Lotus japonicus, an autogamous, diploid legume species for classical and molecular genetics

In the Leguminosae plant family, few of the individual plant species have been used for plant molecular biology research. Among the species investigated no obvious representative ‘model’ legume has emerged. Here a member of the tribe Loteae, Lotus japonicus (Regel) Larsen is proposed as a candidate. L. japonicus is a diploid, autogamous species, with a good seed set, and a generation time of approximately 3 months. The haploid genome consists of six chromosomes and the genome size was estimated to be relatively small (0.5 pg per haploid complement). L. japonicus is susceptible to Agrobacterium tumefaciens and transgenic plants can be regenerated after hygromycin or kanamycin selection. Tissue culture conditions and procedures for transformation and regeneration are described. Stable transformation is demonstrated by segregation of the hygromycin selectable marker after selfing of transgenic plants or test crosses. The possibility of mapping polymorphic DNA markers inbred lines of L. japonicus is also discussed.     

Inhibition of the biosynthesis ofN-acetylneuraminic acid by metal ions and seleniumin vitro

In liver homogenate the biosynthesis ofN-acetylneuraminic acid usingN-acetylglucosamine as precursor can be followed stepwise by applying different chromatographic procedures. In this cell-free system 16 metal ions (Zn²⁺, Mn²⁺, La³⁺, Co²⁺, Cu²⁺, Hg²⁺, VO ₃ ^– , Pb²⁺, Ce³⁺, Cd²⁺, Fe²⁺, Fe³⁺, Al³⁺, Sn²⁺, Cs⁺ and Li⁺) and the selenium compounds, selenium(IV) oxide and sodium selenite, have been checked with respect to their ability to influence a single or possible several steps of the biosynthesis ofN-acetylneuraminic acid. It could be shown that the following enzymes are sensitive to these metal ions (usually applied at a concentration of 1 mmoll^–1):N-acetylglucosamine kinase (inhibited by Zn²⁺ and vandate), UDP-N-acetylglucosamine-2-epimerase (inhibited by zn²⁺, Co²⁺, Cu²⁺, Hg²⁺, VO ₃ ^– , Pb²⁺, Cd²⁺, Fe³⁺, Cs⁺, Li⁺, selenium(IV) oxide and selenite), andN-acetylmannosamine kinase (inhibited by Zn²⁺, Cu²⁺, Cd²⁺, and Co²⁺). Dose dependent measurements have shown that Zn²⁺, Cu²⁺ and selenite are more efficient inhibitors of UDP-N-acetylglucosamine-2-epimerase than vanadate. As for theN-acetylmannosamine kinase inhibition, a decreasing inhibitory effect exists in the following order Zn²⁺, Cd²⁺, Co²⁺ and Cu²⁺. In contrast, La³⁺, Al³⁺ and Mn²⁺ (1 mmoll^–1) did not interfere with the biosynthesis ofN-acetylneuraminic acid. Thus, the conclusion that the inhibitory effect of the metal ions investigated cannot be regarded as simply unspecific is justified.Dedicated to Professor Theodor Günther on the occasion of his 60th birthday     

Expression of polysialylated N-CAM during rat heart development

Developmental patterns of immunoreactivity for the neural cell adhesion molecule (N-CAM) and alpha 2.8-linked polysialic acid (PSA) were identified in embryonic and postnatal rat heart by immunocytochemistry and immunoblotting. Polyclonal antibodies against N-CAM and a monoclonal antibody which recognises only polymers of PSA with a chain length greater than eight units were used. Gold- and alkaline-phosphatase-labelled antibodies were used for detection. The N-CAM polypeptide isoform pattern seen by immunoblotting after endoneuraminidase treatment changed as development progressed. During embryonic development a 160-kDa polypeptide isoform was predominant. Around birth, 130-, 160- and 170-kDa polypeptide isoforms were found. The expression of the 130- and 170-kDa isoforms diminished until finally, in the adult, weak immunoreactivity for bands of 120-, 130- and 160-kDa was seen. In general the extent and intensity of PSA and N-CAM immunostaining in rat heart increased until birth and declined thereafter. Early in development prominent immunostaining for PSA and N-CAM was seen in the epicardium while later in development this area was only weakly stained. Initially myocardial cells, endocardial cells and some cells in the atrioventricular cushions were immunoreactive for both PSA and N-CAM. Later in development N-CAM immunostaining was more prominent than PSA immunoreactivity, reflecting a decrease in N-CAM polysialylation, which was also seen by immunoblotting. During innervation of the heart, nerve fibres were strongly immunostained for PSA and N-CAM, and this was the only immunostaining seen in adult heart.     

