Stronger Error Disturbance Relations for Incompatible Quantum Measurements

We formulate a new error-disturbance relation, which is free from explicit dependence upon variances in observables. This error-disturbance relation shows improvement over the one provided by the Branciard inequality and the Ozawa inequality for some initial states and for particular class of joint measurements under consideration. We also prove a modified form of Ozawa's error-disturbance relation. The later relation provides a tighter bound compared to the Ozawa and the Branciard inequalities for a small number of states.Comment: 5+pages, 3 figure

Optimization and scale-up of fermentation of glucansucrase and branched glucan by Pediococcus pentosaceus CRAG3 using Taguchi methodology in bioreactor

The present investigation focuses on screening and optimization of media components to enhance glucansucrase and glucan production by Pediococcus pentosaceus CRAG3 at shake-flask and bioreactor level using Taguchi orthogonal array design. A three-level Taguchi orthogonal array layout of L27 (33) was employed, in which six variables were studied for their influence on glucansucrase and glucan production. The results showed that sucrose, K2HPO4 and Tween-80 were the most significant factors to improve glucansucrase production while the glucan production was mostly affected by sucrose, peptone and K2HPO4. The optimized medium composition for maximum glucansucrase and glucan production were: sucrose 3.5% and 5%; yeast extract 0.2% and 2.0%; beef extract 0.5% and 0.5%; peptone 3.0% and 1.0%; K2HPO4 0.2% and 0.2%, and Tween-80 1.0 and 0.1%, respectively. The optimized medium gave 10.1 U/ml and 10.2 U/ml glucansucrase activity while glucan concentrations were 56 mg/ml and 80 mg/ml in shake flask and bioreactor level, respectively which were in good agreement with predicted values (10.1 U/ml and 54.5 mg/ml). The optimized medium gave 2 fold enhancement in enzyme activity and 4 fold increase in glucan concentration as compared to non-optimized medium (4.5 U/ml and 15 mg/ml, respectively) at shake flask level

Comments on the Refractive Index of Tin Sulphide Nano-crystalline Thin Films

The refractive indices of nano-crystalline thin films of Tin (IV) Sulphide (SnS) were investigated here. The experimental data conformed well with the single oscillator model for refractive indices. Based on the this, we explain the increasing trend of refractive index to the improvement in crystal ordering with increasing grain size.Comment: Nine figure

Facebook Role Play Addiction – A Comorbidity with Multiple Compulsive–Impulsive Spectrum Disorders

Problematic Internet use (PIU) is an emerging entity with varied contents. Behavioral addictions have high comorbidity of attention deficit hyperactivity disorder and obsessive–compulsive spectrum disorders. Social networking site (SNS) addiction and role playing game (RPG) addiction are traditionally studied as separate entities. We present a case with excessive Internet use, with a particular focus on phenomenology and psychiatric comorbidities. Case presentation Fifteen-year-old girl with childhood onset attention deficit disorder, obsessive–compulsive disorder, adolescent onset trichotillomania, and disturbed family environment presented with excessive Facebook use. Main online activity was creating profiles in names of mainstream fictional characters and assuming their identity (background, linguistic attributes, etc.). This was a group activity with significant socialization in the virtual world. Craving, salience, withdrawal, mood modification, and conflict were clearly elucidated and significant social and occupational dysfunction was evident. Discussion This case highlights various vulnerability and sociofamilial factors contributing to behavioral addiction. It also highlights the presence of untreated comorbidities in such cases. The difference from contemporary RPGs and uniqueness of role playing on SNS is discussed. SNS role playing as a separate genre of PIU and its potential to reach epidemic proportions are discussed. Conclusions Individuals with temperamental vulnerability are likely to develop behavioral addictions. Identification and management of comorbid conditions are important. The content of PIU continues to evolve and needs further study

Određivanje strukture netopljivog dekstrana proizvedenog s pomoću bakterije Leuconostoc mesenteroides NRRL B-1149 u prisutnosti maltoze

