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Differentiation between Phaeocystis pouchetii (Har.) Lagerheim and Phaeocystis globosa Scherffel
(1987)
Developing a new production host from a blueprint: Bacillus pumilus as an industrial enzyme producer
(2014)
The metabolic activity of Chinese hamster ovary (CHO) cells was observed using a light-addressable potentiometric sensor (LAPS). The dependency toward different glucose concentrations (17–200 mM) follows a Michaelis–Menten kinetics trajectory with Kₘ = 32.8 mM, and the obtained Kₘ value in this experiment was compared with that found in literature. In addition, the pH shift induced by glucose metabolism of tumor cells transfected with the HPV-16 genome (C3 cells) was successfully observed. These results indicate the possibility to determine the tumor cells metabolism with a LAPS-based measurement device.
The light-addressable potentiometric sensor (LAPS) is a semiconductor-based potentiometric sensor using a light probe with an ability of detecting the concentration of biochemical species in a spatially resolved manner. As an important biomedical sensor, research has been conducted to improve its performance, for instance, to realize high-speed measurement. In this work, the idea of facilitating the device-level simulation, instead of using an equivalent-circuit model, is presented for detailed analysis and optimization of the performance of the LAPS. Both carrier distribution and photocurrent response have been simulated to provide new insight into both amplitude-mode and phase-mode operations of the LAPS. Various device parameters can be examined to effectively design and optimize the LAPS structures and setups for enhanced performance.
Clostridium propionicum is the only organism known to ferment β-alanine, a constituent of coenzyme A (CoA) and the phosphopantetheinyl prosthetic group of holo-acyl carrier protein. The first step in the fermentation is a CoA-transfer to β-alanine. Subsequently, the resulting β-alanyl-CoA is deaminated by the enzyme β-alanyl-CoA:ammonia lyase (Acl) to reversibly form ammonia and acrylyl-CoA. We have determined the crystal structure of Acl in its apo-form at a resolution of 0.97 Å as well as in complex with CoA at a resolution of 1.59 Å. The structures reveal that the enyzme belongs to a superfamily of proteins exhibiting a so called “hot dog fold” which is characterized by a five-stranded antiparallel β-sheet with a long α-helix packed against it. The functional unit of all “hot dog fold” proteins is a homodimer containing two equivalent substrate binding sites which are established by the dimer interface. In the case of Acl, three functional dimers combine to a homohexamer strongly resembling the homohexamer formed by YciA-like acyl-CoA thioesterases. Here, we propose an enzymatic mechanism based on the crystal structure of the Acl·CoA complex and molecular docking. Proteins 2014; 82:2041–2053. © 2014 Wiley Periodicals, Inc.
The Gram-positive endospore-forming bacterium Bacillus licheniformis can be found widely in nature and it is exploited in industrial processes for the manufacturing of antibiotics, specialty chemicals, and enzymes. Both in its varied natural habitats and in industrial settings, B. licheniformis cells will be exposed to increases in the external osmolarity, conditions that trigger water efflux, impair turgor, cause the cessation of growth, and negatively affect the productivity of cell factories in biotechnological processes. We have taken here both systems-wide and targeted physiological approaches to unravel the core of the osmostress responses of B. licheniformis. Cells were suddenly subjected to an osmotic upshift of considerable magnitude (with 1 M NaCl), and their transcriptional profile was then recorded in a time-resolved fashion on a genome-wide scale. A bioinformatics cluster analysis was used to group the osmotically up-regulated genes into categories that are functionally associated with the synthesis and import of osmostress-relieving compounds (compatible solutes), the SigB-controlled general stress response, and genes whose functional annotation suggests that salt stress triggers secondary oxidative stress responses in B. licheniformis. The data set focusing on the transcriptional profile of B. licheniformis was enriched by proteomics aimed at identifying those proteins that were accumulated by the cells through increased biosynthesis in response to osmotic stress. Furthermore, these global approaches were augmented by a set of experiments that addressed the synthesis of the compatible solutes proline and glycine betaine and assessed the growth-enhancing effects of various osmoprotectants. Combined, our data provide a blueprint of the cellular adjustment processes of B. licheniformis to both sudden and sustained osmotic stress.
