[Mental disorders after acquired CNS damage]. / Psychische Störungen nach erworbener ZNS-Schädigung.

Mental disorders are a frequent consequence of acquired central nervous damage. If not recognized and treated early, they have a negative impact on the course of neurological rehabilitation. This article deals with the diagnosis and treatment of mental disorders after acquired damage to the central nervous system.

Enzymatic Synthesis of Lignin-Based Concrete Dispersing Agents.

Lignin is the most abundant aromatic biopolymer, functioning as an integral component of woody materials. In its unmodified form it shows limited water solubility and is relatively unreactive, so biotechnological lignin valorisation for high-performance applications is greatly underexploited. Lignin can be obtained from the pulp and paper industry as a by-product. To expand its application, a new synthesis route to new dispersing agents for use as concrete additives was developed. The route is based on lignin functionalisation by enzymatic transformation. Screening of lignin-modifying systems resulted in functionalised lignin polymers with improved solubility in aqueous systems. Through grafting of sulfanilic acid or p-aminobenzoic acid by fungal laccases, lignin became soluble in water at pH≤4 or pH≤7, respectively. Products were analysed and evaluated in miniaturised application tests in cement paste and mortar. Their dispersing properties match the performance criteria of commercially available lignosulfonates. The study provides examples of new perspectives for the use of lignin.

Continuous monitoring of enzymatic reactions on surfaces by real-time flow cytometry: sortase a catalyzed protein immobilization as a case study.

Only a few techniques, such as quartz crystal microbalance and surface plasmon resonance spectroscopy, enable the analysis of dynamic processes on solid supports. Here we have developed a straightforward assay based on flow cytometry to continuously follow enzymatic reactions directly on microparticle surfaces. We applied this real-time flow cytometry (RT-FCM) approach to study the covalent immobilization of green-fluorescent protein (GFPuv) on triglycine-modified polystyrene microbeads by the transpeptidase sortase A (SrtA) from Staphylococcus aureus. Though commonly treated as functionally identical catalysts, the SrtA variants SrtAΔ59 and SrtAΔ25, in which the N-terminal amino acid residues 1-59 and 1-25 of the native enzyme are truncated, were shown to perform very differently with regard to this particular immobilization reaction. While SrtAΔ59 efficiently catalyzed the covalent attachment of GFPuv to the surface (as indicated by a linear increase of microbead fluorescence), SrtAΔ25 was essentially inactive. Besides the length of the N-terminal amino acid extension on the SrtA construct, the position of the hexahistidine tag at either the N- or C-terminus affected the efficiency of enzymatic protein immobilization. Apart from three enzyme variants containing the native core structure of SrtA, we also included three recently evolved mutants of SrtA in this comparative study. With these mutants we observed a rapid initial attachment of the GFPuv target protein to the microbeads. However, with proceeding reaction time, cleavage of the covalently immobilized target protein from the surface prevailed over the coupling reaction, consequently causing a decline of microbead fluorescence. In general, the RT-FCM approach used herein represents a powerful analytical tool for qualitative dynamic studies of many heterogeneous enzymatic reactions or other binding events that influence the fluorescence properties of microparticle surfaces.

Enzyme-catalyzed protein crosslinking.

The process of protein crosslinking comprises the chemical, enzymatic, or chemoenzymatic formation of new covalent bonds between polypeptides. This allows (1) the site-directed coupling of proteins with distinct properties and (2) the de novo assembly of polymeric protein networks. Transferases, hydrolases, and oxidoreductases can be employed as catalysts for the synthesis of crosslinked proteins, thereby complementing chemical crosslinking strategies. Here, we review enzymatic approaches that are used for protein crosslinking at the industrial level or have shown promising potential in investigations on the lab-scale. We illustrate the underlying mechanisms of crosslink formation and point out the roles of the enzymes in their natural environments. Additionally, we discuss advantages and drawbacks of the enzyme-based crosslinking strategies and their potential for different applications.

Crystal structures of BapA complexes with ß-lactam-derived inhibitors illustrate substrate specificity and enantioselectivity of ß-aminopeptidases.

