Structural analysis and molybdenum-dependent expression of the pAO1-encoded nicotine dehydrogenase genes of Arthrobacter nicotinovorans

The genes of nicotine dehydrogenase (NDH) were identified, cloned and sequenced from the catabolic plasmid pA01 of Arthrobacter nicotinovorans. In immediate proximity to this gene cluster is the beginning of the 6-hydroxy-L-niotine oxidase (6-HLNO) gene. NDH is composed of three subunits (A, B and C) of M_r 30011, 14924 and 87677. It belongs to a family of bacterial hydroxylases with a similar subunit structure; they have molybdopterin dinucleotide, FAD and Fe-S clusters as cofactors. Here the first complete primary structure of a bacterial hydroxylase is provided. Sequence alignments of each of the NDH subunits show similarities to the sequences of eukaryotic xanthine dehydrogenase (XDH) but not to other known molybdenum-containing bacterial enzymes. Based on alignment with XDH it is inferred that the smallest subunit (NDHB) carries an iron-sulphur cluster, that the middle-sized subunit (NDHA) binds FAD, and that the largest NDH subunit (NDHC) corresponds to the molybdopterin-binding domain of XDH. Expression of both the ndh and the 6-hlno genes required the presence of nicotine and molybdenum in the culture medium. Tungsten inhibited enzyme activity but not the synthesis of the enzyme protein. The enzyme was found in A. nicotinovorans cells in a soluble form and in a membrane-associated form. In the presence of tungsten the fraction of membrane-associated NDH increased.     

Sound localisation in crickets

Intracellular recordings were made in the brain of the cricket Gryllus bimaculatus from an ascending auditory interneuron (AN1). Acoustic stimuli with calling song temporal pattern were delivered via earphones in a preparation with the acoustic trachea cut (attenuation of crossing sound > 30 dB). The input-output function of this cell was then determined by recording its responses to stimulation of the ipsilateral ear alone, of the contralateral ear alone and to stimulation of both ears simultaneously with the same or different carrier frequencies and intensities.This interneuron was excited by the ear ipsilateral to its axon and dendritic field and unresponsive to stimuli presented to the axon-contralateral ear alone. However, in binaural stimulation experiments, the response to a constant ipsilateral stimulus was progressively reduced as the intensity of a simultaneous contralateral stimulus was increased, above a threshold intensity.Tuning curves for threshold of this inhibition, determined in binaural stimulation experiments, indicated significant inhibition in the range 3–20 kHz with lowest threshold at 4–5 kHz. The inhibition was unaffected by sectioning of the contralateral circumoesophageal or neck connective, indicating that the inhibitory influence crosses the midline at the level of the prothoracic ganglion. Intracellular recordings from AN1 in the prothoracic ganglion confirmed that it was indeed neurally inhibited by inputs from the contralateral ear.Tuning curves for excitation of an omega neuron (ON1) by the ear ipsilateral to its soma and also the tuning of inhibition of ON1 by its contralateral ON1 partner, closely match the tuning of inhibition of AN1 and to a lesser extent, of AN2. This was taken as evidence that each AN1 is inhibited by the contralateral ON1. The significance of this interaction for directional hearing and phonotaxis is discussed.Abbreviations AP/CHP action potentials per chirp - AN1, AN2 ascending auditory interneurons 1, 2 - ON1 omega neuron 1 - ipsi ipsilateral contra contralateral - PTG prothoracic ganglion loc lateral ocellar nerve - On optic nerve an antennal nerve - coc circum-oesophageal connective so sound off     

Orotidine-5′-monophosphate decarboxylase fromPseudomonas aeruginosa PAO1: Cloning,overexpression, and enzyme characterization