Leuconostoc mesenteroides NRRL B-1149 is known to produce dextran with 52 % α-(1→6) and 40 % α-(1→3) linkages. Low solubility of dextran in water is associated with the presence of high percentage of α-(1→3) linkages. Leuconostoc mesenteroides NRRL B-1149 produces two types of enzymes, dextransucrase and fructansucrase, which are active with sucrose and raffinose, respectively, as confirmed by the activity staining. The insoluble dextran was synthesized using partially purified dextransucrase in the presence of maltose. A water-soluble dextran was also produced by dextransucrase from Leuconostoc mesenteroides NRRL B-1149. The produced insoluble dextran was purified by alcohol precipitation, and then structurally characterized using FTIR, 1H NMR, and 13C NMR spectroscopic techniques. From the spectral analysis, it was confirmed that the insoluble dextran produced by Leuconostoc mesenteroides NRRL B-1149 contained dextran with α-(1→6) linkages and α-(1→3) branched linkages. The surface morphology of dried and powdered dextran studied using scanning electron microscopy revealed its fibrous structure.Leuconostoc mesenteroides NRRL B-1149 proizvodi dekstran s 52 % α-(1→6) i 40 % α-(1→3) veza. Mala topljivost dekstrana u vodi u svezi je s velikim udjelom α-(1→3) veza. Leuconostoc mesenteroides NRRL B-1149 proizvodi dva tipa enzima, dekstran saharazu, koja se aktivira u prisutnosti saharoze, i fruktan saharazu, koja djeluje u prisutnosti rafinoze, što je potvrđeno bojenjem s Coomasie brilijant plavom bojom. Netopljivi je dekstran sintetiziran pomoću djelomično pročišćene dekstran saharaze u prisutnosti maltoze. Pomoću istog je enzima proizveden i dekstran topljiv u vodi. Netopljivi je dekstran pročišćen taloženjem alkoholom i zatim okarakteriziran FTIR, 1H NMR i 13C NMR spektroskopijom. Spektralnim je analizama potvrđeno da je netopljivi dekstran, proizveden s pomoću Leuconostoc mesenteroides NRRL B-1149, povezan α-(1→6) i α-(1→3) vezama. Pomoću skenirajućeg elektronskog mikroskopa (SEM) utvrđeno je da osušeni i mljeveni dekstran ima vlaknastu strukturu

Physiological studies of Leuconostoc mesenteroides strain NRRL B-1149 during cultivation on glucose and fructose media

Glycosyltransferases are extracellular and cell-associated sucrase enzymes produced mainly by lactic acid bacteria Leuconostoc mesenteroides, oral Streptococcus species and also Lactobacillus species. According to the synthesized polymer (glucan or fructan) in the presence of sucrose, these enzymes are divided into two groups: glucosyltransferases (GTFs) and fructosyltransferases (FTFs). Only Streptococcus, Lactobacillus and Leuconostoc strains are known as producers of both GTFs and FTFs. The enzymes from Lactobacillus and Leuconostoc spp. are implicated in the synthesis of polymers and oligosaccharides (OS) important for human health because of their prebiotic properties and immunomodulating activity. In the present work, we studied the production of extracellular and cell-associated glycosyltransferases by Leuconostoc mesenteroides strain NRRL B-1149 during its growth on media containing glucose or fructose as a main carbon source. The enzyme activities, pH and biomass formation were measured and compared during the cultivation. We have shown that glucose and fructose have not an equal role for enzyme production. The highest extracellular activity was detected at the 4th hour during the cultivation of the strain in medium with fructose – 5.45 U/mg. When the strain was cultivated in medium with glucose, the maximum of extracellular enzyme activity was detected at the 5th hour of the cultivation but the measured activity was about 9 times lower compared to these, obtained after cultivation in fructose medium. The studied strain produced mainly extracellular glycosyltransferases in glucose or fructose medium, which were 92.4% and 97.1% of the total enzyme activity, respectively. In order to characterize the produced enzymes, cell-associated and extracellular enzymes were determined using SDS-PAGE and in situ Periodic Acid Schiff′s staining after incubation with 10% sucrose. When the investigated strain was grown in media with sucrose, glucose or fructose, several types of glycosyltransferases were detected – dextransucrase with molecular weight 180 kDa and two fructosyltransferases, corresponding to 120 kDa and 86 kDa molecular weights

Heterologous production, characterization and isolation of selected G protein-coupled receptors for structural studies

