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Alfalfa (Medicago sativa) seeds planted in cold, wet soil are prone to seed rot, pre- and post-emergence damping-off, and seedling root rot. These diseases reduce initial stand density and impact forage yields and winter survival when root systems are stunted by infection. Successful management depends on identifying the causal agents, but isolation in pure culture is time consuming and may fail to recover slow growing organisms or those requiring special growth conditions. Here, the oomycetes in rhizosphere soil and root endospheres from eight locations with a history of poor alfalfa establishment were identified by amplicon sequence analysis. Seedling bioassays were done with bulk soil and quantitative PCR assays were done using DNA from bulk soil, rhizosphere soil, and the root endosphere for Aphanomyces euteiches, Phytophthora medicaginis, Pythium irregulare, P. sylvaticum, and P. ultimum var. ultimum. In the endosphere and rhizosphere, A. euteiches was the dominant taxon, followed by Phytophthora medicaginis and P. sansomeana, a broad host range pathogen not previously known to infect alfalfa. Pythium inflatum and Globisporangium perplexum were also significantly over-represented in roots at five of the eight locations, indicating that these taxa are commonly enriched in alfalfa roots, but had not previously been identified as alfalfa pathogens. The qPCR assays of bulk soil showed that A. euteiches and P. medicaginis were unevenly distributed in the plots and low concentrations of the pathogens led to high levels of root rot disease. These results support development of cultivars with high levels of resistance to the most common oomycete pathogens.

The plant is one of the primary drivers of microbial communities in the rhizosphere. The consistent presence of same plant species over time, such as in monocropping in agriculture can drive significant changes in plant-associated microbiomes. Most of the studies with monocropping have focused on bacteria, which are involved in the natural suppression of a number of soilborne diseases, including Rhizoctonia root rot and take-all. However, few studies have examined how monocropping and root rot pathogens jointly affect the structure of fungal communities in the rhizosphere. In this greenhouse study, rhizosphere fungal communities from successive wheat plantings infected with the fungal pathogen Rhizoctonia solani AG8 were characterized using MiSeq sequencing targeting the ITS1 region of the rRNA gene. Sequence analyses revealed that distinct fungal groups clustered by planting cycles with or without AG8 inoculation, but infection with AG8 enhanced the separation of fungal communities. Clusters of fungal communities were also observed in AG8-infected and non-infected rhizospheres, whereas there was no difference in fungal communities between the rhizosphere with the least root disease and those with the worst root disease. Planting cycles significantly reduced fungal alpha diversity. The most abundant fungal genus was Mortierella which increased in relative abundance with planting cycles in AG8-infected samples. In contrast, a group of fungal genera, including Pseudogymnoascus, Gibberella, Fusarium, Fusicolla, Exophiala, and Waitea, were reduced in relative abundance with successive plantings and AG8 infection. Together, this study revealed how fungal communities change with successive wheat growth under the pressure of a soilborne fungal pathogen.

Trotz deutlicher technologischer Fortschritte wird Predictive Analytics in der Praxis noch immer nur selten für die finanzielle Prognose eingesetzt. Notwendig für eine erfolgreiche Anwendung ist ein ganzheitlicher Ansatz bei der Implementierung, der über die rein technisch „richtige“ Anwendung hinausgeht. Aus der Analyse verschiedener Implementierungsprojekte wurden daher die Erfolgsfaktoren für Predictive Analytics Projekte abgeleitet und in einem ganzheitlichen Konzept zusammengefasst. Damit können Verbesserungen in den Bereichen Prognosegenauigkeit, Ressourceneinsatz und Steuerungswirkung realisiert werden.

