Alternative final steps in berberine biosynthesis in Coptis japonica cell cultures

In Coptis japonica cell cultures an alternative pathway has been discovered which leads from (S)-tetrahydrocolumbamine via (S)-canadine to berberine. The two enzymes involved have been partially purified. (S)-Tetrahydrocolumbamine is stereospecifically transformed into (S)-canadine under formation of the methylenedioxy bridge in ring A. This new enzyme was named (S)-canadine synthase. (S)-Canadine in turn is stereospecifically dehydrogenated to berberine by an oxidase, (S)-canadine oxidase (COX), which was partially purified (25-fold). This enzyme has many physical properties in common with the already known (S)-tetrahydroprotoberberine oxidase from Berberis but grossly differs from the latter enzyme in its cofactor requirement (Fe) and its substrate specificity. Neither (S)-norreticuline nor (S)-scoulerine serves as substrate for the Coptis enzyme, while both substrates are readily oxidized by the Berberis enzyme. The four terminal enzymes catalyzing the pathway from (S)-reticuline to berberine are housed in Berberis as well as in Coptis in smooth vesicles with a density of =1.14 g/ml. These vesicles have been enriched and characterized by electron microscopy

Studies on processing, particle formation, and immunogenicity of the HIV-1 gag gene product: a possible component of a HIV vaccine

Antigens in a particulate conformation were shown to be highly immunogenic in mammals. For this reason, the particle forming capacity of derivatives of the HIV-1 group specific core antigen p55 gag was assayed and compared dependent on various expression systems: recombinant bacteria, vaccinia- and baculoviruses were established encoding the entire core protein p55 either in its authentic sequence or lacking the myristylation consensus signal. Moreover, p55 gag was expressed in combination with the protease (p55-PR) or with the entire polymerase (p55-pol), respectively. Budding of 100-160 nm p55 core particles, resembling immature HIV-virions, was observed in the eucaryotic expression systems only. In comparison to the vaccinia virus driven expression of p55 in mammalian cells, considerably higher yields of particulate core antigen were obtained by infection of Spodoptera frugiperda (Sf9) insect cells with the recombinant Autographa californica nuclear polyhedrosis (AcMNPV) baculovirus. Mutation of the NH2-terminal myristylation signal sequence prevented budding of the immature core particles. Expression of the HIV p55-PR gene construct by recombinant baculovirus resulted in complete processing of the p55 gag precursor molecule in this system. The introduction of an artificial frameshift near the natural frameshift site resulted in constitutive expression of the viral protease and complete processing of p55, both in Escherichia coli and in vaccinia virus infected cells. Interestingly, significant processing of p55 resembling that of HIV infected H9 cells could also be achieved in the vaccinia system by fusing the entire pol gene to the gag gene. Moreover, processing was not found to be dependent on amino-terminal myristylation of the gag procursor molecule, which is in contrast to observations with type C and type D retrovirus. However, complete processing of p55 into p24, p17, p9 and p6 abolished particle formation. Purified immature HIV-virus like particles were highly immunogenic in rabbits, leading to a strong humoral immune response after immunization. Empty immature p55 gag particles represent a noninfectious and attractive candidate for a basic vaccine component

Winter precipitation trends for two selected European regions over the last 500 years and their possible dynamical background

Summary: We analyse winter (DJF) precipitation over the last 500 years on trends using a spatially and temporally highly resolved gridded multi-proxy reconstruction over European land areas. The trends are detected applying trend matrices, and the significance is assessed with the Mann-Kendall-trend test. Results are presented for southwestern Norway and southern Spain/northern Morocco, two regions that show high reconstruction skill over the entire period. The absolute trend values found in the second part of the 20th century are unprecedented over the last 500 years in both regions. During the period 1715-1765, the precipitation trends were most pronounced in southwestern Norway as well as southern Spain/northern Morocco, with first a distinct negative trend followed by a positive countertrend of similar strength. Relating the precipitation time series to variations of the North Atlantic Oscillation Index (NAOI) and the solar irradiance using running correlations revealed a couple of instationarities. Nevertheless, it appears that the NAO is responsible in both regions for most of the significant winter precipitation trends during the earlier centuries as well as during recent decades. Some of the significant winter precipitation trends over southwestern Norway and southern Spain/northern Morocco might be related to changes in the solar irradianc

How Does the Choice of the Lower Boundary Conditions in Large-Eddy Simulations Affect the Development of Dispersive Fluxes Near the Surface?