G protein-coupled receptors (GPCRs) play regulatory roles in many different physiological processes and they represent one of the most important class of drug targets. However, due to the lack of three-dimensional structures, structure based drug design has not been possible. The major bottleneck in getting three-dimensional crystal structure of GPCRs is to obtain milligram quantities of pure, homogenous and stable protein. Therefore, during my Ph.D. thesis, I focused on expression, characterization and isolation of three GPCRs namely human bradykinin receptor subtype 2 (B2R), human angiotensin II receptor subtype 1 (AT1aR), and human neuromedin U receptor subtype 2 (NmU2R). These receptors were heterologously produced in three different expression systems (i.e. Pichia pastoris, insect cells and mammalian cells), biochemically characterized and subsequently solubilized and purified for structural studies The human bradykinin receptor subtype 2 (B2R) is constitutively expressed in a variety of cells, including endothelial cells, vascular smooth muscle cells and cardiomyocytes. Activation of B2R is important in pathogenesis of inflammation, pain, tissue injury and cardioprotective mechanisms. During this study, recombinant B2R was produced in methylotrophic yeast Pichia pastoris (3.5 pmol/mg), insect cells (10 pmol/mg) and mammalian cells (60 pmol/mg). The recombinant receptor was characterized in terms of [3H] bradykinin binding, G protein coupling, localization, and glycosylation. Subsequently, it was solubilized and purified using affinity chromatography. Homogeneity and stability of purified B2R was monitored by gel filtration analysis. Milligram amounts of pure and stable receptor were obtained from BHK cells and Sf9 cells, which were used for three-dimensional crystallization attempts. The second receptor, which I worked on, is human angiotensin II receptor subtype 1 (AT1aR). AT1aR is distributed in smooth muscle cells, liver, kidney, heart, lung and testis. Activation of AT1aR is implicated in the regulation of blood pressure, hypertension and cardiovascular diseases. Recombinant AT1aR was produced at high levels in Pichia pastoris (167 pmol/mg), while at moderate levels in insect cells (29 pmol/mg) and mammalian cells (32 pmol/mg). The recombinant receptor was characterized in terms of [3H] angiotensin II binding, localization, and glycosylation. Subsequently, the receptor was solubilized and purified using affinity chromatography. Homogeneity and stability of purified AT1aR was monitored by gel filtration analysis. Milligram amounts of pure and stable receptor were obtained from Pichia pastoris, which were used for threedimensional crystallization attempts. In addition to B2R and AT1aR, I also attempted to produce and isolate the human neuromedin U receptor subtype 2 (NmU2R), which was deorphanized recently. It is found in highest abundance in the central nervous system, particularly the medulla oblongata, spinal cord and thalamus. The distribution of this receptor suggests its regulatory role in sensory transmission and modulation. During this study, recombinant NmU2R was produced in Pichia pastoris (6 pmol/mg) and BHK cells (9 pmol/mg). Recombinant receptor was characterized with regard to [125I] NmU binding, localization and glycosylation. Subsequently, the receptor was solubilized and purified using affinity chromatography. Due to its low expression level, further expression optimization is required in order to obtain milligram amounts for structural studies. The long-term goal of this study was to obtain three-dimensional crystal structure of recombinant GPCRs. However, 3-dimensional crystallization of human recombinant membrane proteins still remains a difficult task. On the other hand, recent advances in the solid-state NMR spectroscopy offer ample opportunities to study receptor-ligand systems, provided milligram quantities of purified receptor are available. Therefore, in parallel to 3-dimensional crystallization trials, purified B2R was also used for solid-state NMR analysis in order to investigate the receptor bound conformation of bradykinin. Preliminary results are promising and indicate significant structural changes in bradykinin upon binding to B2R. Further experiments are ongoing and will hopefully result in the structure of receptor bound bradykinin. One of the challenges in GPCR crystallization is the small hydrophilic surface area that is available to make crystal contacts. One possibility to overcome this problem can be the reconstitution of a GPCR complex with an interacting protein for cocrystallization. For this purpose, I coexpressed B2R and AT1aR, which form a stable heterodimer complex, in BHK cells. I could successfully isolate the heterodimer complex by using two-step affinity purification. Unfortunately, this complex was not stable over time and disassociates within three days of purification. However, during coexpression of B2R and AT1aR in BHK cells, I observed that B2R was localized in the plasma membrane in coexpressing cells while it was retained intracellularly when expressed alone. This coexpression of AT1aR with B2R resulted in a four-fold increase in [3H] bradykinin binding sites on the cell surface. In addition, these two receptors were cointernalized in response to their individual specific ligands. Interestingly, colocalization of B2R and AT1aR was also found in human foreskin fibroblasts (which endogenously express both receptors), in line with the possibility that heterodimerization may be required for surface localization of B2R in native tissues as well. This is the first report where surface localization of a peptide GPCR is triggered by a distantly related