Activation of the T cell receptor (TCR) results in binding of the adapter protein Nck (noncatalytic region of tyrosine kinase) to the CD3ϵ subunit of the TCR. The interaction was suggested to be important for the amplification of TCR signals and is governed by a proline-rich sequence (PRS) in CD3ϵ that binds to the first Src homology 3 (SH3) domain of Nck (Nck-SH3.1). Inhibition of this protein/protein interaction ameliorated inflammatory symptoms in mouse models of multiple sclerosis, psoriasis, and asthma. A small molecule, AX-024, was reported to inhibit the Nck/CD3ϵ interaction by physically binding to the Nck1-SH3.1 domain, suggesting a route to develop an inhibitor of the Nck1/CD3ϵ interaction for modulating TCR activity in autoimmune and inflammatory diseases. We show here that AX-024 reduces T cell proliferation upon weak TCR stimulation but does not significantly affect phosphorylation of Zap70 (ζ chain of T cell receptor–associated protein kinase 70). We also find that AX-024 is likely not involved in modulating the Nck/TCR interaction but probably has other targets in T cells. An array of biophysical techniques did not detect a direct interaction between AX-024 and Nck-SH3.1 in vitro. Crystal structures of the Nck-SH3.1 domain revealed its binding mode to the PRS in CD3ϵ. The SH3 domain tends to generate homodimers through a domain swap. Domain swaps observed previously in other SH3 domains indicate a general propensity of this protein fold to exchange structural elements. The swapped form of Nck-SH3.1 is unable to bind CD3ϵ, possibly representing an inactive form of Nck in cells.

Therapeutic approaches to fight Alzheimer's disease include anti-Amyloidβ (Aβ) antibodies and secretase inhibitors. However, the blood-brain barrier (BBB) limits the brain exposure of biologics and the chemical space for small molecules to be BBB permeable. The Brain Shuttle (BS) technology is capable of shuttling large molecules into the brain. This allows for new types of therapeutic modalities engineered for optimal efficacy on the molecular target in the brain independent of brain penetrating properties. To this end, we designed BACE1 peptide inhibitors with varying lipid modifications with single-digit picomolar cellular potency. Secondly, we generated active-exosite peptides with structurally confirmed dual binding mode and improved potency. When fused to the BS via sortase coupling, these BACE1 inhibitors significantly reduced brain Aβ levels in mice after intravenous administration. In plasma, both BS and non-BS BACE1 inhibitor peptides induced a significant time- and dose-dependent decrease of Aβ. Our results demonstrate that the BS is essential for BACE1 peptide inhibitors to be efficacious in the brain and active-exosite design of BACE1 peptide inhibitors together with lipid modification may be of therapeutic relevance., Highlights • Dual active and exosite designed BACE1 inhibitors with cell and in vivo activity. • X-ray structures explain the potency and selectivity of the BACE1 inhibitors. • The BACE1 peptide inhibitors become active within the brain mediated by Brain Shuttle transport across the blood brain barrier. Alzheimer's disease (AD) is a devastating brain disorder that affects many people. This work describe how specifically designed inhibitors against the enzyme BACE1, which is known to produce toxic molecules that is claimed to be involved in causing AD. The BACE1 inhibitors are very effective and selective as they bind to two sites at the same time to this enzyme and accumulate at the cell membrane where the enzyme is located. As these inhibitors are too big to penetrate into the brain we use the Brain Shuttle technology. Potentially this approach could be used to treat people with the risk of getting AD.

There are no data-supported recommendations on how proximal is too proximal for retrograde nailing (RGN). At six level 1 trauma centers, patients with femur fractures within the proximal one-third of the femur treated with RGN were included. This article describes a proximal segment capture ratio (PSCR) and nail segment capture ratio to evaluate RGN of proximal fractures. The study included 107 patients. The average follow-up was 44 weeks. There were two nonunions and three malunions. There was no significant difference between PSCR of 0.3 or less and need for secondary procedures or time to full weight bearing (p.05). In this study, a smaller (0.3) PSCR was not associated with an increased number of complications. A higher Orthopaedic Trauma Association classification was predictive of malunion and increased time to union. These data demonstrate that retrograde nailing is safe and effective for the treatment of supraisthmal femur fractures.

