The use of azidoarylimidoesters in RNA-protein cross-linking studies with Escherichia coli ribosomes

A series of related hetero-bifunctional RNA-protein cross-linking reagents has been prepared, carrying an imidoester or N-hydroxysuccinimide ester function at one end of the molecule, and a phenylazido function at the other. These compounds have been applied to RNA-protein cross-linking studies with ribosomal subunits, and one of them, p-azido-phenylacetic imidoester, has proved to be a particularly useful reagent for this purpose. The reagent first reacts specifically with protein amino groups, and subsequent photolysis of the azide group leads to cross-linking to the RNA in yields of up to 8% of the total protein. The whole reaction takes place under very mild conditions in aqueous solution.The individual proteins concerned in the cross-links have been identified by two-dimensional gel electrophoresis, and the existence of a covalent cross-link was confirmed by the isolation by two different methods of protein-oligonucleotide complexes carrying a ³²P label. Although most of the ribosomal proteins could be cross-linked to their corresponding ribosomal RNA within the individual subunits, RNA-protein cross-links at the ribosomal subunit interface were only detectable in vanishingly small amounts.The advantages of this type of genuine hetero-bifunctional reagent in RNA-protein cross-linking studies are discussed.     

Delayed onset of midline netrin expression in Artemia franciscana coincides with commissural axon growth and provides evidence for homology of midline cells in distantly related arthropods

Although many similarities in arthropod central nervous systems (CNS) development exist, differences in midline cell formation and ventral nerve cord axonogenesis have been noted in arthropods. It is possible that changes in the expression of axon guidance molecules such as Netrin, which functions during commissural axon guidance in Drosophila and many other organisms, may parallel these differences. In this investigation, we analyze this hypothesis by examining Netrin accumulation during development of the brine shrimp Artemia franciscana, a branchiopod crustacean. An Artemia franciscana netrin (afrnet) orthologue was cloned. An antibody to the afrNet protein was generated and used to examine the pattern of afrNet accumulation during Artemia development. Despite differences between Drosophila and Artemia nerve cord development, examination of afrNet accumulation suggests that this protein functions to regulate commissure formation during Artemia CNS development. However, detection of afrNet at the midline and on commissural axons occurs at a relatively later time point in Artemia as compared with Drosophila. Detection of afrNet in a subset of midline cells that closely resemble Netrin-expressing cells at the Drosophila midline provides evidence for homology of midline cells in arthropods. Expression of Netrins in many other tissues is comparable, suggesting that Netrin proteins may play many conserved roles during arthropod development.     

Literaturberichte

Ohne Zusammenfassung     

Fluvastatin prevents glutamate-induced blood-brain-barrier disruption in vitro

Glutamate is an important excitatory amino acid in the central nervous system. Under pathological conditions glutamate levels dramatically increase. Aim of the present study was to examine whether the HMG-CoA inhibitor fluvastatin prevents glutamate-induced blood-brain-barrier (BBB) disruption. Measurements of transendothelial electrical resistance (TEER) were performed to analyze BBB integrity in an in vitro co-culture model of brain endothelial and glial cells. Myosin light chain (MLC) phosphorylation was detected by immunohistochemistry, or using the in-cell western technique. Intracellular Ca2+ and reactive oxygen species (ROS) levels were analyzed using the fluorescence dyes Ca-green or DCF. Glutamate induced a time- (1-3 h) and concentration- (0.25-1 mmol/l) dependent decrease of TEER values that was blocked by the NMDA-receptor antagonist MK801, the Ca2+ chelator BAPTA, the NAD(P)H-oxidase inhibitor apocynin and the MLC-kinase inhibitor ML-7. Furthermore we observed a concentration-dependent increase of intracellular Ca2+ and ROS after glutamate application. Glutamate caused an increase of MLC phosphorylation that was antagonized by apocynin, or BAPTA, indicating that Ca2+ and ROS signaling is involved in the activation of the contractile machinery. Fluvastatin (10-25 micromol/l) completely abolished the glutamate-induced barrier disruption and oxidative stress. The BBB-protecting effect of fluvastatin was completely lost if the cells were treated with the nitric oxide (NO) synthase inhibitor L-NMMA (300 micromol/l). In the present study we demonstrated that glutamate-induced BBB disruption involves Ca2+ signalling via NMDA receptors, which is followed by an increased ROS generation by the NAD(P)H-oxidase. This oxidative stress then activates the MLC kinase. Fluvastatin preserves barrier function in a NO-dependent way and reduces glutamate-induced oxidative stress.     