The composition and physiochemical properties of aquatic-phase natural organic matter (NOM) are most important problems for both environmental studies and water industry. Laser desorption/ionization (LDI) mass spectrometry facilitated successful examinations of NOM, as humic and fulvic acids in NOM are readily ionized by the nitrogen laser. In this study, hydrophobic NOMs (HPO NOMs) from river, reservoir and waste water were characterized by this technique. The effect of analytical variables like concentration, solvent composition and laser energy was investigated. The exact masses of small molecular NOM moieties in the range of 200–1200 m/z were determined in reflectron mode. In addition, spectra of post-source-decay experiments in this range showed that some compounds from different natural NOMs had the same fragmental ions. In the large mass range of 1200–15 000 Da, macromolecules and their aggregates were found in HPO NOMs from natural waters. Highly humic HPO exhibited mass peaks larger than 8000 Da. On the other hand, the waste water and reservoir water mainly had relatively smaller molecules of about 2000 Da. The LDI-MS measurements indicated that highly humic river waters were able to form large aggregates and membrane foulants, while the HPO NOMs from waste water and reservoir water were unlikely to form large aggregates. Copyright © 2014 John Wiley & Sons, Ltd.
Bacillus pumilus reveals a remarkably high resistance to hydrogen peroxide provoked oxidative stress
(2014)
Bacillus pumilus is characterized by a higher oxidative stress resistance than other comparable industrially relevant Bacilli such as B. subtilis or B. licheniformis. In this study the response of B. pumilus to oxidative stress was investigated during a treatment with high concentrations of hydrogen peroxide at the proteome, transcriptome and metabolome level. Genes/proteins belonging to regulons, which are known to have important functions in the oxidative stress response of other organisms, were found to be upregulated, such as the Fur, Spx, SOS or CtsR regulon. Strikingly, parts of the fundamental PerR regulon responding to peroxide stress in B. subtilis are not encoded in the B. pumilus genome. Thus, B. pumilus misses the catalase KatA, the DNA-protection protein MrgA or the alkyl hydroperoxide reductase AhpCF. Data of this study suggests that the catalase KatX2 takes over the function of the missing KatA in the oxidative stress response of B. pumilus. The genome-wide expression analysis revealed an induction of bacillithiol (Cys-GlcN-malate, BSH) relevant genes. An analysis of the intracellular metabolites detected high intracellular levels of this protective metabolite, which indicates the importance of bacillithiol in the peroxide stress resistance of B. pumilus.
Synthesis of derivatives of the peptide sequence L-pyroglutamyl-L-phenylalanyl-L-aspartyl-glycyl-L-lysyl-glycyl-glycyl-glycine as the antigenic determinant representing the N-terminal non-helical region of the α-2-chain of rabbit skin collagen, and conjugation to two different polypeptide carriers, are described.
Die tert.-Butyloxycarbonyl-Gruppe (Boc) läßt sich mittels reiner Trifluoressigsäure nicht selektiv neben dem Benzyloxycarbonyl-Rest (Z) abspalten. Das gelingt auch nicht mit Lösungen von Trifluoressigsäure bzw. Chlorwasserstoff in organischen Lösungsmitteln. Kern-substituierte Z-Gruppen wie Z(pCl), Z(mCl) oder Z(pNO₂) sind zwar stabiler, werden aber von den obengenannten Reagenzien ebenfalls angegriffen bzw. sind nicht mehr acidolytisch abspaltbar. – Mit 70proz. wäßriger Trifluoressigsäure gelingt die Abspaltung von Boc neben Z dagegen fast selektiv; dabei werden aber Benzylester, besonders Glutaminsäure-γ-benzylester, teilweise hydrolysiert, während Methyl- sowie Äthylester nahezu beständig sind. Die Brauchbarkeit des Abspaltungsverfahrens wird anhand der schrittweise durchgeführten Synthese zweier Heptapeptid-Derivate gezeigt. – Ähnlich spezifisch gelingt die Abspaltung von Boc mit Bortrifluorid-ätherat in Eisessig; Benzylester sind gegenüber diesem Reagenz stabiler als gegen wäßrige Trifluoressigsäure. Das Bortrifluorid-Verfahren eignet sich besonders für die Abspaltung von Boc-Gruppen neben säurelabilen Thiol-Schutzgruppen (Tetrahydropyranyl- bzw. Trityl-Rest) sowie neben dem Cyclocystinyl-Rest. Die Leistungsfähigkeit der Methode wird durch die Synthese zweier Peptid-Derivate mit S-Trityl-Schutzgruppen belegt. Als Nebenreaktion ist die Acetylierung von aliphatischen Hydroxylgruppen möglich. Sie läßt sich vermeiden, wenn man die Spaltung in anderen Lösungsmitteln durchführt. Die als Modellverbindungen für Stabilitätsuntersuchungen verwendeten Nε-acylierten Lysin-Derivate werden mit dem Aminosäureanalysator quantitativ neben Lysin bestimmt.