Phase I trial of intravenous infusion of ex-vivo-activated autologous blood-derived macrophages in patients with non-small-cell lung cancer: Toxicity and immunomodulatory effects

Summary The purpose of this phase I study was to evaluate the toxicity and biological activity of autologous blood-derived macrophages activated ex-vivo with recombinant human interferon (rhuIFN) [monokine-activated killer (MAK) cells] and administered intravenously to 11 lung cancer patients once a week for 6 consecutive weeks. Peripheral blood monocytes were collected by leukapheresis and then purified by counterflow elutriation. The MAK cells were generated by culturing the purified monocytes in Teflon bags for 7 days and adding rhuIFN to the cultured cells for the last 18 h. These MAK cells expressed differentiation-associated surface antigen MAX1, and were cytotoxic in vitro against tumour cell line U937. The MAK cells were infused at dose levels from 1 × 10⁷ to 5 × 10⁸ on an intrapatient dose-escalating schedule. No severe adverse side-effects occurred. Toxicity was mild to moderate [primarly fever (75%) and chills (32%)], non-dose-dependent, and non-cumulative. No consistent change in haemostatic function, or liver or renal function was observed. Dose-limiting toxicity was not reached at 5 × 10⁸ cells (optimal dose reproduced for each patient). The maximum tolerated dose was not determined. The immunomodulatory activity of i.v. infused MAK cells was demonstrated both in vivo by significant increases in granulocyte count and neopterin level in the patients' peripheral blood postinfusion and in vitro by secretory products (IL-1. TNF, neopterin, and thromboplastin-like substance) in the culture supernatants. The in vivo traffic patterns of autologous MAK cells labelled ex-vivo with¹¹¹In oxine were studied in 7 patients. Gamma imaging showed an immediate but transient lung uptake (<24 h), and a progressive uptake of radioactivity in the liver and spleen was seen from 6 h to 72 h post-infusion. Our results indicate that the preparation of high numbers of autologous, blood-derived MAK cells is a feasible procedure, and their transfusion is safe for patients. This immunotherapeutic approach seems to be encouraging from the point of view of establishing an adjuvant therapeutic modality in cancer patients with minimal residual disease.This work was supported in part by a grant 6911 from the Association pour la Recherche contre le Cancer (ARC), grants from the Ligue Nationale contre le cancer and the Ligues Regionales (Bas-Rhin, Haut-Rhin) contre le cancer, and contract 891013 from the Institut National pour la Santé et la Recherche Médicale (INSERM), France     

Resistance in the wild crop relatives Avena macrostachya and Hordeum bogdani to the aphid Rhopalosiphum padi

In previous screening tests the two wild crop relatives Avena macrostachya (Bal., ex Coss. et Dur.) and Hordeum bogdani (Wil.) demonstrated a high degree of resistance to the aphid Rhopalosiphum padi (L.). In a choice situation using wild and cultivated oats and barley, alate aphids settled in lower numbers on the wild species. The results were, however, variable in the Avena combination. Nymph production was significantly higher, development time shorter and adult weight higher on the cultivated varieties. From the third instar and onwards the excretion of honeydew was significantly lower on the resistant plants. In general the honeydew contained less than 1% free amino acids although excreta from H. vulgare contained 3.5%. The percentage of free amino acids found in the honeydew was similar for all plant species (5.2–7.6%) except for H. vulgare, on which the aphids excreted 22% of the amounts ingested. Amino acids excreted in high proportions on all plants included asparagine, -aminobutyric acid, glutamic acid, and glycine. Tissue sectioning did not reveal any obvious mechanical barriers to stylet penetration. The potential use of these wild species as sources for aphid resistance breeding in oats and barley is considered.

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Résumè Lors d'examens systématiques antérieurs, Avena macrostachya (Bal. ex Coss. & Dur.) et Hordeum bogdani (Wil.) ont présenté une résistance élevée au puceron Rhopalosiphum padi (L.). Lorsqu'ils avaient un choix comprenant de l'avoine et de l'orge cultivés, les pucerons ailés ont atterri en nombres moins importants sur les espèces sauvages. Les résultats étaient cependant variables dans le complexe avoine. La production de nymphes et le poids des adultes étaient plus élevés sur espèces cultivées, ainsi que la durée du développement était plus longue sur les espèces sauvages. A partir du troisième stade, l'excrétion de miellat a été significativement plus faible sur les espèces résistantes. En général, le miellat y contenait moins de 1% d'acides aminés bien que sur H. vulgare il en contînt 3,5%. Les pourcentages d'acides aminés libres du miellat étaient semblables sur toutes les plantes (5,2–7,6%), à l'exception de H. vulgare sur lequel les pucerons excrétaient 22% des taux ingérés. Les acides aminés excrétés en fortes quantités sur les différentes plantes, comprenaient l'asparagine, l'acide -aminobutyrique, l'acide glutamique et la glycine. Des coupes de tissus n'ont révélé aucun obstacle mécanique clair à la pénétration des stylets. Les possibilités d'utiliser ces espèces sauvages comme source de résistance aux pucerons dans la sélection de l'avoine et de l'orge ont été examinées.