Witterungseinflüsse auf die Pigmentbildung der Wiesenpflanzen und den Vitamin A-Gehalt der Milchfette

Zusammenfassung Der Carotin-, Chlorophyll a- und b-Gehalt (Pigment) in Gras von Weiden in 600 m Höhe am nördlichen Alpenrand (Südbayern) und der Vitamin A-Gehalt im Milchfett von Kühen im Stall nach Fütterung mit dem Gras wurde bestimmt und mit den Ergebnissen der meteorologischen Beobachtungen verglichen. Die Pigmentbildung in den Pflanzen wurde durch warme strahlungsreiche Witterung gefördert und durch starke TemperaturrückgÄnge und Bodenfröste gehemmt. Sie war ausserdem vom Standort abhÄngig: an S-HÄngen und auf Höhen in strahlungsgünstiger Lage war in sonnigen Perioden der Carotingehalt hoch, in Niederungen mit hÄufiger Nebelbildung, auf schattigen HÄngen und in Tallagen mit Kaltluft tief. In Witterungsperioden mit Bewölkung glich der höhere Anteil der Himmelsstrahlung die Expositionsunterschiede wieder aus. Extrem grosse Licht- und WÄrmeeinwirkungen sommerlicher Hitzeperioden hemmten die Pigmentbildung; der Milch Vitamin A-Gehalt zeigte gleichsinnige Schwankungen.

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Carotine, chlorophyll a and b (pigment) in some types of meadow grass (600 m) at the northern rim of the Alps and the vitamin A content in milk from cows in stables fed on this grass was determined and correlated with meteorological data. The pigment formation in the grass was enhanced by radiation-rich warm weather and was impeded by a fall in temperature and ground frosts. The carotine content was high in grass from south slopes and on hills in a favourable position for radiation during sunny periods and low in grass from low sites with cold air.During periods of cloudy weather the greater amount of sky radiation compensated for the smaller amount of exposure in the less favourable sites. Extremely strong effects of light and warmth during summer heat periods inhibited the pigment formation and there was also a similar change in the vitamin A content of the milk.

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Résumé La teneur en carotène et en chlorophylle a et b (pigment) de quelques herbes de prairies du nord des Alpes (Bavière) et la teneur en vitamine A du lait des vaches qui en furent nourries en étables ont été comparées à des données méteorologiques. La formation des pigments des herbages était accrue par un temps chaud et ensoleillé et était diminué par les chutes de température et le refroidissement du sol. Le taux de carotène des herbes des pentes exposées au sud et sur les collines était élevé durant les périodes de soleil, et bas dans les fonds de vallées exposées à l'air froid et sur les pentes à l'ombre. Pendant les périodes nuageuses, la plus grande luminosité du ciel compensait les différences dans 1'exposition.La formation de pigments était ralentie en été pendant les périodes de grandes chaleurs et d'irradiation extrèmes. Le contenu du lait en vitamine A montrait des variations dans le mÊme sens.