peptide GPCR. These data support the hypothesis that heterodimerization may be a prerequisite for cell surface localization of some GPCRs. A second approach that I followed to stabilize the purified B2R was to reconstitute the B2R-β-arrestin complex. β-arrestin is a cytosolic protein that participates in agonist mediated desensitization of GPCRs and therefore dampens the cellular responses initiated by the activation of GPCRs. I tried to reconstitute B2R-β-arrestin complex in vitro by mixing purified B2R and purified β-arrestin. But, no interaction of these two proteins was observed in the pull-down assays. However, a C-terminal mutant of B2R (where a part of the C-terminus of the B2R is exchanged with that of the vasopressin receptor) was found to interact with β-arrestin in vitro as revealed by pull-down assays. In conclusion, this work establishes the production, characterization and isolation of three recombinant human GPCRs. Recombinant receptors were produced in milligram amounts and therefore, pave the way for structural analysis. The heterodimer complex of B2R-AT1aR and B2R-β-arrestin complex can be of great help during crystallization. In addition, it was also found for the first time that the surface localization of a peptide GPCR can be triggered by heterodimerization with a distantly related peptide GPCR.Die G-Protein-gekoppelten Rezeptoren (GPCRs) stellen die größte Familie der Zelloberflächenrezeptoren dar. 1-5% des Wirbeltiergenoms kodiert für diese Rezeptorfamilie. Im Humangenom sind etwa 800-1000 Gene vertreten, die für GPCRs kodieren. Trotz der großen Unterschiede in ihrer Sequenz und Aktivierung haben alle GPCRs zwei Gemeinsamkeiten: (1) Ihre Architektur wird durch sieben Transmembranhelices beschrieben. (2) Ihre Funktion in der Signaltransduktion üben alle durch Aktivierung der heterotrimeren Guanylnukleotid-Bindeproteine (GProteine) aus. Die GPCRs sind an der Regulierung einer Vielzahl von physiologischen Prozessen beteiligt und stellen daher wichtige Ziele für die Medikamentenentwicklung dar. Bisher gibt es kaum Möglichkeiten zur strukturbasierenden Medikamentenentwicklung, da, bis auf das Rinder-Rhodopsin, nur sehr wenige Informationen zur dreidimensionalen Struktur von GPCRs verfügbar sind. Das Rinder-Rhodopsin nimmt allerdings unter den GPCRs eine Sonderstellung ein. Im Gegensatz zu allen übrigen GPCRs bindet es seinen Liganden, 11-cis Retinal, kovalent und liegt dann in der nicht-aktivierten Form vor. Zudem kann Rhodopsin in großen Mengen aus Rinderretina isoliert werden, wohingegen die übrigen GPCRs nur in geringen Mengen in ihren natürlichen Geweben vorkommen. Die vorliegende Arbeit verfolgt drei Ziele: Erstens sollen GPCRs durch heterologe Expression in hohen Ausbeuten hergestellt und biochemisch charakterisiert werden. Die Etablierung eines Solubilisierungs- und Aufreinigungsprotokolls stellt das zweite Ziel dar. Drittens soll die Interaktion von Ligand und Rezeptor mittels verschiedener Techniken untersucht werden. Grund für die erste Zielsetzung ist die geringe Verfügbarkeit reinen, homogenen und stabilen Proteins im Milligramm-Maßstab, welches die größte Hürde für strukturelle Untersuchungen von GPCRs darstellt. Hier wurden verschiedene Expressionssysteme zur heterologen Produktion von Membranproteinen etabliert. Die Wahl des Expressionssystems ist hierbei entscheidend, um posttranslationale Modifikationen wie Glykosylierung sowie die korrekte Faltung des Rezeptors zu gewährleisten. Neben E. coli haben sich hierbei vor allem eukaryotische Expressionssystems wie Pichia pastoris bewährt. In der vorliegenden Arbeit wurden drei GPCRs hergestellt und analysiert: der humane Bradykinin Rezeptor Typ 2 (B2R), der humane Angiotensin II Rezeptor Typ 1 (AT1aR) und der humane Neuromedin U Rezeptor Typ 2 (NmU2R). Diese drei Rezeptoren wurden in drei Expressionsystemen (Pichia pastoris, Insektenzellen und Säugerzellen) heterolog produziert und biochemisch charakterisiert. Für jedes der drei Proteine wurden Solubilisierungs- und Aufreinigungsprotokolle etabliert. Die aufgereinigten Proteine wurden anschließend für Kristallisationsexperimente, für Festkörper NMR Untersuchungen und weitere Experimente eingesetzt. Der erste untersuchte Rezeptor, B2R, kann vor allem in Endothelzellen, vaskulären glatten Muskelzellen und Kardiomyozyten nachgewiesen werden. Seine Aktivierung spielt bei der Entstehung von Entzündungen, Schmerz, Gewebsverletzung sowie herzschützenden Mechanismen eine Rolle. Im Rahmen der Doktorarbeit wurde B2R in der Hefe Pichia pastoris (3,5 pmol/mg), in BHK-Zellen (10 pmol/mg) und in Sf9-Zellen (60 pmol/mg) erfolgreich rekombinant produziert. Zur Charakterisierung wurde die Bindung des Liganden [3H] Bradykinin, die GProtein- Kopplung, zelluläre Lokalisierung sowie die Glykosylierung des Rezeptors untersucht. Der heterolog produzierte Rezeptor konnte in hoher Reinheit isoliert werden. Homogenität und Stabilität des aufgereinigten Proteins wurden mittels Gelfiltration analysiert. Aus BHK und Sf9 Zellen konnten Milligramm-Mengen reinen und stabilen Rezeptors isoliert werden, die zu Kristallisationsexperimenten verwendet wurden. Hier zeigten sich kristallartige Strukturen, die zur Zeit weiter charakterisiert werden. Der zweite untersuchte Rezeptor, AT1aR, kann in glatten Muskelzellen, Leber, Nieren, Herz, Lunge und Hoden nachgewiesen werden. Die Aktivierung dieses Rezeptors spielt eine Rolle bei der Regulation des Blutdrucks und bei cardiovaskulären Erkrankungen. Rekombinanter AT1aR konnte mit hoher Ausbeute (167 pmol/mg) in Pichia pastoris hergestellt werden. Die Ausbeute bei Produktion in Insektenzellen (29 pmol/mg) und Säugerzellen (32 pmol/mg) lag im mittleren Bereich. Der rekombinante