Cloning and characterization of an Arabidopsis thaliana Nudix hydrolase homologous to the mammalian GFG protein

In a search for a plant antimutator MutT protein, an Arabidopsis thaliana Nudix hydrolase with homology to the mammalian GFG protein was expressed as a hexahistidine fusion polypeptide in Escherichia coli and purified to homogeneity. Unlike the GFG protein, the A. thaliana homolog could not complement the mutT mutation in a MutT-deficient E. coli strain nor was it able to hydrolyze 8-oxo-dGTP, the main substrate of the MutT protein. Instead the recombinant protein hydrolyzed a variety of nucleoside diphosphate derivatives showing a preference for ADP-ribose, with Km and k(cat) values of 1.2 mM and 2.7 s(-1) respectively. The products of ADP-ribose hydrolysis were AMP and ribose-5-phosphate. The optimal activity was at alkaline pH (8.5) with Mg2+ (5 mM) ions as the cofactor. The protein exists as a dimmer in solution.     

Association between Ag1-CA Alleles and Severity of Autosomal Recessive Proximal Spinal Muscular Atrophy

The gene for autosomal recessive proximal spinal muscular atrophy (SMA) has been mapped to an 850-kb interval on 5q11.2-q13.3, between the centromeric D5S823 and telomeric D5S557 markers. We report a new complex marker, Ag1-CA, that lies in this interval, whose primers produce one, two, or rarely three amplification-fragment-length variants (AFLVs) per allele. Class I chromosomes are those which amplify a single AFLV allele, and class II chromosomes are those which amplify an allele with two or three AFLVs. Ag1-CA shows highly significant allelic association with type I SMA in both the French Canadian (Hôpital Sainte-Justine [HSJ]) and American (Ohio State University [OSU]) populations (P<.0001). Significant association between the Ag1-CA genotype and disease severity was also observed. Type I patients were predominantly homozygous for class I chromosomes (P=.0003 OSU; P=.0012 HSJ), whereas the majority of type II patients were heterozygous for class I and II chromosomes (P=.0014 OSU; P=.001 HSJ). There was no significant difference in Ag1-CA genotype frequencies between type III patients (P=.5 OSU; P=.25 HSJ) and the paired normal chromosomes from both carrier parents. Our results indicate that Ag1-CA is the most closely linked marker to SMA and defines the critical candidate-gene region. Finally, we have proposed a model that should be taken into consideration when screening candidate SMA genes.     

Photocleavable biotin phosphoramidite for 5'-end-labeling, affinity purification and phosphorylation of synthetic oligonucleotides.

We report the design, synthesis and evaluation of a non-nucleosidic photocleavable biotin phosphoramidite (PCB-phosphoramidite) which provides a simple method for purification and phosphorylation of oligonucleotides. This reagent introduces a photocleavable biotin label (PCB) on the 5'-terminal phosphate of synthetic oligonucleotides and is fully compatible with automated solid support synthesis. HPLC analysis shows that the PCB moiety is introduced predominantly on full-length sequences and is retained during cleavage of the synthetic oligonucleotide from the solid support and during subsequent deprotection with ammonia. The full-length 5-PCB-labeled oligonucleotide can then be selectively isolated from the crude oligonucleotide mixture by incubation with immobilized streptavidin. Upon irradiation with 300-350 nm light the 5'-PCB moiety is cleaved with high efficiency in <4 min, resulting in rapid release of affinity-purified 5'-phosphorylated oligonucleotides into solution. 5'-PCB-labeled oligonucleotides should be useful in a variety of applications in molecular biology, including cassette mutagenesis and PCR. As an example, PCB-phosphoramidite has been used for the synthesis, purification and phosphorylation of 50-and 60mer oligonucleotides.     