Es wird die Synthese der Sequenz A 6–9 des Schafinsulins in der geschützten Form Boc-Cys-Cys-Ala-Gly-OBuᵗ (5) sowie das Verhalten dieses monomeren cyclischen Cystinpeptidderivates gegenüber den in der Peptidchemie gebräuchlichen Reagenzien Bortrifluorid/Eisessig, Triäthylamin und Hydrazinhydrat beschrieben.
Die Darstellung der N-[2-(p-Biphenylyl)isopropyloxycarbonyl]-Derivate (Bpoc-Derivate) des Cysteins unter Verwendung der Thiolschutzgruppen Tetrahydropyranyl (Thp) für 1, Diphenylmethyl (Dpm) für 2, Trityl (Trt) für 3 und S-tert.-Butyl (SBut) für 4 sowie die Synthese von aktivierten Estern der Bpoc-Derivate des Glycins (5), Isoleucins (6) und Prolins (7) werden beschrieben. An einem Beispiel wird die Möglichkeit aufgezeigt, die Bpoc-Gruppe über das Bpoc-Azid nachträglich in den Peptidverband einzuführen.
Die Synthese der Sequenzen A2—21 (13) und A1—21 (15) der Schafinsulin-A-Kette als monomere cyclische Dicystinpeptidderivate wird beschrieben. Die intrachenaren Cystinbrücken A6—7 und A 11 —20 vermitteln die Löslichkeit dieser Derivate in Dimethylformamid und ermöglichen erstmalig die Reindarstellung vollgeschützter Insulin-A-Kettenderivate. Die während der Synthese eingesetzten Schutzgruppen lassen sich mittels Trifluoressigsäure und 2-Mercaptoäthanol quantitativ entfernen.
Thermal synthesis of the optical pure pentapeptide derivative Z-(L)-Ala-(L)-Phe-Gly-(L)-Phe-Gly-OMe
(1980)
Synthese der Sequenz 71—86 des Humanproinsulins, III : Synthese über die Fragmente 71—78 und 79—86
(1979)
Zur Synthese der Hühnerinsulin-A-Kette, I : Darstellung der Fragmente A1–8, A9–15, A1–7 und A8–15
(1979)
Poly(vinyl acetate), PVAc, with a degree of polymerization Xn = 10 was prepared by chain-transfer radical polymerization using carbon tetrachloride and used as oligomeric plasticizer for commercial PVAc. However, the chlorinated chain ends cause a low thermal stability requiring mild Cl/H substitution. The product exhibits high thermal stability and excellent melt-compounding properties. Blends of oligomeric and commercial PVAc show single glass transition temperatures which decrease with higher oligomer content and exhibit small negative deviations from Fox' linear additivity rule. This indicates plasticization and miscibility being mainly due to entropic effects. Injection-moulded thick specimens show ductile behaviour at oligomer contents >10 wt %, while sheets with a thickness of 0.2–0.5 mm appear flexible already at 7.5 wt %. The oxygen permeability coefficients are an order of magnitude lower than those of low-density polyethylene. Due to the sum of their properties, the plasticized sheets present a promising alternative in the preparation of barrier materials.