Sicherheit ist oberstes Gebot Die Gewährleistung der Sicherheit für Versammlungsstätten und Veranstaltungen ist eine anspruchsvolle und vielschichtige Aufgabe. Welche Risiken sind zu beachten, welche Schutzziele adäquat? Welches Regelwerk ist tatsächlich einschlägig und welche Maßnahmen sind noch verhältnismäßig? Geeignete Sicherheitskonzepte entwickeln Diese und weitere Fragen stellen sich bei der Sicherheitskonzeption für jede Veranstaltung in unterschiedlicher Intensität. Die Antworten sind dabei häufig so heterogen und komplex wie der Veranstaltungsmarkt selbst, der unter anderem exklusive Konzernveranstaltungen, emotionsgeladene Nachbarschaftsduelle rivalisierender Fußballvereine oder Open-Air-Musikfestivals mit über 100.000 Zuschauern vereint. Mit dem einzigartigen Leitfaden für die Praxis Das Handbuch "Sicherheit für Versammlungsstätten und Veranstaltungen" ist ein thematisch geordnetes Nachschlagewerk zu allen sicherheitsrelevanten Fragen bei Veranstaltungen. Es beschreibt in dieser Zusammenstellung erstmals die • besonderen Merkmale des Veranstaltungsmarktes und der Veranstaltungssicherheit, • grundlegenden Sicherheitsmaßnahmen für Versammlungsstätten und Veranstaltungen, • Spezifika einzelner Veranstaltungsarten, • Sicht der Behörden und Organisationen mit Sicherheitsaufgaben sowie beteiligter Dienstleister auf die Veranstaltungssicherheit. Umfassendes Expertenwissen nutzen Insgesamt 18 erfahrene Experten des jeweiligen Fachbereichs haben ihr Wissen sowie ihre berufliche Erfahrung in das Werk eingebracht. Sie ermöglichen es, die grundlegenden Zusammenhänge und Entwicklungen der Veranstaltungssicherheit aus unterschiedlichen Perspektiven zu betrachten. Fazit Das Handbuch gibt dem Leser einen umfassenden Überblick über die einzelnen Handlungsfelder und Maßnahmen der Veranstaltungssicherheit aus unterschiedlichen Perspektiven.

Crystal structures of mouse, rat and human catechol-O-methyltransferase were determined in apo, semi-holo, holo and Michaelis forms under a variety of conditions. Domain swaps and large conformational changes in the active sites are observed, which testify to why this enzyme is a difficult drug target., Methylation catalysed by catechol-O-methyltransferase (COMT) is the main pathway of catechol neurotransmitter deactivation in the prefrontal cortex. Low levels of this class of neurotransmitters are held to be causative of diseases such as schizophrenia, depression and Parkinson’s disease. Inhibition of COMT may increase neurotransmitter levels, thus offering a route for treatment. Structure-based drug design hitherto seems to be based on the closed enzyme conformation. Here, a set of apo, semi-holo, holo and Michaelis form crystal structures are described that define the conformational space available to COMT and that include likely intermediates along the catalytic pathway. Domain swaps and sizeable loop movements around the active site testify to the flexibility of this enzyme, rendering COMT a difficult drug target. The low affinity of the co-substrate S-adenosylmethionine and the large conformational changes involved during catalysis highlight significant energetic investment to achieve the closed conformation. Since each conformation of COMT is a bona fide target for inhibitors, other states than the closed conformation may be promising to address. Crystallographic data for an alternative avenue of COMT inhibition, i.e. locking of the apo state by an inhibitor, are presented. The set of COMT structures may prove to be useful for the development of novel classes of inhibitors.