Specific [3H]quinuclidinyl benzilate binding activity in Digitonin-solubilized preparations from bovine brain

Receptors for the specific muscarinic radioligand [³H]quinuclidinyl benzilate ([³H]QNB) were solubilized by digitonin from a particulate preparation of bovine brain without significant alteration in binding affinities for muscarinic antagonists. Electron microscopy and sucrose density gradient sedimentation analysis confirmed the solubility of these receptors in aqueous solutions of digitonin. Equilibrium and kinetic studies of [³H]QNB binding to solubilized receptors indicated that binding was stereoselective and was blocked by muscarinic compounds. These tests permit tentative identification of digitonin-solubilized [³H]QNB binding sites as muscarinic acetylcholine receptors. Digitonin-solubilized receptors were homogeneous with respect to sedimentation behavior and binding affinities for agonist and antagonist drugs, unlike membrane-bound receptors. Enzyme digestion studies and treatment with group-specific reagents indicated that muscarinic receptors are proteins whose binding activity could be disrupted by reduction with dithiothreitol or by modification of sulfhydryl residues.     

Ecology of bottom biocoenoses in the possjet bay (the Sea of Japan) and the peculiarities of their distribution in connection with physical and chemical conditions of the habitat

Summary 1. By employing autonomous diving techniques, direct observations on the biocoenoses of the upper marine zones were carried out. The population of each biotope was studied quantitatively in areas from 100 m² to 1/10 or 1/100 m² depending on size and density of the organisms studied.2. The quality and quantity of bottom organisms depend not only on the nature of the ground, other physico-chemical environmental factors or the quantity of food available, but also on the biocoenotic background, i. e. conditions created by aggregations of certain species whose presence create additional, new niches.3. In estuaries and lagoons, salinity greatly affects biocoenosis composition. The effect of salinity changes increases from phytal and epifauna to infauna elements, a fact which may be associated with the relatively higher salinity inside the bottom substratum.4. The most highly effective force in determining distribution, composition and biogeographical structure of biocoenoses is the water temperature (absolute values as well as fluctuations).5. In half-closed areas of bays the patchiness of biocoenoses tends to increase and the territories occupied become smaller. These changes may be associated with (a) higher fluctuations of physico-chemical conditions, (b) their increased variety and (c) accelerated succession.6. There is the closest connection between the distribution of organisms and the position of the layers of definite water masses in the system of vertical marine zones. In the Bay of Possjet the low border of the particular vertical zone lies at a depth of 5 m (0 to 5 m); its location is determined by the position of the upper layer of the surface Japanese marine water mass.7. When carrying out biogeographical studies, in addition to comparing similar vertical zones, one should consider bionomically similar areas.

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Ökologie der Bodenbiozönosen in der Posjet-Bucht (Japanisches Meer) und die Besonderheiten ihrer Verbreitung in Verbindung mit physikalischen und chemischen Bedingungen des Habitats

Kurzfassung Verbreitung und Zusammensetzung der Fauna und Flora der reichbesiedelten, oberen marinen Wasserzonen sind nur ungenügend bekannt, weil große Forschungsschiffe mit geeigneten Einrichtungen zum Sammeln und zur Datenverarbeitung in küstennahen Gewässern wegen der geringen Wassertiefe nicht arbeiten können. Die Anwendung autonomer Tauchausrüstungen hat es uns ermöglicht, direkte Beobachtungen an litoralen Biozönosen durchzuführen. Je nach Größe und Besiedlungsdichte der angetroffenen Organismen haben wir Areale von 100 m² bis zu 1/100 m² untersucht. Qualität und Quantität der Boden-organismen hängen nicht nur von der Beschaffenheit des Untergrundes ab, sondern auch von physiko-chemischen Umweltfaktoren sowie vom Nahrungsangebot und dem biozönotischen Hintergrund (biotischen Faktoren). In Ästuarien und Lagunen beeinflußt der Salzgehalt in starkem Maße die artliche Zusammensetzung der Biozönose, wobei der Salzgehaltseinfluß in der Endofauna geringer ist als im Phytal und bei der Epifauna — ein Umstand, der vermutlich mit dem relativ höheren Salzgehalt innerhalb des Bodens zusammenhängt. Verbreitung, Komposition und biographische Struktur der Biozönosen werden primär von der Wassertemperatur (absolute Intensität und Fluktuationsmuster) beeinflußt. In halb abgeschlossenen Meeresbuchten und flachen Litoralgebieten neigen die Biozönosen immer mehr zu kleinflächigen Verbreitungsmustern. Diese Veränderungen hängen möglicherweise zusammen mit (a) intensiveren Fluktuationen der physiko-chemischen Verhältnsse, (b) deren zunehmende Variabilität und (c) beschleunigter Sukzession. Die Verbreitung der Organismen ist sehr eng gebunden an die Positionen bestimmter Wassermassen. Bei der Durchführung biogeographischer Studien sollten — zusätzlich zu Vergleichen ähnlicher Vertikalzonen — bionomisch ähnliche Gebiete analysiert werden. Die Einwanderung von Warmwasserorganismen von einer Warmwasserzone in die andere erfolgt vermutlich während Perioden, in denen die intermediären Gebiete starken Oberflächenerwärmungen ausgesetzt sind.