Orotidine-5-monophosphate decarboxylase (OMPdecase) catalyzes the final step in pyrimidine biosynthesis, the conversion of orotidine-5-monophosphate (OMP) to uridine-5-monophosphate. ThepyrF gene, encoding OMPdecase, was isolated from a chromosomal library ofPseudomonas aeruginosa PAO1 by screening for complementation of anEscherichia coli and aP. aeruginosa pyrF mutant. The nucleotide sequence of a 2510-bp chromosomal DNA fragment, complementing both strains, was determined (EMBL accession number X65613). On this a 696-bp open reading frame capable of encoding the 24 kDa OMPdecase was identified. Despite a generally good correspondence to other OMPdecase sequences, theP. aeruginosa gene was unique in that it did not constitute part of an operon. ThepyrF gene was amplified by polymerase chain reaction, overexpressed in the pT7-7/E. coli BL21(DE3) system and purified to near electrophoretic homogeneity by anion exchange chromatography. Characterization of the purified enzyme revealed the following data, aK _m value for OMP of 9.91 M and an isoelectric point of 6.65. No major decrease in enzyme activity was observed in a pH range between 7.8 and 10.2. Gel electrophoresis under nondenaturing conditions suggested that the native form of OMPdecase is the dimer.     

Dynamics of the immune system response in cerebrospinal fluid and blood of SIVmac-infected rhesus monkeys

Paired sera and CSF samples were collected from SIV_mac-infected macaques. Animals infected with SIV_mac251 maintained low gag and high env-specific antibody levels in plasma. Increasing env-specific antibody titers in CSF were associated in one animal with strong intrathecal synthesis. SIV_mac239-infected monkeys revealed high antibody titers of gag and env-specificity, in one animal accompanied by weak intrathecal synthesis of virus-specific antibodies. In all animals, the CD4/CD8 ratio in CSF decreased faster compared to blood.     

Cyanobacteria of Rocks and Soils of the Orinoco Lowlands and the Guayana Uplands,Venezuela

The occurrence of 23 cyanobacterial species, belonging to 9 different genera and 5 cyanobacterial lichen species of 5 different genera on exposed, open rock surfaces of inselbergs and on soil in savannas of the Orinoco lowlands and the Guayana uplands is described. Their distribution patterns and frequency within the different habitats are given. The filamentous procaryotic blue-green algae/cyanobacteria Stigonema ocellatum and Scytonema crassum, together with the unicellular cyanobacterium Gloeocapsa sanguinea were the most frequent species on rocks, whereas the filamentous cyanobacterium, Schizothrix telephoroides, dominated in cyanobacterial mats on the savanna soil. All species showed intensively coloured sheaths, either brown or yellow in the case of Stigonema ocellatum and Scytonema crassum, or red in Gloeocapsa sanguinea and Schizothrix telephoroides. In addition, a number of cyanobacterial lichens occurred.     

Neuronal responses to purinoceptor agonists in the leech central nervous system

Exracellular nucleotides like ATP and its derivatives are possible chemical messengers in vertebrate nervous systems. In invertebrate nervous systems, however, little is known about their role in neurotransmission. We have studied the reponse of identified neurones of the leech Hirudo medicinalis to the purinoceptor agonist ATP, ADP, AMP, and adenosine using conventional intracellular microelectrodes and whole-cell patch-clamp recording. Bath application of the agoinsts depolarized the different neurons, but not neuropil glial cells. The most effective responses (up to 10 mV) were observed with ATP (100 μM) or ADP (100 μM) in the noxious and touch cells. In most neurons the nonhydrolyzable ATP derivative ATP-γ-S (5 μM) induced larger depolarizations that 100 μM ATP, indicating that most of the potency of ATP is lost presumably due to its degradation by ectonucleotidases. In medial noxios cells, ATP (100 μM) induced an inward current of 1.7 ± 1.1 nA at a holding potential of ?60 mV. The ATP-induced current-voltage relationship showed an inward rectification and a reversal potential close to 0 m V. In a Na⁺-free extracellular solution, the ATP-induced inward current decreased and in a Na⁺- and Ca²⁺-free saline only a small residual current persisted. The possible P₂ purinoceptor antagonist suramin did not antagonize the ATP-induced current, but itself evoked an inward current and a conductance increase. We conclude that ATP activates nonselective cation channels in medial noxious cells of the leech with the order of potency of purinoceptor agonists ATP ≥ ADP > AMP. The results suggest that these cells express purinoceptors of the P₂ type. 1994 John Wiley & Sons, Inc.     