The serine protease chymase (EC = 3.4.21.39) is expressed in the secretory granules of mast cells, which are important in allergic reactions. Fynomers, which are binding proteins derived from the Fyn SH3 domain, were generated against human chymase to produce binding partners to facilitate crystallization, structure determination and structure-based drug discovery, and to provide inhibitors of chymase for therapeutic applications. The best Fynomer was found to bind chymase with a KD of 0.9 nM and koff of 6.6x10 (-4) s (-1) , and to selectively inhibit chymase activity with an IC 50 value of 2 nM. Three different Fynomers were co-crystallized with chymase in 6 different crystal forms overall, with diffraction quality in the range of 2.25 to 1.4 Å resolution, which is suitable for drug design efforts. The X-ray structures show that all Fynomers bind to the active site of chymase. The conserved residues Arg15-Trp16-Thr17 in the RT-loop of the chymase binding Fynomers provide a tight interaction, with Trp16 pointing deep into the S1 pocket of chymase. These results confirm the suitability of Fynomers as research tools to facilitate protein crystallization, as well as for the development of assays to investigate the biological mechanism of targets. Finally, their highly specific inhibitory activity and favorable molecular properties support the use of Fynomers as potential therapeutic agents.

An X-ray-guided design approach led to the identification of a novel, balanced class of alpha-ethoxy-phenylpropionic acid-derived dual PPARalpha/gamma agonists. The series shows a wide range of PPARalpha/gamma ratios within a rather narrow structural space. Advanced compounds possess favorable physicochemical and pharmacokinetic profiles and show a high efficacy in T2D and dyslipidemia animal models.

Design, synthesis, and SAR of novel alpha-alkoxy-beta-arylpropionic acids as potent and balanced PPARalphagamma coagonists are described. One representative thereof, Aleglitazar ((S)-2Aa), was chosen for clinical development. Its X-ray structure in complex with both receptors as well as its high efficacy in animal models of T2D and dyslipidemia are also presented.

To explore guinea pigs as models of chymase biology, we cloned and expressed the guinea pig ortholog of human chymase. In contrast to rats and mice, guinea pigs appear to express just one chymase, which belongs to the α clade, like primate chymases and mouse mast cell protease-5. The guinea pig enzyme autolyzes at Leu residues in the loop where human chymase autolyzes at Phe. In addition, guinea pig α-chymase selects P1 Leu in a combinatorial peptide library and cleaves Ala-Ala-Pro-Leu-4-nitroanilide but has negligible activity toward substrates with P1 Phe and does not cleave angiotensin I. This contrasts with human chymase, which cleaves after Phe or Tyr, prefers P1 Phe in peptidyl 4-nitroanilides, and avidly hydrolyzes angiotensin I at Phe8 to generate bioactive angiotensin II. The guinea pig enzyme also is inactivated more effectively by α1-antichymotrypsin, which features P1 Leu in the reactive loop. Unlike mouse, rat, and hamster α-chymases, guinea pig chymase lacks elastase-like preference for P1 Val or Ala. Partially humanized A216G guinea pig chymase acquires human-like P1 Phe- and angiotensin-cleaving capacity. Molecular models suggest that the wild type active site is crowded by the Ala216 side chain, which potentially blocks access by bulky P1 aromatic residues. On the other hand, the guinea pig pocket is deeper than in Val-selective chymases, explaining the preference for the longer aliphatic side chain of Leu. These findings are evidence that chymase-like peptidase specificity is sensitive to small changes in structure and provide the first example of a vertebrate Leu-selective peptidase.

The full-length and ectodomain forms of beta-site APP cleavage enzyme (BACE) have been cloned, expressed in Sf9 cells, and purified to homogeneity. This aspartic protease cleaves the amyloid precursor protein at the beta-secretase site, a critical step in the Alzheimer's disease pathogenesis. Comparison of BACE to other aspartic proteases such as cathepsin D and E, napsin A, pepsin, and renin revealed little similarity with respect to the substrate preference and inhibitor profile. On the other hand, these parameters are all very similar for the homologous enzyme BACE2. Based on a collection of decameric substrates, it was found that BACE has a loose substrate specificity and that the substrate recognition site in BACE extends over several amino acids. In common with the aspartic proteases mentioned above, BACE prefers a leucine residue at position P1. Unlike cathepsin D etc., BACE accepts polar or acidic residues at positions P2'0 and P1 but prefers bulky hydrophobic residues at position P3. BACE displays poor kinetic constants toward its known substrates (wild-type substrate, SEVKM/DAEFR, K(m) = 7 microm, K(cat) = 0.002 s(-1); Swedish mutant, SEVNL/DAEFR, K(m) = 9 microm, K(cat) = 0.02 s(-1)). A new substrate (VVEVDA/AVTP, K(m) = 1 microm, K(cat) = 0.004) was identified by serendipity.

