Effect of cytokinins on plastid development and photosynthetic polypeptides during organogenesis ofPinus ponderosa Dougl. cotyledons culturedin vitro

InPinus ponderosa Dougl., application of the cytokinins, benzyladenine and 2-isopentenyl adenine, to excised cotyledons, promoted thein vitro formation of meristematic centers which led to bud and shoot production. Meristematic cells showed plastids with poorly developed thylakoid membranes and rudimentary grana, whereas cells in non-meristematic tissues and in growth regulator free medium, had chloroplasts with well developed inner membranes, and more thylakoid membranes and grana than plastids of meristematic cells. Chlorophyll and six polypeptides associated with photosynthesis were present in lower concentrations in cytokinin-treated cotyledons than in those cultured in growth regulator free medium. Both benzyladenine and 2-isopentenyl adenine are effective in inhibiting the accumulation of at least two photosynthetic polypeptides in the first 24 h in culture. The ability of cotyledons to respond in this way to cytokinins is lost after three days in culture in growth regulator free medium prior to treatment with cytokinin.     

In vitro characterisation of a monovalent and bivalent form of a fully human anti Ep-CAM phage antibody

 Antibodies to tumour-associated antigens are increasingly being used as targeting vehicles for the visualisation and for therapy of human solid tumours. The epithelial cell adhesion molecule (Ep-CAM) is an antigen that is overexpressed on a variety of human solid tumours and constitutes an attractive target for immunotargeting. We set out to obtain fully human antibodies to this antigen by selecting from a large antibody repertoire displayed on bacteriophages. Two single-chain variable antibody fragments (scFv) were identified that specifically bound recombinant antigen in vitro. One of the selected antibodies (VEL-1) cross-reacted with extracellular matrix components in immunohistochemistry of colon carcinoma, whereas the other scFv (VEL-2) specifically recognised colon cancer cells. The latter antibody was further characterised with respect to epitope specificity and kinetics of antigen-binding. It showed no competition with the well-characterised anti Ep-CAM MOC-31 monoclonal antibody and had an off-rate of 5 × 10⁻² s⁻¹. To obtain an antibody format more suitable for in vivo tumour targeting and to increase the apparent affinity through avidity, the genes of scFv VEL-2 were re-formatted by fusion to a human (γ1) hinge region and CH3 domain. This “minibody” was expressed in Escherichia coli, specifically bound the Ep-CAM antigen and showed a 20-fold reduced off-rate in surface plasmon resonance analysis. These results show that phage antibody selection, combined with antibody engineering, may result in fully human antibody molecules with promising characteristics for in vivo use in tumour targeting. Received: 13 July 2000 / Accepted: 12 October 2000     

Mutations in cell division proteins FtsZ and FtsA inhibit φX174 protein-E-mediated lysis of Escherichia coli

Electron microscopic studies emphasized that the protein-E-specific transmembrane tunnel structure, which permeabilizes Escherichia coli, is not randomly distributed over the cell envelope but is restricted to areas of potential division sites. These sites were located predominantly in the middle of the cell, but approximately one-third of these structures are found at the polar sites. Therefore, E. coli mutant strains with defects in cell division components were tested for their sensitivity to protein-E-mediated lysis. The ftsZ84 and the ftsA12 cell division mutant strains of E. coli were tolerant to protein-E-mediated lysis, whereas the ftsA3 mutant strain was lysed by protein E under conditions nonpermissive for division. The protein-E-tolerant phenotype of ftsZ84 and ftsA12 and the lysis-sensitive phenotype of other components of the septosome (e.g., ftsA3, ftsQ, and ftsI) suggest that initiation of cell division – rather than specific functions of cell division – plays an essential role in protein-E-mediated lysis. SulA-overproducing cells had a lysis-positive phenotype, the ring structure – but not the GTPase function - of FtsZ was impaired. Received: 14 April 1998 / Accepted: 9 June 1998     

Identification of MK-5710 ((8aS)-8a-methyl-1,3-dioxo-2-[(1S,2R)-2-phenylcyclo- propyl]-N-(1-phenyl-1H-pyrazol-5-yl)hexahydro-imidazo[1,5-a]pyrazine-7(1H)-carboxamide), a potent smoothened antagonist for use in Hedgehog pathway dependent malignancies, part 2

The Hedgehog (Hh-) signaling pathway is a key developmental pathway which gets reactivated in many human tumors, and smoothened (Smo) antagonists are emerging as novel agents for the treatment of malignancies dependent on the Hh-pathway, with the most advanced compounds demonstrating encouraging results in initial clinical trials. A novel series of potent bicyclic hydantoin Smo antagonists was reported in the preceding article, these have been resolved, and optimized to identify potent homochiral derivatives with clean off-target profiles and good pharmacokinetic properties in preclinical species. While showing in vivo efficacy in mouse allograft models, unsubstituted bicyclic tetrahydroimidazo[1,5-a]pyrazine-1,3(2H,5H)-diones were shown to epimerize in plasma. Alkylation of the C-8 position blocks this epimerization, resulting in the identification of MK-5710 (47) which was selected for further development.     