ß-Aminopeptidases have exclusive biocatalytic potential because they react with peptides composed of ß-amino acids, which serve as building blocks for the design of non-natural peptidomimetics. We have identified the ß-lactam antibiotic ampicillin and the ampicillin-derived penicilloic acid as novel inhibitors of the ß-aminopeptidase BapA from Sphingosinicella xenopeptidilytica (K(i) values of 0.69 and 0.74 mM, respectively). We report high-resolution crystal structures of BapA in noncovalent complexes with these inhibitors and with the serine protease inhibitor 4-(2-aminoethyl)benzenesulfonyl fluoride. All three inhibitors showed similar binding characteristics; the aromatic moiety extended into a hydrophobic binding pocket of the active site, and the free amino group formed a salt bridge with Glu133 of BapA. The exact position of the inhibitors and structural details of the ligand binding pocket illustrate the specificity and the enantioselectivity of BapA-catalyzed reactions with ß-peptide substrates.

Bacterial ß-aminopeptidases: structural insights and applications for biocatalysis.

ß-Aminopeptidases comprise a class of enzymes with functional and structural similarities. All members of the ß-aminopeptidases described to date were isolated from bacterial sources. Uniquely, they catalyze the hydrolysis of ß(3) - and/or ß(2) -amino acid residues from amides and peptides that are otherwise considered proteolytically stable. Due to this unusual reactivity with ß-peptide substrates, ß-aminopeptidases have potential to be used as biocatalysts for ß-peptide synthesis and for the resolution of enantiomerically pure ß-amino acids from racemic substrate mixtures. ß-Aminopeptidases are formed from an inactive precursor by posttranslational autoproteolytic cleavage, exposing the catalytic nucleophile at the N-terminus of the newly formed ß-polypeptide chain. Such an activation step is a characteristic trait of enzymes of the N-terminal nucleophile (Ntn) hydrolase superfamily. However, classical Ntn hydrolases and ß-aminopeptidases differ by the fold of their catalytic cores and are hence likely to originate from distinct evolutionary ancestors. In this contribution, we review the existing literature on ß-aminopeptidases, including biochemical and functional studies, as well as structural investigations that recently allowed insights into the catalytic mechanisms of precursor processing and ß-peptide conversion.

Beta-aminopeptidase-catalyzed biotransformations of beta(2)-dipeptides: kinetic resolution and enzymatic coupling.

We have previously shown that the beta-aminopeptidases BapA from Sphingosinicella xenopeptidilytica and DmpA from Ochrobactrum anthropi can catalyze reactions with non-natural beta(3)-peptides and beta(3)-amino acid amides. Here we report that these exceptional enzymes are also able to utilize synthetic dipeptides with N-terminal beta(2)-amino acid residues as substrates under aqueous conditions. The suitability of a beta(2)-peptide as a substrate for BapA or DmpA was strongly dependent on the size of the C(alpha) substituent of the N-terminal beta(2)-amino acid. BapA was shown to convert a diastereomeric mixture of the beta(2)-peptide H-beta(2)hPhe-beta(2)hAla-OH, but did not act on diastereomerically pure beta(2),beta(3)-dipeptides containing an N-terminal beta(2)-homoalanine. In contrast, DmpA was only active with the latter dipeptides as substrates. BapA-catalyzed transformation of the diastereomeric mixture of H-beta(2)hPhe-beta(2)hAla-OH proceeded along two highly S-enantioselective reaction routes, one leading to substrate hydrolysis and the other to the synthesis of coupling products. The synthetic route predominated even at neutral pH. A rise in pH of three log units shifted the synthesis-to-hydrolysis ratio (v(S)/v(H)) further towards peptide formation. Because the equilibrium of the reaction lies on the side of hydrolysis, prolonged incubation resulted in the cleavage of all peptides that carried an N-terminal beta-amino acid of S configuration. After completion of the enzymatic reaction, only the S enantiomer of beta(2)-homophenylalanine was detected (ee>99 % for H-(S)-beta(2)-hPhe-OH, E>500); this confirmed the high enantioselectivity of the reaction. Our findings suggest interesting new applications of the enzymes BapA and DmpA for the production of enantiopure beta(2)-amino acids and the enantioselective coupling of N-terminal beta(2)-amino acids to peptides.