Fluorogenic substrates for assaying novel proteolytic enzymes could be rapidly identified using an easy, solid-phase combinatorial assay technology. The methodology was validated with leader peptidase of Escherichia coli using a subset of an intramolecularly quenched fluorogenic peptide library. The technique was extended toward the discovery of substrates for a new aspartic protease of pharmaceutical relevance (human napsin A). We demonstrated for the first time known to us that potent fluorogenic substrates can be discovered using extracts of cells expressing recombinant enzyme to screen the peptide library. The straightforward and rapid optimization of protease substrates greatly facilitates the drug discovery process by speeding up the development of high throughput screening assays and thus helps more effective exploitation of the enormous body of information and chemical structures emerging from genomics and combinatorial chemistry technologies.

Recombinant human napsin A expressed in human embryonic kidney 293 cells was purified to homogeneity by a single-step procedure using part of napsin A propeptide as affinity ligand. N-Terminal amino-acid sequencing of the purified enzyme identified the mature form of napsin A. Treatment of purified napsin A with endoglycosidases F and H resulted in a decrease in its molecular mass from 39 kDa to ≈ 37 kDa, confirming that napsin A is glycosylated. The kinetic properties were analyzed by using two fluorogenic synthetic substrates K(Dabsyl)-TSLLMAAPQ–Lucifer yellow (DS1) and K(Dabsyl)-TSVLMAAPQ–Lucifer yellow (DS3). The Km values obtained were 1.7 µm and 6.2 µm, respectively. A substrate-specificity study using a napsin A-targeted peptide library confirmed the preference of napsin A for hydrophobic residues at positions P1 and P1′. Adjacent positions, P2–P4 and P2′–P4′, appeared less restricted in distribution of amino acids. A pH optimum between 4.0 and 5.5 at room temperature was determined. The purified enzyme was fully active for more than 10 h at pH 5.0 and 6.0, while a half-life of 4 h was determined at pH 7.0 and 37 °C.

The binding reaction between purified human platelet glycoprotein IIb-IIIa and fibrinogen was investigated by real-time measurements using the surface-plasmon-resonance sensor technology. In these experiments, either glycoprotein IIb-IIIa or fibrinogen was immobilized on a sensor surface. The time-dependent change in surface coverage that occurred immediately upon contact with a solution of the complementary protein was then detected. The ability to record this dynamic event from its initiation allowed the collection of kinetic and thermodynamic data over an extended time period. These data indicated that initially, in fast reaction, a reversible low-affinity complex with an equilibrium dissociation constant, Kd, of 155-180 nM was formed. In a subsequent slower reaction this complex was transformed into a more stable high-affinity complex with a Kd of 20-70 nM. Efficient dissociation of the high-affinity complex could only be induced in the presence of a competitive inhibitor such as RGDV. These data demonstrate that the binding between glycoprotein IIb-IIIa and fibrinogen is not a single monophasic reaction, but is composed of at least two consecutive processes both with their own kinetics.