Rezeptor wurde hinsichtlich der Bindung von [3H] Angiotensin II, der zellulären Lokalisierung und Glykosylierung charakterisiert. Im Anschluss wurde er erfolgreich mittels Affinitätschromatographie gereinigt. Homogenität und Stabilität des gereinigten AT1aR wurden mittels Gelfiltration analysiert. Aus Pichia pastoris konnte das Protein im Milligramm-Maßstab isoliert werden, so dass Kristallisationsexperimente möglich waren. Dem dritten Rezeptor, NmU2R, konnte erst kürzlich sein Ligand, Neuromedin U, zugeordnet werden. Der Rezeptor ist im zentralen Nervensystem, und hier insbesondere in der Medulla oblongata, dem Rückenmark und dem Thalamus lokalisiert. Aufgrund dieser Verteilung wird angenommen, dass er eine Rolle in der Regulation der Weiterleitung sensorischer Nervenimpulse sowie deren Modulation spielt. Während meiner Arbeit konnte ich bei der heterologen Produktion des Rezeptors Ausbeuten von 6 pmol/mg in Pichia pastoris und 9 pmol/mg in BHK Zellen erzielen. Der rekombinante Rezeptor wurde mittels Bindung eines Radioliganden ([125I] NmU) charakterisiert. Weiterhin wurde die zellulärer Lokalisierung und Glykosylierung des GPCRs untersucht. Obwohl der rekombinante NmU2R erfolgreich isoliert werden konnte, sind auf Grund der geringen Produktionsmengen zur Zeit keine Struktur untersuchungen möglich. Zur Analyse der pharmakologisch wichtigen Ligand-Rezeptor- Wechselwirkung wurde Festkörper NMR Spektroskopie eingesetzt. Durch die Verwendung von selektiv mit 13C und 15N markierten Peptiden können Konformationsänderung des Peptidliganden beim Binden des Rezeptors untersucht werden. Die Bestimmung der genauen Konformation des gebunden Liganden ist für die Medikamentenentwicklung von Bedeutung. In der vorliegenden Arbeit wurde mittels der Festkörper NMR Spektroskopie die Konformation des rezeptorgebunden Liganden, Bradykinin, untersucht. Die ersten Ergebnisse weisen auf signifikante Strukturänderungen Bradykinins hin, sobald es an den B2R bindet. Untersuchungen bezüglich Wechselwirkung von GPCRs mit anderen Protein sind auch für die Kristallisation relevant. Eine der Herausforderungen in der Kristallisation von GPCRs ist die kleine hydrophile Oberfläche, die zur Bildung von Kristallkontakten im Kristallgitter oft nicht ausreichend ist. Eine Möglichkeit, dieses Problem zu lösen, ist die Bildung eines stabilen Komplexes aus dem Rezeptor und einem interagierenden Protein. Zusätzlich kann der Rezeptor durch die Interaktion in eine weniger flexible Form überführt werden, was die Kristallisation und die spätere Strukturbestimmung erleichtern kann. Basierend auf diesem Ansatz wurden B2R und AT1aR, die einen stabilen heterodimeren Komplex bilden, in BHK Zellen ko-exprimiert. Bemerkenswert war hierbei dass B2R im Komplex mit AT1aR in der Plasmamembran vorzufinden war, während B2R alleine hauptsachlich in intrazellulären Membranen exprimiert wurde. Weiterhin führte die Koexpression der beiden Rezeptoren zu einem vierfachen Anstieg der [3H] Bradykinin Bindungsstellen auf der Zelloberfläche. Es konnte ebenfalls nachgewiesen werden, dass nach der Stimulation mit nur einem der beiden rezeptorspezifischen Liganden beide GPCRs zusammen internalisiert wurden. Dieses Phänomen wurde auch in menschlichen Vorhaut- fibroblasten nachgewiesen, in denen beide Rezeptoren vorkommen. Die erhaltenen Ergebnisse deuten darauf hin, dass auch in nativen Geweben die Anwesenheit des AT1aR für die Expression und den Transfer des B2R zur Plasmamembran nötig ist. Diese Daten unterstützen die Hypothese, dass Heterodimerisierung eine Voraussetzung für die Zelloberflächenlokalisierung bestimmter GPCRs ist. Der Ko-Komplex aus B2R und AT1aR konnte mittels dualer Affinitätschromatographie isoliert, wie durch SDS-PAGE Analyse, analytische Gelfiltration und Bindung von Radioliganden gezeigt werden konnte. „Pull-down“ Experimente, die drei Tage nach der Reinigung durchgeführt wurden, wiesen darauf hin, dass der Ko-Komplex nicht stabil war und zerfiel. Bei der Reinigung von Membranproteinen verursacht der Verlust von Lipiden während des Isolationsprozeßes oft eine Beeinträchtigung der Stabilität des Proteins. Auch das verwendete Detergenz beeinflusst die Stabilität von Membranproteinen. Experimente zur Verbesserung der Langzeitstabilität des Komplexes durch Zugabe von Lipiden und anderen Detergenzien sind in Vorbereitung. Die Bildung von Ko-Komplexen wurde zusätzlich mit Beta-Arrestin, einem Inhibitor der Kopplung von G-Proteinen und ihren Rezeptoren untersucht. Beta- Arrestin ist ein zytosolisches Protein, dass an der Desensibilisierung der Agoniststimulierten GPCRs beteiligt ist. Versuche, eine Ko-Komplexbildung aus gereinigtem B2R und gereinigtem Beta-Arrestin in vitro zu erzielen, schlugen fehl. In „Pull-Down“ Experimenten konnte keine Interaktion nachweisen werden. Wurde anstatt des nativen B2R eine C-terminale Mutante, bei welcher der C-Terminus des B2R gegen den des Vasopressinrezeptors ausgetauscht worden war, verwendet, konnte in vitro Ko- Komplexbildung mit Beta-Arrestin festgestellt werden. Mit Experimenten zur Bestimmung der Langzeitstabilität des Ko-Komplexes sowie zur Ko-Kristallisations wurde begonnen. Im Rahmen der vorliegenden Arbeit wurde die Produktion, Charakterisierung und Aufreinigung von drei rekombinanten humanen GPCRs etabliert. Die rekombinanten Rezeptoren wurden im Milligramm-Maßstab produziert. Damit ist die erste, wesentliche Hürde zur Strukturanalyse genommen. Der B2R-β-Arrestin Komplex kann sich als vorteilhaft für die Kristallisation herausstellen. Zusätzlich konnte zum ersten Mal gezeigt werden, dass der Transfer eines GPCRs an die Zelloberfläche von der Heterodimerisierung mit einem anderen nicht-verwandten GPCR abhängig sein kann