The Role of Native T1 Mapping in the Diagnosis of Myocarditis in a Real-World Setting.

BACKGROUND: This prospective single-center study sought to investigate the impact of cardiovascular magnetic resonance (CMR) on the diagnosis of myocarditis, with special attention given to absolute T1 values and defined cutoff values. METHODS: All patients referred to our center with the suspicion of an inflammatory myocardial disease were diagnosed by a consensus expert consortium blinded to CMR findings. Classical Lake Louise criteria were then used to confirm or change the diagnosis. RESULTS: Of a total of 149 patients, 15 were diagnosed with acute myocarditis without taking CMR findings into account. Acute myocarditis was excluded in 91 patients, whereas 42 cases were unclear. Using classical Lake Louise criteria, an additional 35 clear diagnoses were made, either confirming or excluding myocarditis. In the remaining patients, there was no further increase in definitive diagnoses using T1 measurements. The diagnostic performance of T1 mapping in distinguishing acute myocarditis patients from healthy controls was good (area under the curve (AUC) 0.835, cutoff value 1019 ms, sensitivity 73.7%, specificity 72.4%). In the group of patients with suspected and then excluded myocarditis, the cutoff value had a false-positive rate of 56.6%. CONCLUSIONS: Acute myocarditis should be diagnosed on the basis of clinical and imaging factors, whereas T1 mapping could be helpful, especially for excluding acute myocarditis.

Kinetic resolution of aliphatic beta-amino acid amides by beta-aminopeptidases.

The growing demand for enantiomerically pure beta-amino acids to be used in the pharmaceutical industry and as fine chemicals requires the development of new strategies for their synthesis. The beta-aminopeptidases BapA from Sphingosinicella xenopeptidilytica 3-2W4, BapA from Sphingosinicella microcystinivorans Y2, and DmpA from Ochrobactrum anthropi LMG7991 are hydrolases that possess the unique ability of cleaving N-terminal beta-amino acids from peptides and amides. Hydrolysis of racemic beta(3)-amino acid amides catalyzed by these enzymes displays enantioselectivity with a strong preference for substrates with the L-configuration and gives access to various aliphatic beta(3)-amino acids of high enantiopurity. This approach presents a new access to enantiopure beta(3)-amino acids under mild reaction conditions and complements chemical asymmetric synthesis strategies.

Enzyme-catalyzed formation of beta-peptides: beta-peptidyl aminopeptidases BapA and DmpA acting as beta-peptide-synthesizing enzymes.

In recent studies, we discovered that the three beta-peptidyl aminopeptidases, BapA from Sphingosinicella xenopeptidilytica 3-2W4, BapA from S. microcystinivorans Y2, and DmpA from Ochrobactrum anthropi LMG7991, possess the unique feature of cleaving N-terminal beta-amino acid residues from beta- and alpha/beta-peptides. Herein, we investigated the use of the same three enzymes for the reverse reaction catalyzing the oligomerization of beta-amino acids and the synthesis of mixed peptides with N-terminal beta-amino acid residues. As substrates, we employed the beta-homoamino acid derivatives H-beta hGly-pNA, H-beta3 hAla-pNA, H-(R)-beta3 hAla-pNA, H-beta3 hPhe-pNA, H-(R)-beta3 hPhe-pNA, and H-beta3 hLeu-pNA. All three enzymes were capable of coupling the six beta-amino acids to oligomers with chain lengths of up to eight amino acid residues. With the enzyme DmpA as the catalyst, we observed very high conversion rates, which correspond to dimer yields of up to 76%. The beta-dipeptide H-beta3 hAla-beta3 hLeu-OH and the beta/alpha-dipeptide H-beta hGly-His-OH (carnosine) were formed with almost 50% conversion, when a five-fold excess of beta3-homoleucine or histidine was incubated with H-beta3 hAla-pNA and H-beta hGly-pNA, respectively, in the presence of the enzyme BapA from S. microcystinivorans Y2. BapA from S. xenopeptidilytica 3-2W4 turned out to be a versatile catalyst capable of coupling various beta-amino acid residues to the free N-termini of beta- and alpha-amino acids and even to an alpha-tripeptide. Thus, these aminopeptidases might be useful to introduce a beta-amino acid residue as an N-terminal protecting group into a 'natural' alpha-peptide, thereby stabilizing the peptide against degradation by other proteolytic enzymes.