     

Environmental assessment of municipal wastewater discharges: a comparative study of evaluation methods

Purpose

Wastewater treatment plants produce high environmental impacts on receiving water bodies and pose economic burdens on municipalities or industrial facilities. Their overall operational costs and achieved effluent quality depend very much on the influent type, presence of priority pollutants, treatment technology and required effluent quality (for discharge or for recycle/reuse). Life cycle assessment (LCA), environmental impact quantification (EIQ) and water footprint (WF) are important instruments for sustainability assessments applied for products, production and consumption evaluations in connection with natural resources depletion and pollution threats. This study focuses on the environmental assessment of a municipal wastewater treatment plant (MWWTP) discharges by means of these three evaluation methods with the purpose to understand their (methodological) weak and strong points in capturing the impacts. Such a comparative analysis is necessary to understand how the (individual) advantages provided by each of these instruments can be complimentarily used to improve an assessment framework for various stakeholders concerned with water use cycle management (regional water operators, water management authority, public authorities, research entities, societal organizations, etc.).

Methods

The three assessment methodologies (LCA, EIQ, WF) are presented, implemented and critically analysed based on a unitary set of data concerning the MWWTP of Iasi city (a municipality of approx. 300,000 inhabitants situated in the North Eastern region of Romania), the wastewater and river water quality indicators as well as all the other relevant data being collected for the year 2012.

Results and discussion

Although the three methodologies have different principles for environmental impact quantification, the results have shown that most impacts induced to surface waters due to Iasi MWWTP effluents are given by the nutrients (nitrogen and phosphorous compounds), which could induce an eutrophication impact, and to a lesser extent by pollutants responsible for toxicity impacts (such as heavy metals).

Conclusions

Based on the results of this comparative study, a critical analysis of these three methods was realized by considering the data requirements, their development and integration status. Furthermore, the strong and weak points that are relevant for each method implementation and their subsequent use by decision-makers and Water Authorities are discussed, in the context of legislative requirements (including the Water Framework Directive), actual development of regional water operators and stakeholders' interests.

     

Methionine Changes in Bacteriorhodopsin Detected by FTIR and Cell-Free Selenomethionine Substitution

Bacteriorhodopsin (BR) is an integral membrane protein, which functions as a light-driven proton pump in Halobacterium salinarum. We report evidence that one or more methionine residues undergo a structural change during the BR→M portion of the BR photocycle. Selenomethionine was incorporated into BR using a cell-free protein translation system containing an amino acid mixture with selenomethionine substituted for methionine. BR→M FTIR difference spectra recorded for unlabeled and selenomethionine-labeled cell-free expressed BR closely resemble the spectra of in vivo expressed BR. However, reproducible changes occur in two regions near 1284 and 900 cm⁻¹ due to selenomethionine incorporation. Isotope labeled tyrosine was also co-incorporated with selenomethionine in order to confirm these assignments. Based on recent x-ray crystallographic studies, likely methionines which give rise to the FTIR difference bands are Met-118 and Met-145, which are located inside the retinal binding pocket and in a position to constrain the motion of retinal during photoisomerization. The assignment of methionine bands in the FTIR difference spectrum of BR provides a means to study methionine-chromophore interaction under physiological conditions. More generally, combining cell-free incorporations of selenomethionine into proteins with FTIR difference spectroscopy provides a useful method for investigating the role of methionines in protein structure and function.     