Male-male pairs in Greylag Geese (Anser anser)

Summary For almost two decades a flock of 130 free-flying Greylag Geese (Anser anser) has been the focus of detailed ethological investigations at the Konrad Lorenz Institut in Grünau im Almtal, Austria. Gander pairs, i. e. male-male pairs, represent a prominent social unit in this flock and were the subject of a detailed behavioral investigation. Analysis of the composition and dynamics of the flock over a 15 year period indicated that the incidence of homosexual pairings closely paralleled the male bias of the sex ratio. The behavior of ganders in gander pairs was investigated and compared to that of gander and goose in heterosexual pairs. The behavior of the two males in a gander pair (1) was comparable in most aspects, (2) was similar to the behavior of the gander in heterosexual pairs, and (3) differed greatly from that of the heterosexually paired goose. Therefore, pseudo-female behavior in one partner cannot account for the formation of a pairbond between two males. As a unit, gander pairs were characterized by a higher frequency of offensive agonistic behavior compared to heterosexual pairs and spent significantly more time peripheral to, and away from the flock than did heterosexual pairs.

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Zusammenfassung Das Sozialgefüge einer Schar Graugänse ist weitaus komplizierter, als es das monogame Fortpflanzungssystem erwarten ließe (Collias &Jahn 1959,Fischer 1965,Kalas 1979,Rutschke 1982). Ganterpaare, die häufig über Jahre hinweg bestehen bleiben, sind für tiersoziologische Untersuchungen interessant, weil ihre Funktion nicht im Rahmen der Fortpflanzung gesehen werden kann. Welche Bedingung begünstigen die Bildung von Ganterpaaren, und welche Verhaltensmechanismen tragen zum Entstehen und zur Aufrechterhaltung dieser Verbindung bei? Die Zusammensetzung der Grünauer Graugansschar 1973–1988 zeigt, daß die Anzahl der Ganterpaare von einem Überschuß von Männchen in der Schar abhängt. Das Verhalten von 6 Ganterpaaren wurde untersucht und mit dem von heterosexuellen Paaren verglichen. Innerhalb eines Ganterpaares entsprachen sich die Partner in der Häufigkeit von agonistischem sowie sozial-bindendem Verhalten. Homo- und heterosexuell verpaarte Ganter zeigten sich im Verhalten vergleichbar. Der Ganter eines Ganterpaares unterschied sich jedoch in der Häufigkeit aller untersuchten Verhaltensweisen von dem der heterosexuell verpaarten Gans. Folgende Schlußfolgerungen und Hypothesen bieten sich an: (1) Pseudo-weibliches Verhalten bei einem der Ganter scheint nicht die Bildung von Ganterpaaren erklären zu können. Beide Ganter verhalten sich rein männlich und behandeln den Partner so, als ob dieser ein Weibchen wäre. (2) Ein Mangel an gegengeschlechtlichen Schargenossen fördert die Bildung von homosexuellen Paaren und aufzuchtsbekannte Vögel werden dabei vorgezogen. (3) Ein Zusammenschluß mit einem gleichgeschlechtlichen Artgenossen sollte, verglichen mit der Möglichkeit alleine zu bleiben, eine überlegene Strategie darstellen, da unverpaarte Gänse geringeren Zugang zu Futterquellen haben und eher Raubtieren zum Opfer fallen. (4) Homosexuelle Paare könnten als ein Puffersystem für Ganter angesehen werden, vor allem zu Zeiten in denen das Geschlechtsverhältnis in Richtung der Männchen verschoben ist. (5) Aggression des Ganters richtet sich generell gegen andere männliche Schargenossen. Die Bildung von Ganterpaaren, also besonders aggressiven Paaren, könnte daher dazu beitragen, Ganter aus der Schar zu vertreiben und ein Übermaß von Männchen in der Schar zu verhindern. (6) Da wir zeigen konnten, daß sich homosexuelle Paare oft am Rande der Schar aufhalten und dabei häufig sichern, könnte solchen Paaren eine Art Wächterfunktion zukommen. (7) Andererseits ist es durchaus möglich, daß Ganterpaare bloß ein Epiphenomen einer Graugansschar mit einem Überschuß an Männchen darstellen.

     

Detection of enteroviruses in groundwater with the polymerase chain reaction.