     

Isolation and partial characterization of basic proteinases from stem bromelain

Crude bromelain extracts from pineapple stems (Ananas comosus) were fractionated by two-step FPLC-cation-exchange chromatography. At least eight basic proteolytically active components were detected. The two main components F4 and F5 together with the most active proteinase fraction F9 were characterized by SDS-PAGE, mass spectroscopy, multizonal cathodal electrophoresis, partial amino acid sequence, and monosaccharide composition analysis. F9 amounts to about 2% of the total protein and has a 15 times higher specific activity against the substratel-pyroglutamyl-l-phenylanalyl-l-leucine-p-nitroanilide (PFLNA) than the main component F4. The molecular masses of F4, F5, and F9 were determined to 24,397, 24,472, and 23,427, respectively, by mass spectroscopy. Partial N-terminal amino acid sequence analysis (20 amino acids) revealed that F9 differs from the determined sequence of F4 and F5 by an exchange at position 10 (tyrosineserine) and position 20 (asparagine glycine). F4 and F5 contained fucose, N-acetylglucosamine, xylose, and mannose in ratio of 1.02.01.02.0, but only 50% of the proteins seem to be glycosylated, whereas F9 was found to be unglycosylated. Polyclonal antibodies (IgG) against F9 detected F4 and F5 with tenfold reduced reactivity. ThepH optimum of F4 and F5 was betweenpH4.0 and 4.5 and for F9 close to neutralpH. The kinetic parameters for PFLNA hydrolysis were similar for F4 (K _m 2.30 mM,k _cat 0.87 sec^–1 and F5 (K _m 2.42 mM,k _cat 0.68 sec^–1), and differed greatly from F9 (K _m 0.40 mM,k _cat 3.94 sec^–1).Dedicated to H. Tschesche, Bielefeld, Germany, on behalf of his 60th anniversary.     

Detection of a novel variant human hemoglobin by electrospray ionization mass spectrometry

A novel hemoglobin variant was detected by electrospray ionization mass spectrometry. Hb Zurich-Hottingen is characterized by an Asn --> Ser replacement in the alpha-chain at position 9 as confirmed by DNA analysis. This hemoglobin variant is silent in isoelectric focusing, reversed-phase chromatography, and cation-exchange chromatography. The mutant alpha-chain was detectable only with electrospray mass spectrometry by its mass shift of -27 Da. The carrier was found to be heterozygous for the new hemoglobin variant. These results illustrate the power of ESI mass spectrometry for hemoglobin analysis.     

Genomic pedigree reconstruction identifies predictors of mating and reproductive success in an invasive vertebrate

The persistence of an invasive species is influenced by its reproductive ecology, and a successful control program must operate on this premise. However, the reproductive ecology of invasive species may be enigmatic due to factors that also limit their management, such as cryptic coloration and behavior. We explored the mating and reproductive ecology of the invasive Brown Treesnake (BTS: Boiga irregularis) by reconstructing a multigenerational genomic pedigree based on 654 single nucleotide polymorphisms for a geographically closed population established in 2004 on Guam (N = 426). The pedigree allowed annual estimates of individual mating and reproductive success to be inferred for snakes in the study population over a 14‐year period. We then employed generalized linear mixed models to gauge how well phenotypic and genomic data could predict sex‐specific annual mating and reproductive success. Average snout–vent length (SVL), average body condition index (BCI), and trappability were significantly related to annual mating success for males, with average SVL also related to annual mating success for females. Male and female annual reproductive success was positively affected by SVL, BCI, and trappability. Surprisingly, the degree to which individuals were inbred had no effect on annual mating or reproductive success. When juxtaposed with current control methods, these results indicate that baited traps, a common interdiction tool, may target fecund BTS in some regards but not others. Our study emphasizes the importance of reproductive ecology as a focus for improving BTS control and promotes genomic pedigree reconstruction for such an endeavor in this invasive species and others.     

Ecological and evolutionary consequences of biotic homogenization

Biotic homogenization, the gradual replacement of native biotas by locally expanding non-natives, is a global process that diminishes floral and faunal distinctions among regions. Although patterns of homogenization have been well studied, their specific ecological and evolutionary consequences remain unexplored. We argue that our current perspective on biotic homogenization should be expanded beyond a simple recognition of species diversity loss, towards a synthesis of higher order effects. Here, we explore three distinct forms of homogenization (genetic, taxonomic and functional), and discuss their immediate and future impacts on ecological and evolutionary processes. Our goal is to initiate future research that investigates the broader conservation implications of homogenization and to promote a proactive style of adaptive management that engages the human component of the anthropogenic blender that is currently mixing the biota on Earth.