Matrix-assisted laser desorption/ionization mass spectrometry of DNA using photocleavable biotin

Oligonucleotides containing a photocleavable biotin (5'-PC-biotin) were analyzed by matrix assisted laser desorption/ionization mass spectrometry (MALDI-MS) with wavelengths in the ultraviolet (UV) and infrared (IR) from solution and after capture on streptavidin-coated agarose or magnetic beads. The analysis was used to monitor the release of the oligonucleotides as a result of photochemical cleavage of the biotinylated linker. Near-UV pulses (UV-MALDI) led to predominant release of the photocleaved product. In contrast, only the uncleaved analyte was detected using IR pulses (IR-MALDI). Results from MALDI analysis are also presented for DNA containing a photocleavable 5'-amino group which can be covalently linked to a variety of activated surfaces and marker molecules. In a demonstration of this approach, a 5'-PC-biotinylated 49 nt RNA oligonucleotide was enzymatically synthesized using a PC-biotin-r(AG) dinucleotide primer, captured on streptavidin coated magnetic beads and analyzed by UV-MALDI. Potential applications of photocleavable linkers combined with MALDI for the analysis of nucleic acids are discussed.     

Rosavin as a product of glycosylation by Rhodiola rosea (roseroot) cell cultures

The paper discusses glycosylation of trans-cinnamyl alcohol to obtain the biologically active compound rosavin and possibly other cinnamoylglycosides. Cell suspension cultures of Rhodiola rosea were established from callus of leaf origin cultured under light in a modified Murashige and Skoog medium. Under these conditions, no rosavin was formed. However, when trans-cinnamyl alcohol (2.5 mM; in MeOH) was added to the medium, after 72 h incubation cells transformed over 90% of the cinnamyl alcohol into a number of unidentified products. The structure of potential rosavin accumulated in intracellular spaces was elucidated as [3-phenyl-2-propenyl-O-(6'-O-α-L-arabinopyranosyl) -β-D-glucopyranoside] by means of chemical and spectral analysis using TLC, HPLC, UV, LSIMS and NMR methods. Rosavin yields of 0.03–1.01% dry weight were obtained. The actual amount depended on the cell strain cultured and the biotransformation period. This revised version was published online in June 2006 with corrections to the Cover Date.     

The influence of LBG addition to carrageenan on the mechanical stability of the gel and the fermentative activity of immobilized propionic acid bacteria

In the first part of the experiments, the mechanical properties of 1%, 2% and 3% carrageenan and 1%, 2% and 3% carrageenan/locust bean gum (LBG) gels stored in various concentrations of propionic and acetic acids and their mixtures were examined. The stability of these materials was measured by uniaxial compression between two parallel plates using the Instron Universal Testing Machine. A mathematical model explaining the dependence of the destroying force on the storage time was chosen for data analysis. Using this model, the average rate of gel deterioration was calculated. The structural properties of the examined gels were most influenced by the highest concentration of propionic and acetic acids and their mixtures (1% acetic acid and 2% propionic acid). The addition of LBG to carrageenan decreased the gel destroying force and increased its resistance to acids. In the second part of the experiments, the Propionibacterium freudenreichii subsp. shermanii NCFB 1081 and NCFB 566 were immobilized in a living state in 1%, 2% and 3% carrageenan and 1%, 2% and 3% carrageenan/LBG gels. The ammonia consumption, glucose utilization, production of propionic and acetic acids and the biosynthesis of vitamin B₁₂ were examined. An increase in the productivity of propionic acid and a significant decrease in the vitamin B₁₂ produced in the biosynthesis were observed when immobilized cells were used. The immobilization of cells enhanced the productivity of propionic acid by up to 40% compared to free cells. The best results were obtained for the second and third applications of immobilized cells in all concentrations of carrageenan gels and 2% and 3% carrageenan/LBG gels The results showed that carrageenan/LBG is a better support material for the immobilization of propionic acid bacteria than the pure carrageenan.