Standard methods for the detection of enteroviruses in environmental samples involve the use of cell culture, which is expensive and time-consuming. The polymerase chain reaction (PCR) is an attractive method for the detection of enteroviruses in water because primary cell culture is not needed and the increased sensitivity of PCR allows detection of the low numbers of target DNAs and RNAs usually found in environmental samples. However, environmental samples often contain substances that inhibit PCR amplification of target DNA and RNA. Procedures that remove substances that interfere with the amplification process need to be developed if PCR is to be successfully applied to environmental samples. An RNA-PCR assay for the detection of enteroviruses in water was developed and used to test a variety of groundwater concentrates and humic acid solutions seeded with poliovirus type 1. The groundwater samples and humic acid solutions were treated with Sephadex G-50, Sephadex G-100, Sephadex G-200, Chelex-100 resin, and a mixed bed resin to remove PCR-inhibitory material from the samples. Sephadex G-100 in combination with Chelex-100 was found to be very effective in removing inhibitory factors for the detection of enteroviruses in groundwater concentrates by PCR. Viruses were detected in two of the groundwater concentrates by the RNA-PCR assay after treatment with Sephadex G-100 plus Chelex-100. This was confirmed by tissue culture, suggesting that the treatment protocol and, subsequently, the RNA-PCR assay are applicable for the detection of enteroviruses in environmental samples.     

A strategy for the characterization of minute chromosome rearrangements using multiple color fluorescence in situ hybridization with chromosome-specific DNA libraries and YAC clones

The identification of marker chromosomes in clinical and tumor cytogenetics by chromosome banding analysis can create problems. In this study, we present a strategy to define minute chromosomal rearrangements by multicolor fluorescence in situ hybridization (FISH) with whole chromosome painting probes derived from chromosome-specific DNA libraries and Alu-polymerase chain reaction (PCR) products of various region-specific yeast artificial chromosome (YAC) clones. To demonstrate the usefulness of this strategy for the characterization of chromosome rearrangements unidentifiable by banding techniques, an 8p+ marker chromosome with two extra bands present in the karyotype of a child with multiple anomalies, malformations, and severe mental retardation was investigated. A series of seven-color FISH experiments with sets of fluorochrome-labeled DNA library probes from flow-sorted chromosomes demonstrated that the additional segment on 8p+ was derived from chromosome 6. For a more detailed characterization of the marker chromosome, three-color FISH experiments with library probes specific to chromosomes 6 and 8 were performed in combination with newly established telomeric and subtelomeric YAC clones from 6q25, 6p23, and 8p23. These experiments demonstrated a trisomy 6pter6p22 and a monosomy 8pter8p23 in the patient. The present limitations for a broad application of this strategy and its possible improvements are discussed.Dedicated to Professor Dr. U. Wolf on the occasion of his 60th birthday     

Faster induction of photosynthesis increases biomass and grain yield in glasshouse-grown transgenic Sorghum bicolor overexpressing Rieske FeS

Sorghum is one of the most important crops providing food and feed in many of the world's harsher environments. Sorghum utilizes the C₄ pathway of photosynthesis in which a biochemical carbon-concentrating mechanism results in high CO₂ assimilation rates. Overexpressing the Rieske FeS subunit of the Cytochrome b₆f complex was previously shown to increase the rate of photosynthetic electron transport and stimulate CO₂ assimilation in the model C₄ plant Setaria viridis. To test whether productivity of C₄ crops could be improved by Rieske overexpression, we created transgenic Sorghum bicolor Tx430 plants with increased Rieske content. The transgenic plants showed no marked changes in abundances of other photosynthetic proteins or chlorophyll content. The steady-state rates of electron transport and CO₂ assimilation did not differ between the plants with increased Rieske abundance and control plants, suggesting that Cytochrome b₆f is not the only factor limiting electron transport in sorghum at high light and high CO₂. However, faster responses of non-photochemical quenching as well as an elevated quantum yield of Photosystem II and an increased CO₂ assimilation rate were observed from the plants overexpressing Rieske during the photosynthetic induction, a process of activation of photosynthesis upon the dark–light transition. As a consequence, sorghum with increased Rieske content produced more biomass and grain when grown in glasshouse conditions. Our results indicate that increasing Rieske content has potential to boost productivity of sorghum and other C₄ crops by improving the efficiency of light utilization and conversion to biomass through the faster induction of photosynthesis.