Asymptotic analysis and reflection photoelasticity for the study of transient crack propagation in graded materials

The behavior of a rapidly moving transient crack in functionally graded materials (FGMs) is investigated theoretically and experimentally. First, a systematic theoretical analysis is presented for the development of the transient elastodynamic local stress, strain, and displacement field expansions near a growing mixed mode crack tip in FGMs. The crack propagation direction is assumed to be inclined to the direction of the property variation. The displacement potential approach in conjunction with asymptotic analysis is utilized to derive explicit expressions for stress, strain, and in-plane displacement fields. The transient crack growth is assumed to include processes in which both the crack tip speed and the dynamic stress intensity factor are differentiable functions of time. These stress fields are used to generate the contours of constant maximum shear stress (isochromatics fringes) and the effect of transient crack growth on these contours is discussed. To further understand the transient crack growth behavior, a series of dynamic fracture experiments are performed with functionally graded material fabricated in-house. The phenomenon of transition from a static crack to a dynamic mode I crack is examined in these experiments. The full-field stress data around the crack is recorded using dynamic photoelasticity and high-speed digital photography. Due to opaqueness of FGMs, birefringent coatings are employed to obtain the full-field isochromatics around the crack tip. The stress field expansions developed in the first part of the study are used to interpret the experimental observations. The results of the experiments showed that the higher order transient expansion provides an accurate representation of crack tip fields under severe transient conditions