Tissue factor as a link between wounding and tissue repair.

The initial phase of wound repair involves inflammation, induction of tissue factor (TF), formation of a fibrin matrix, and growth of new smooth muscle actin (alpha-SMA)-positive vessels. In diabetes, TF induction in response to cutaneous wounding, which ordinarily precedes increased expression of vascular endothelial growth factor (VEGF) and alpha-SMA transcription, is diminished, though not to a degree causing excessive local bleeding. Enhanced TF expression in wounds of diabetic mice caused by somatic TF gene transfer increased VEGF transcription and translation and, subsequently, enhanced formation of new blood vessels and elevated blood flow. Furthermore, increased levels of TF in wounds of diabetic mice enhanced wound healing; the time to achieve 50% wound closure was reduced from 5.5 days in untreated diabetic mice to 4.1 days in animals undergoing TF gene transfer (this was not statistically different from wound closure in nondiabetic mice). Thus, cutaneous wounds in diabetic mice display a relative deficiency of TF compared with nondiabetic controls, and this contributes to delayed wound repair. These data establish TF expression as an important link between the early inflammatory response to cutaneous wounding and reparative processes.

Bacterial beta-peptidyl aminopeptidases with unique substrate specificities for beta-oligopeptides and mixed beta,alpha-oligopeptides.

We previously discovered that BapA, a bacterial beta-peptidyl aminopeptidase, is able to hydrolyze two otherwise metabolically inert beta-peptides [Geueke B, Namoto K, Seebach D and Kohler H-PE (2005) J Bacteriol 187, 5910-5917]. Here, we describe the purification and characterization of two distinct bacterial beta-peptidyl aminopeptidases that originated from different environmental isolates. Both bapA genes encode a preprotein with a signal sequence and were flanked by ORFs that code for enzymes with similar predicted functions. To form the active enzymes, which had an (alphabeta)(4) quaternary structure, the preproteins needed to be cleaved into two subunits. The two beta-peptidyl aminopeptidases had 86% amino acid sequence identity, hydrolyzed a variety of beta-peptides and mixed beta/alpha-peptides, and exhibited unique substrate specificities. The prerequisite for peptides being accepted as substrates was the presence of a beta-amino acid at the N-terminus; peptide substrates with an N-terminal alpha-amino acid were not hydrolyzed at all. Both enzymes cleaved the peptide bond between the N-terminal beta-amino acid and the amino acid at the second position of tripeptidic substrates of the general structure H-betahXaa-Ile-betahTyr-OH according to the following preferences with regard to the side chain of the N-terminal beta-amino acid: aliphatic and aromatic > OH-containing > hydrogen, basic and polar. Experiments with the tripeptides H-d-betahVal-Ile-betahTyr-OH and H-betahVal-Ile-betahTyr-OH demonstrated that the two BapA enzymes preferred the peptide with the l-configuration of the N-terminal beta-homovaline residue as a substrate.

Enzymatic degradation of beta- and mixed alpha,beta-oligopeptides.

One of the main and most astonishing characteristics of peptides comprised of beta-amino acids with proteinogenic side chains is their extraordinarily high stability towards enzymatic degradation. So far, only certain microbial enzymes have been shown to cleave N-terminal beta(3)-homoamino acid residues from peptides. In this work, the L-aminopeptidase-D-amidase/esterase (DmpA) from Ochrobactrum anthropi LMG7991 is compared to two closely related beta-peptidyl aminopeptidases (BapA), which originate from Sphingosinicella strains, and to microsomal leucine aminopeptidase (LAP) as a reference. All four enzymes are aminopeptidases cleaving N-terminal amino acids from small peptides. Degradation experiments reveal that DmpA and both BapA enzymes exhibit unique, but clearly distinct substrate specificities and preferences. DmpA also cleaves beta- and mixed alpha,beta-peptides and amides, but a short side chain of the N-terminal beta-amino acid residue seems to be a prerequisite, since only peptides carrying N-terminal betahGly and beta(3)hAla are hydrolyzed with good efficiencies. Both beta-peptidyl aminopeptidases cleave beta-amino acids from a variety of beta-peptides and mixed alpha,beta-peptides, but they do not accept alpha-amino acids in the N-terminal position. Astonishingly, DmpA exhibited much higher catalytical rates for the mixed dipeptide carnosine (H-betahGly-His-OH) than for any other substrate described until now.