     

Cowpea aphid performance and behaviour on two resistant cowpea lines

In a series of laboratory and climate chamber tests we compared the growth and behaviour of Aphis craccivora on one susceptible (ICV-1) and two aphid-resistant (ICV-11 and ICV-12) cowpea lines. The aphids' growth rates were much lower on the resistant cowpea lines than on the susceptible one, indicating strong antibiosis. In addition, the aphids invariably settled in higher numbers on the susceptible line than on either of the resistant. Compared to ICV-1, damaged leaves of the resistant line ICV-12 were settled upon to a higher degree than undamaged leaves, and leaf discs from the same line were even less resistant.On resistant lines individual aphids waited a significantly longer time before making their first test probe. Total probing time as well as the time preceding a decision to stay or leave was also longer.These results are discussed in relation to the possible mechanisms involved, and we also consider the effects of previous leaf feeding on the expression of resistance in the field.

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Résumé Au cours d'expériences au laboratoire et en chambres climatisées nous avons comparé la croissance et le comportement de A. craccivora sur une lignée sensible (ICV-1) et deux lignées résistantes (ICV-11 et ICV-12) de V. unguiculata. Les vitesses de croissance des pucerons ont été beaucoup plus lentes sur les lignées résistantes que sur la lignée sensible, ce qui révèle une forte antibiose. De plus, les pucerons atterrissent invariablement en plus grand nombre sur la variété sensible. Par comparaison avec ICV-1, les atterrissages sur lignée résistante ICV-12 étaient plus nombreux sur les feuilles endommagées que sur les feuilles intactes; les disques de feuilles de cette même lignée étaient encore moins résistants.Les pucerons ont séjourné individuellement un temps plus long sur les lignées résistantes avant de faire leur premier sondage. Le temps consacré aux sondages ainsi que le temps précédant de choix entre départ ou maintien sur la feuille étaient plus longs avec les lignées résistantes.Ces résultats ont été discutés en fonction des mécanismes impliqués. Nous avons aussi examiné les effets de la consommation antérieure sur les manifestations de la résistance dans la nature.

     

Cloning of Trametes versicolor Genes Induced by Nitrogen Starvation

We have screened a genomic library of Trametes versicolor for genes whose expression is associated with nitrogen starvation, which has been shown to induce ligninolytic activity. Using two different approaches based on differential expression, we isolated 29 clones. These were shown by restriction mapping and cross-hybridization to code for 11 distinct differentially expressed genes. Northern analysis of the kinetics of expression of these genes revealed that at least four of them have kinetics of induction that parallel kinetics of induction of ligninolytic activity.     

Behaviour of drifting insect larvae

The larval drift behaviour of 23 species representing Ephemeroptera, Plecoptera and Trichoptera was investigated in the laboratory using different current regimes. Mayfly nymphs often performed swimming, while caddis larvae were reluctant to do so. Stonefly nymphs were intermediate. In mayflies swimming seemed to be used to reach the substrate as soon as possible. In contrast most stonefly nymphs by swimming prolonged the time spent in the water column. Modes of swimming and sinking posture differed markedly between the orders. Living passively sinking animals often reached bottom faster than dead control specimens, so consequently behaviour did not always express itself in activity. Some caddis larvae spun adherent anchor lines. Differences among taxa seemed more important in explaining swimming activity compared to preferred habitats (as stream, river and lake) in each species. However, observed differences among closely related species indicated subtle differences related to microhabitat to be of profound importance in explaining the alternative behavioural strategies used.     

Virulence genes are carried by a megaplasmid of the plant pathogen Pseudomonas solanacearum

Summary A class of avirulent mutants of the plant pathogenic bacterium Pseudomonas solanacearum, strain GMI1000, resistant to acridine orange (Acr^r), harbour a deletion of over 85 kb in their genome. This deletion affects, a1,000 kb megaplasmid which has previously been shown to be present in most of the strains of this species. In addition at least 11 out of 13 independent Tn5 insertions, leading to loss of virulence, are located on the megaplasmid. Nine of them are present in the region which is deleted from the Acr^r mutants. These results suggest that the majority of virulence genes identified so far are plasmid borne.