin and penicillin V were determined. This method was suitable for determining the kinetic constants of penicillin acylases in periplasmic extracts from Escherichia coli, Alcaligenes faecalis,and Kluyvera citrophila.The use of chromogenic reference substrates thus appears to be a rapid and reliable method for determining kinetic constants with various substrates and enzymes.

Epoxide hydrolases catalyze the cofactor-independent hydrolysis of reactive and toxic epoxides. They play an essential role in the detoxification of various xenobiotics in higher organisms and in the bacterial degradation of several environmental pollutants. The first x-ray structure of one of these, from Agrobacterium radiobacterAD1, has been determined by isomorphous replacement at 2.1-Å resolution. The enzyme shows a two-domain structure with the core having the α/β hydrolase-fold topology. The catalytic residues, Asp107and His275, are located in a predominantly hydrophobic environment between the two domains. A tunnel connects the back of the active-site cavity with the surface of the enzyme and provides access to the active site for the catalytic water molecule, which in the crystal structure, has been found at hydrogen bond distance to His275. Because of a crystallographic contact, the active site has become accessible for the Gln134side chain, which occupies a position mimicking a bound substrate. The structure suggests Tyr152/Tyr215as the residues involved in substrate binding, stabilization of the transition state, and possibly protonation of the epoxide oxygen.

Studies the catabolic route of 1,2-dichloroethane as an example of a pathway for the conversion of chlorinated xenobiotic compounds. Isolation of Xanthobacter and Ancylobacter; Catalysis by a hydrolytic haloalkane dehalogenase.

Discusses motivations and methods for the translation of uncertainty representation formats between knowledge bases in different expert system building tools (ESBTs). Knowledge Canonical Form; Methods for translation; Major benefits from the research; Brief survey on some commercial and some characteristics of their uncertainty management system (UMSs).

Methods of studing programmer productivity are difficult to find. The classical methods of observation and statistical analysis are in many cases inappropriate. This paper describes a simulation approach in which programmers are' considered to be individual processors. Relative group productivity is then measured based upon the productivity levels and communications relationships of the processors.

This study was conducted to assess the effect of a plaster cast on the mobility of the lumbosacral joint in 10 patients with chronic low back pain. During static and dynamic exercises, movements between the proximal vertebra (L4 or L5) and the sacrum were registered in 10 patients without a support and after the application of a plaster cast, and with and without unilateral hip immobilization, respectively.

A strain of Pseudomonas putida was isolated that was able to degrade 2-chloroethanol. The degradation proceeded via 2-chloroacetaldehyde and chloroacetate to glycolate. In crude extracts the enzymes for this degradation pathway could be detected. All enzymes proved to be inducible. The dehalogenase that catalyzed the dehalogenation of chloroacetate to glycolate was further characterized. It consisted of a single polypeptide chain with a molecular mass of 28 kDa. After induction the dehalogenase was expressed at a high level. In a mutant resistant to high concentrations of 2-chloroethanol the dehalogenase was no longer expressed. The mechanism of resistance seemed to be due to the inability to convert chloroacetate and export of this compound out of the cell.

In Pseudomonas aeruginosa the formation of urease, histidase and some other enzymes involved in nitrogen assimilation is repressed by ammonia in the growth medium. The key metabolite in this process appears to be glutamine or a product derived from it, since ammonia and glutamate did not repress urease and histidase synthesis in a mutant lacking glutamine synthetase activity when growth was limited for glutamine. The synthesis of these enzymes was repressed in cells growing in the presence of excess glutamine. High levels of glutamine were also required for the derepression of NADP-dependent glutamate dehydrogenase formation in the glutamine synthetase-negative mutant.

Glutamine synthetase from Pseudomonas aeruginosa is regulated by repression/derepression of enzyme synthesis and by adenylylation/deadenylylation control. High levels of deadenylylated biosynthetically active glutamine synthetase were observed in cultures growing with limiting amounts of nitrogen while synthesis of the enzyme was repressed and that present was adenylylated in cultures with excess nitrogen.

Aniline-Nen and aniline-Arn (n=1 and 2) complexes were synthesized in continuous and in pulsed supersonic expansions and interrogated by laser-induced fluorescence. Information on the formation kinetics and excited state energetics was obtained.