This paper describes the application of a novel sensor technology for determining the concentration of biologically and diagnostically relevant analytes with high sensitivity and investigating biological recognition reactions such as antibody-antigen reactions. On the basis of the interaction between an anti-human IgG and a human IgG, a broad dynamic concentration range extending over six orders of magnitude and a detection limit of 1 × 10−11 m dissolved antibody are demonstreated to be relevant for the determination of concentrations with this sensor system. The effective surface concentration obtained at this dilution is 19 pg mm−2, which corresponds to less than 4 parts per thousand of a closely packed monolayer of these molecules. Measurements of the clinically relevant hepatitis B surface antigen show the same high sensitivity: 2 X 10−13 m antigen could be detected even in undiluted human serum. The most interesting feature of biospecific interaction analysis with this sensor system is the fact that one can observe the course of a binding reaction between two molecules in real time. On the basis of the anti-human IgG/human IgG model system, we demonstrate the determination of equilibrium as well as rate constants of such reactions directly from the binding data.

L-Dopa, the standard therapeutic for Parkinson's disease, is inactivated by the enzyme catechol-O-methyltransferase (COMT). COMT catalyzes the transfer of an activated methyl group from S-adenosylmethionine (SAM) to its catechol substrates, such as L-dopa, in the presence of magnesium ions. The molecular recognition properties of the SAM-binding site of COMT have been investigated only sparsely. Here, we explore this site by structural alterations of the adenine moiety of bisubstrate inhibitors. The molecular recognition of adenine is of special interest due to the great abundance and importance of this nucleobase in biological systems. Novel bisubstrate inhibitors with adenine replacements were developed by structure-based design and synthesized using a nucleosidation protocol introduced by Vorbruggen and co-workers. Key interactions of the adenine moiety with COMT were measured with a radiochemical assay. Several bisubstrate inhibitors, most notably the adenine replacements thiopyridine, purine, N-methyladenine, and 6-methylpurine, displayed nanomolar IC(50) values (median inhibitory concentration) for COMT down to 6 nM. A series of six cocrystal structures of the bisubstrate inhibitors in ternary complexes with COMT and Mg(2+) confirm our predicted binding mode of the adenine replacements. The cocrystal structure of an inhibitor bearing no nucleobase can be regarded as an intermediate along the reaction coordinate of bisubstrate inhibitor binding to COMT. Our studies show that solvation varies with the type of adenine replacement, whereas among the adenine derivatives, the nitrogen atom at position 1 is essential for high affinity, while the exocyclic amino group is most efficiently substituted by a methyl group.

Surface plasmon resonance (SPR) technology has emerged as a new and powerful technique to investigate the interaction between low-molecular-weight molecules and target proteins. In the present work, the authors assemble from a large compound collection a library of 2226 molecules (fragments having low molecular weights between 100 and 300 Da) to screen them for binding to chymase, a serine protease. Both the active chymase and a zymogen-like form of the protein were used in parallel to distinguish between specific and unspecific binding. The relative ligand-binding activity of the immobilized protein was periodically measured with a reference compound. The screening experiments were performed at 25 degrees C at a fragment concentration of 200 microM in the presence of 2% DMSO. Applying the filter cascade, affinity-selectivity-competition (competition with reference compounds and cross-competition with fragments), 80 compounds show up as positive screening hits. Competition experiments between fragments show that they bind to different parts of the active site. Of 36 fragments co-crystallized for X-ray studies, 12 could be located in the active site of the protein. These results validate the authors' library and demonstrate that the application of SPR technology as a filter in fragment screening can be achieved successfully.

Starting from a hit identified by focused screening, 3-aminopyrrolidine factor Xa inhibitors were designed. The binding mode as determined by X-ray structural analysis as well as the pharmacokinetic behaviour of selected compounds is discussed.

Fragment screening revealed that tyramine binds to the active site of the Alzheimer's disease drug target BACE-1. Hit expansion by selection of compounds from the Roche compound library identified tyramine derivatives with improved binding affinities as monitored by surface plasmon resonance. X-ray structures show that the amine of the tyramine fragment hydrogen-bonds to the catalytic water molecule. Structure-guided ligand design led to the synthesis of further low molecular weight compounds that are starting points for chemical leads.