Monitoring of dynamic microbiological processes using real-time flow cytometry.

We describe a straightforward approach to continuously monitor a variety of highly dynamic microbiological processes in millisecond resolution with flow cytometry, using standard bench-top instrumentation. Four main experimental examples are provided, namely: (1) green fluorescent protein expression by antibiotic-stressed Escherichia coli, (2) fluorescent labeling of heat-induced membrane damage in an autochthonous freshwater bacterial community, (3) the initial growth response of late stationary E. coli cells inoculated into fresh growth media, and (4) oxidative disinfection of a mixed culture of auto-fluorescent microorganisms. These examples demonstrate the broad applicability of the method to diverse biological experiments, showing that it allows the collection of detailed, time-resolved information on complex processes.

Autoproteolytic and catalytic mechanisms for the ß-aminopeptidase BapA--a member of the Ntn hydrolase family.

The ß-aminopeptidase BapA from Sphingosinicella xenopeptidilytica belongs to the N-terminal nucleophile (Ntn) hydrolases of the DmpA-like family and has the unprecedented property of cleaving N-terminal ß-amino acid residues from peptides. We determined the crystal structures of the native (αß)4 heterooctamer and of the 153 kDa precursor homotetramer at a resolution of 1.45 and 1.8 Å, respectively. These structures together with mutational analyses strongly support mechanisms for autoproteolysis and catalysis that involve residues Ser250, Ser288, and Glu290. The autoproteolytic mechanism is different from the one so far described for Ntn hydrolases. The structures together with functional data also provide insight into the discriminating features of the active site cleft that determine substrate specificity.

Simple enzymatic procedure for L-carnosine synthesis: whole-cell biocatalysis and efficient biocatalyst recycling.

ß-Peptides and their derivates are usually stable to proteolysis and have an increased half-life compared with α-peptides. Recently, ß-aminopeptidases were described as a new enzyme class that enabled the enzymatic degradation and formation of ß-peptides. As an alternative to the existing chemical synthesis routes, the aim of the present work was to develop a whole-cell biocatalyst for the synthesis and production of ß-peptides using this enzymatic activity. For the optimization of the reaction system we chose the commercially relevant ß,α-dipeptide L-carnosine (ß-alanine-L-histidine) as model product. We were able to show that different recombinant yeast and bacteria strains, which overexpress a ß-peptidase, could be used directly as whole-cell biocatalysts for the synthesis of L-carnosine. By optimizing relevant reaction conditions for the best-performing recombinant Escherichia coli strain, such as pH and substrate concentrations, we obtained high l-carnosine yields of up to 71%. Long-time as well as biocatalyst recycling experiments indicated a high stability of the developed biocatalyst for at least five repeated batches. Application of the recombinant E. coli in a fed-batch process enabled the accumulation of l-carnosine to a concentration of 3.7 g l(-1).

CD8+ T-cell response against MUC1-derived peptides in gastrointestinal cancer survivors.

The tumor-associated antigens CEA, MUC1 and Her2/neu are broadly expressed in gastrointestinal tumors, and are attractive candidates for targeting by T-cell-based immunotherapy. However, little is known about the natural cytotoxic T-cell response of patients suffering from colorectal or gastric carcinoma against these three as well as other antigens. Using a quantitative reverse transcription-polymerase chain reaction-based assay for IFN-gamma, we analyzed the CD8+ T-cell repertoire present in the blood of HLA-A2+ gastrointestinal tumor survivors against five known epitopes derived from CEA, MUC1 and Her2/neu. The results show that most of the patients (16 from 22 tested) have detectable, peripheral CD8+ T cells directed against at least one of these three proteins. Interestingly, the majority of these patients reacts to the two MUC1-derived HLA-A*0201 epitopes tested (14 from 16), demonstrating that this protein represents one dominant target for CD8+ T cells in gastrointestinal cancer.