Large-eddy simulations (LES) are an important tool for investigating the longstanding energy-balance-closure problem, as they provide continuous, spatially-distributed information about turbulent flow at a high temporal resolution. Former LES studies reproduced an energy-balance gap similar to the observations in the field typically amounting to 10–30% for heights on the order of 100 m in convective boundary layers even above homogeneous surfaces. The underestimation is caused by dispersive fluxes associated with large-scale turbulent organized structures that are not captured by single-tower measurements. However, the gap typically vanishes near the surface, i.e. at typical eddy-covariance measurement heights below 20 m, contrary to the findings from field measurements. In this study, we aim to find a LES set-up that can represent the correct magnitude of the energy-balance gap close to the surface. Therefore, we use a nested two-way coupled LES, with a fine grid that allows us to resolve fluxes and atmospheric structures at typical eddy-covariance measurement heights of 20 m. Under different stability regimes we compare three different options for lower boundary conditions featuring grassland and forest surfaces, i.e. (1) prescribed surface fluxes, (2) a land-surface model, and (3) a land-surface model in combination with a resolved canopy. We show that the use of prescribed surface fluxes and a land-surface model yields similar dispersive heat fluxes that are very small near the vegetation top for both grassland and forest surfaces. However, with the resolved forest canopy, dispersive heat fluxes are clearly larger, which we explain by a clear impact of the resolved canopy on the relationship between variance and flux–variance similarity functions

The role of working memory and contextual constraints in children's processing of relative clauses

An auditory sentence comprehension task investigated the extent to which the integration of contextual and structural cues was mediated by verbal memory span with 32 English-speaking 6- to 8-year old children. Spoken relative clause sentences were accompanied by visual context pictures which fully (depicting the actions described within the relative clause) or partially (depicting several referents) met the pragmatic assumptions of relativisation. Comprehension of the main and relative clauses of centre-embedded and right-branching structures was compared for each context. Pragmatically-appropriate contexts exerted a positive effect on relative clause comprehension, but children with higher memory spans demonstrated a further benefit for main clauses. Comprehension for centre-embedded main clauses was found to be very poor, independently of either context or memory span. The results suggest that children have access to adult-like linguistic processing mechanisms, and that sensitivity to extra-linguistic cues is evident in young children and develops as cognitive capacity increases

Optimizing the parameters for hydro-jet dissection in fatty tissue — A morphological Ex Vivo analysis

Summary: Background: The advantage of water-jet dissection is the preservation of vessels and nerves. Especially in liver surgery, blood loss can be significantly decreased. The use of water-jet dissectors in other fields of surgery is currently under investigation. The preparation of vessels in fatty tissue is of special interest for plastic surgeons. The optimal technical parameters were investigated. Methods: Abdominal fat tissue of fresh cadavers was cut under standardized conditions with different parameters of the water-jet dissector. Results: One single pass at a cutting pressure between 20 and 60 Bar makes an incision of 8 mm. Deeper cuts can be achieved by repeated application on the same cut. Five passes at 40 Bar results in a depth of 1.7 cm without vessel damage. If the applied pressure is 50 or 60 Bar, up to 7% damaged vessels can be found. The water-jet dissection leads to a water uptake of the cut tissue. Conclusions: The optimal pressure for water-jet dissection of fatty tissue lies between 30 and 40 Bar. The effect of the mechanical irritation of the vessels has to be investigatedin vivo before using the water-jet dissector for preparation of blood vessels in humans, e.g. for flap dissectio

Charged-Surface Instability Development in Liquid Helium; Exact Solutions

The nonlinear dynamics of charged-surface instability development was investigated for liquid helium far above the critical point. It is found that, if the surface charge completely screens the field above the surface, the equations of three-dimensional (3D) potential motion of a fluid are reduced to the well-known equations describing the 3D Laplacian growth process. The integrability of these equations in 2D geometry allows the analytic description of the free-surface evolution up to the formation of cuspidal singularities at the surface.Comment: latex, 5 pages, no figure