In the pharmaceutical industry, knowledge of the three-dimensional structure of a specific target facilitates the drug-discovery process. Despite possessing favoured analytical properties such as high purity and monodispersion in light scattering, some proteins are not capable of forming crystals suitable for X-ray analysis. Cyclophilin D, an isoform of cyclophilin that is expressed in the mitochondria, was selected as a drug target for the treatment of cardiac disorders. As the wild-type enzyme defied all attempts at crystallization, protein engineering on the enzyme surface was performed. The K133I mutant gave crystals that diffracted to 1.7 A resolution using in-house X-ray facilities and were suitable for soaking experiments. The crystals were very robust and diffraction was maintained after soaking in 25% DMSO solution: excellent conditions for the rapid analysis of complex structures including crystallographic fragment screening.

An immobilization procedure for protein on surface plasmon resonance sensor (SPR) chips is described. The target protein, cyclophilin D, is thereby genetically linked to a mutant of the human DNA repair protein O 6 -alkylguanine-DNA-alkyltransferase (hAGT). The procedure includes the immobilization of an alkylguanine derivative on the surface by amine coupling and contact of the surface with a solution of the fusion protein (TCypD-hAGT). TCypD-hAGT could be immobilized using buffer solutions of purified protein or cell extracts. High densities of covalently linked proteins were achieved by either procedure. Binding experiments performed with the ligand cyclosporin A indicate relative binding activities close to 100%. The K D value (12 nM) and the kinetic rate constants k on (3 × 10 5 M −1 s −1 ) and k off (4 × 10 −3 s −1 ) are given and compared to values determined for cyclophilin D linked to the surface by amide coupling chemistry. The K D value is in excellent agreement with the K D value determined in solution by fluorescence titration.

Murine monoclonal antibodies (mAbs) are described that recognize the extracellular human interferon gamma receptor alpha-chain (IFN gamma R) and inhibit the binding to it of interferon gamma. The inhibitory activities (IC50s) of these mAbs, quantified by radioimmunoassay using native receptor on human Raji cells, lie in the range 0.5-24 nM, whereas their relative affinities for the immobilised recombinant extracellular receptor, determined using surface plasmon resonance technology, are in the range 0.6-40.9 nM. Nine mAbs derived from one immunization, were shown by variable region cDNA sequencing to be clonally related, with mAb A6 from this group showing the highest affinity for the receptor. Another two mAbs, gamma R38 and gamma R99, derived from a separate immunization, are clonally unrelated to each other and to those in the A6 family. From the V-region sequences, the L-chains of mAbs A6, gamma R38 and gamma R99 were shown to belong to the V kappa 34C, V kappa 34C and V kappa 1 families, whereas the H-chains belong to the 3069, J606 and J558 families, respectively. The mAbs A6 and gamma R38 recognize overlapping epitopes on the N-terminal Ig-like domain of the IFN gamma R, whereas the gamma R99 epitope is located largely in the membrane proximal Ig-like domain. Sequence comparisons with Ig structures solved by X-ray diffraction allowed deductions concerning likely CDR canonical conformations. These studies provide essential information for crystallographic and mutagenesis experiments aimed at understanding the molecular basis of the interactions of these mAbs with the extracellular IFN gamma R.

The extracellular portion of the human interferon-gamma receptor (IFN-gamma R) is predicted to adopt two Ig-like domains each with Ig superfamily type C2 topology. Surface plasmon resonance technology has been used here to compare the equilibrium and kinetic constants for binding reactions between these and several mutant domains, and neutralising anti-IFN-gamma R monoclonal antibodies. The biosensor measurements provide a sensitive method for monitoring the effects of mutations on the functional epitopes recognized by the neutralising antibodies. Thus, using recombinant native-like proteins made in E. coli, the ten N-terminal residues of the receptor were found not to be essential for domain folding, nor for recognition by the antibodies A6, D2 and gamma R38. In a similar way, residues in the interdomain region were found to play an important functional role in the epitope recognized by the antibody gamma R99.