16S rRNA-Based Identification of a Glucan-Hyperproducing Weissella confusa

A gram-positive, nonmotile, irregular, short, rod-shaped new strain of Weissella confusa bacterium was isolated from fermented cabbage. The isolate was physiologically and biochemically characterised. The 16S rDNA was amplified by polymerase chain reaction (PCR). The isolate was identified as Weissella confusa (GenBank accession number: GU138518.1) based on nucleotide homology and phylogenetic analysis. The isolate produces glucansucrase when grown in sucrose-supplemented culture medium which catalyses glucan formation. This novel isolate possesses high capacity of industrial use due to its high productivity of glucan (34 mg/mL) as compared to other strains reported. The optimum temperature for glucansucrase production was 25°C. The shaking condition gave an enzyme activity of 6.1 U/mL which was 1.5 times higher than that given by static condition (4.1 U/mL). The temperature 35°C, pH 5.4, and ionic strength 10–20 mM were optimum for enzyme assay. This investigation unraveled the abundance of industrially valuable microflora of the north east India

STABILITY AND MOLECULAR CHARACTERIZATION TO SCREEN OUT HEAT TOLERANT GENOTYPES OF CHICKPEA (CICER ARIETINUM L.)

Thirty diverse genotypes sown in three different dates were screened using twenty SSR primers. Field observations were recorded for 14 phenological and morphological characters. Among which days to 50% flowering has less susceptibility for these genotypes against environmental fluctuations. The component G × E interaction were found significant for harvest index, total seeds per pod and effective pods per plant, hundred seed weight, biological yield and seed yield. The highest gene diversity was found in TA 146 (0.898), followed by primer TR 19 (0.884) and ICCM 0249 (0.877). Based on a dendrogram all the 30 genotypes were grouped into two clusters A and B. The genotype JG 19 showed highest gene similarities (96.77%) with JG 21, similarly MP-JG 99-115 showed 96.77% resemblance with JG 17 and JG14-11 which are observed in same cluster. The genotypes, ICCV 07102, JG 21 and JG 22 were found suitable for all the dates of sowing while genotypes JG 16, JG 21, GG 2, ICC 4958, and JG 22 were suitable for late and very late sowing