Smog nitrogen and the rapid acidification of forest soil, San Bernardino Mountains, southern California

We report the rapid acidification of forest soils in the San Bernardino Mountains of southern California. After 30 years, soil to a depth of 25 cm has decreased from a pH (measured in 0.01 M CaCl2) of 4.8 to 3.1. At the 50-cm depth, it has changed from a pH of 4.8 to 4.2. We attribute this rapid change in soil reactivity to very high rates of anthropogenic atmospheric nitrogen (N) added to the soil surface (72 kg ha(-1) year(-1)) from wet, dry, and fog deposition under a Mediterranean climate. Our research suggests that a soil textural discontinuity, related to a buried ancient landsurface, contributes to this rapid acidification by controlling the spatial and temporal movement of precipitation into the landsurface. As a result, the depth to which dissolved anthropogenic N as nitrate (NO3) is leached early in the winter wet season is limited to within the top approximately 130 cm of soil where it accumulates and increases soil acidity.     

Towards the Generation of B-Cell Receptor Retrogenic Mice

Transgenic expression of B- and T-cell receptors (BCRs and TCRs, respectively) has been a standard tool to study lymphocyte development and function in vivo. The generation of transgenic mice is time-consuming and, therefore, a faster method to study the biology of defined lymphocyte receptors in vivo would be highly welcome. Using 2A peptide-linked multicistronic retroviral vectors to transduce stem cells, TCRs can be expressed rapidly in mice of any background. We aimed at adopting this retrogenic technology to the in vivo expression of BCRs. Using a well characterised BCR specific for hen egg lysozyme (HEL), we achieved surface expression of the retrogenically encoded BCR in a Rag-deficient pro B-cell line in vitro. In vivo, retrogenic BCRs were detectable only intracellularly but not on the surface of B cells from wild type or Rag2-deficient mice. This data, together with the fact that no BCR retrogenic mouse model has been published in the 7 years since the method was originally published for TCRs, strongly suggests that achieving BCR-expression in vivo with retrogenic technology is highly challenging if not impossible.     

Tit for tat? A mycorrhizal fungus accumulates phosphorus under low plant carbon availability

The exchange of carbohydrates and mineral nutrients in the arbuscular mycorrhizal (AM) symbiosis must be controlled by both partners in order to sustain an evolutionarily stable mutualism. Plants downregulate their carbon (C) flow to the fungus when nutrient levels are sufficient, while the mechanism controlling fungal nutrient transfer is unknown. Here, we show that the fungus accumulates nutrients when connected to a host that is of less benefit to the fungus, indicating a potential of the fungus to control the transfer of nutrients. We used a monoxenic in vitro model of root organ cultures associated with Glomus intraradices, in which we manipulated the C availability to the plant. We found that G. intraradices accumulated up to seven times more nutrients in its spores, and up to nine times more in its hyphae, when the C pool available to the associated roots was halved. The strongest effect was found for phosphorus (P), considered to be the most important nutrient in the AM symbiosis. Other elements such as potassium and chorine were also accumulated, but to a lesser extent, while no accumulation of iron or manganese was found. Our results suggest a functional linkage between C and P exchange.     

Embryologische und histogenetische Studien an „ monokotylen Dikotylen“ III.Anemone apennina L.

Zusammenfassung Anemone apennina gehört in die kleine Grappe der einkeimblättrigen Dikotylen-Arten.Der einzige Kotyledo wird lateral angelegt, drängt aber den Sproßvegetationspunkt später etwas zur Seite. Von der Anlage eines zweiten Kotyledos fehlt jede Spur.Weder der Embryogenese noch der Entwicklungsgeschichte und der Anatomie der Keimpflanzen sind Hinweise für das Vorliegen einer kongenitalen Verwachsung zweier Kotyledonen (Synkotylie) zu entnehmen.Der Kotyledo ist in gerader Linie über dem einen Pol der Xylemplatte der diarchen Keimachsenstele inseriert. Der Kotyledonarstiel wird von einem einfachen Leitstrang durchzogen, der im basalen Teil als diarche Stele und im apikalen als kollaterales Bündel ausgebildet ist. Die Nervatur der zweiteiligen Kotyledonarspreite macht wahrscheinlich, daß es sich um ein asymmetrisch fiederteiliges Blatt handelt.Daraus ergibt sich, daß bei A.apennina eine echte Einkeimblättrigkeit vorliegt, in dem Sinne, daß das einzige Keimblatt einem der beiden Keimblätter verwandter Arten homolog ist.Wir danken der Deutschen Forschungsgemeinschaft für die Unterstützung unserer Arbeiten.     

Photobiont association and genetic diversity of the optionally lichenized fungus Schizoxylon albescens

The fungus Schizoxylon albescens occurs both as lichen and as saprobe. Lichenized colonies grow on the bark of Populus tremula; saprotrophic morphs grow on dead Populus branches. We wanted to (1) test whether lichenized and saprotrophic S. albescens are genetically distinct, (2) investigate photobiont association and diversity, (3) investigate the interactions between fungi and algae that occur during co-cultivation and (4) test whether Schizoxylon shows algal selectivity during lichenization. Fungal and algal genetic diversity were investigated for three markers. Algae from lichenized thalli were isolated in axenic cultures, and isolate sequence diversity was compared with algae amplified directly from thallus fragments. Co-culture experiments of fungi and algae were performed to study the morphological interaction patterns. Two distinct phylogenetic units are revealed in S. albescens, which are interpreted as phenotypically cryptic species. The algae are related to Coccomyxa and Pseudococcomyxa, and form two distinct sister clades separating samples isolated in cultures from those amplified directly from thallus fragments, indicating that more easily cultured strains of algae are not necessarily major components of the lichens. Schizoxylon albescens interacts with isolated algal strains, similar to fungal-Coccomyxa symbioses in nature. As the system is maintained without difficulty in culture, it can potentially be an easily controlled lichen symbiosis study system under laboratory conditions.