This paper describes the application of a novel sensor technology for the concentration determination of biologically and diagnostically relevant analytes with high sensitivity and for the investigation of biological recognition reactions such as antibody antigen reactions. On the basis of the interaction between an anti human IgG and a human IgG a broad dynamic concentration range extending over six orders of magnitude and a detection limit of 1×10-11 M dissolved antibody are demonstrated to be relevant for the determination of concentrations with this sensor system. The effective surface concentration obtained at this dilution is 19 pg/mm2, which corresponds to less than 4 per mille of a possible monolayer. Measurements of the clinically relevant hepatitis B surface antigen show the same high sensitivity - 2×10-13 M antigen could be detected even in undiluted human serum. The most interesting feature of biospecific interaction analysis with this sensor system is the fact that one can observe the course of a binding reaction between two molecules in real time. Based on the anti human IgG / human IgG model system we demonstrate the determination of equilibrium as well as rate constants of such reactions directly from the binding data.

Two genes encoding for L-lactate dehydrogenase (LDH) from the psychrophilic bacterium Bacillus psychrosaccharolyticus (DSM 6) were cloned and their nucleotide sequence determined using a pEMBL vector and gene hybridization probes. The deduced amino-acid sequence of the gene from clone pLDH(X), which is located on a 5.87-kb HindIII-fragment, shows an identity of 86% as compared with the sequence of the wildtype LDH(P) from B. psychrosaccharolyticus and consists of 319 amino acids. Clone pLDH(P) contained a gene on a 4-kb HindIII-EcoRI fragment, of which the amino-acid sequence is identical with the enzyme isolated from B. psychrosaccharolyticus. The nucleotide sequences of LDH(P) and LDH(X) show 77% identity. Both genes are expressed in E. coli and the proteins could be isolated as shown by enzyme activity tests and determination of the N-terminal amino-acid sequence. However no expression of LDH(X) could be detected in B. psychrosaccharolyticus itself under the conditions chosen for oxygen induction of LDH. The function of the additional, non-expressed enzyme is not known.

L-lactate dehydrogenase of the psychrophilic bacterium B. psychrosaccharolyticus was isolated by a three-step procedure and its total amino-acid sequence determined by automated Edman degradation. The protein consists of 318 amino-acid residues and its calculated molecular mass is 35,254 Da. Most of the primary structure could be established by sequencing large peptide fragments obtained by chemical cleavages, namely with BNPS-skatole and with CNBr. Further fragmentations of two tryptophan peptides with the endoproteinase Lys-C and with diluted HCl resulted in shorter overlapping peptides, the analysis of which completed the sequence. The C-terminal sequence Glu-Gln was established by carboxypeptidase A experiments and was then verified by the analysis of short C-terminal tryptic and chymotryptic peptides. The first lactate dehydrogenase sequenced so far of a psychrophilic bacillus shows sequence homologies between 60% and 75% to the enzymes from the mesophilic B. megaterium and B. subtilis and the thermophilic B. stearothermophilus, B. caldolyticus and B. caldotenax. Within the 50 N-terminal residues, three additional sequences could be included in our comparisons. In this part of the molecule, sequence homologies between 56% and 74% were calculated.

Ferredoxin was isolated from the aerobic, thermophilic and acidophilic bacterium Bacillus acidocaldarius and its sequence of 78 amino acids completely determined by automated Edman degradation of the protein and of peptides derived from chemical cleavage between aspartic acid and proline and from enzymatic digestions. The optical spectrum of the oxidized protein has a broad maximum around 400 nm. The ferredoxin is thermostable: its absorbance begins to decrease only at incubation over 71 degrees C. The number of iron and inorganic sulphur atoms per molecule was determined to be 5.3 and 5.0, respectively. The calculated molar extinction coefficient was 23 000 M-1 X cm-1, the molecular mass of the apoferredoxin 8 872 Da. Contrary to all expectations, the sequence of B. acidocaldarius ferredoxin shows very little homology to that of B. stearothermophilus but closely resembles that of Thermus thermophilus.