UDP-glucose:digitoxin 16′-O-glucosyltransferase from suspension-cultured Digitalis lanata cells

Suspension cultures from several cell lines of Digitalis lanata, as well as cultures from 6 other plant species were checked for their ability to form purpurea-glycoside A from digitoxin. An in-vitro assay for the UDP-glucose:digitoxin 16-O-glucosyltransferase (DGT, EC 2.4.1.-) has been established based on an HPLC method. The enzyme is located in the soluble fraction. Its pH optimum is at 7.4. No enzyme activity was found in either purified vacuole preparations or lysed vacuoles. Ascorbate (10 mM) increased the transferase activity about 4-fold. Of the sugar nucleotides tested, only UDP-glucose served as a glucosyl donor. Digitoxin, digoxin, -acetyldigitoxin, and -acetyldigoxin are substrates for the glucosyltransferase. The role of the DGT during the biotransformation of cardenolides in Digitalis lanata cell suspension cultures is discussed.Abbreviation DGT UDP-glucose:digitoxin 16-C-glucosyltransferase     

Paleoenvironmental analysis

New analysis has been carried out concerning the palaeoenvironmental reconstruction of some Italian sites dating from the Middle Pleistocene to the Bronze Age. Different aspects have been investigated on each site considering the data collected. The following sites have been analyzed: Isernia La Pineta (Molise); Visogliano and Caverna degli Orsi (Tieste); Toirano Caves (Liguria); Grotta Paglicci (Gargano); Riparo del Molare (Salerno); Grotta del Cavallo (Lecce); Castellaro Lagusello (Monzambano, Mantova).     

Phylogeny of novel naked Filose and Reticulose Cercozoa: Granofilosea cl. n. and Proteomyxidea revised

Naked filose and reticulose protozoa were long lumped as proteomyxids or left outside higher groups. We cultivated eight naked filose or reticulose strains, did light microscopy, 18S rDNA sequencing and phylogeny (showing all are Cercozoa), and sequenced 80 environmental 18S-types. Filose species belong in subphylum Filosa and reticulose ones in subphylum Endomyxa, making proteomyxids polyphyletic. We therefore transfer the classically mainly reticulose Proteomyxidea to Endomyxa, removing evident filosans as new class Granofilosea (including Desmothoracida, Acinetactis and new heliomonad family Heliomorphidae (new genus Heliomorpha (=Dimorpha)). Five new species of Limnofila gen. n. (L. mylnikovi; L. anglica; L. longa; L. oxoniensis; L. borokensis, previously misidentified as Biomyxa (=Gymnophrys) cometa) form a large freshwater clade (new order Limnofilida). Mesofila limnetica gen., sp. n. and Nanofila marina gen., sp. n. group separately in Granofilosea (Cryptofilida ord. n.). In Endomyxa, a new genus of reticulose proteomyxids (Filoreta marina, F. japonica, F. turcica spp. n., F. (=Corallomyxa) tenera comb. n.) forms a clade (Reticulosida) related to Gromiidea/Ascetosporea. Platyreta germanica gen., sp. n. and Arachnula impatiens are related vampyrellids (Aconchulinida) within a large clade beside Phytomyxea. Biomyxidae and Rhizoplasmidae fam. n. remain incertae sedis within Proteomyxidea. Gymnophrydium and Borkovia are revised. The reticulose Corallomyxa are unlike Filoreta and possibly Amoebozoa, not Cercozoa.     

Production of doubled haploid lines in frequencies sufficient for barley breeding programs

Winter barley (Hordeum vulgare L.) anthers were cultured on different liquid and on starch-solidified media. The optimal embryo and callus formation with different F₁-lines and the cv. Igri was obtained on a liquid medium with 20% Ficoll, 20 g/l maltose and barley starch. But the influence of the growth conditions of the donor plants and the genotypical differences are still enormous. The procedure has been optimized to such an extent that it can be used economically.     

The Binding Properties of the Particulate and Solubilized Sulfonylurea Receptor from Cerebral Cortex Are Modulated by the Mg2+ Complex of ATP

Glibenclamide closes an ATP-sensitive K+ channel (K-ATP channel) by interaction with the sulfonylurea receptor in the plasma membrane of pancreatic B cells and thereby initiates insulin release. Previous studies demonstrated that the Mg2+ complex of ATP decreases glibenclamide binding to the sulfonylurea receptor from pancreatic islets. The aim of the present study was to examine the effect of adenine and guanine nucleotides on binding of sulfonyl-ureas to the cerebral sulfonylurea receptor. For this purpose, binding properties of the particulate and solubilized site from rat or pig cerebral cortex were analyzed. Maximum recovery of receptors in detergent extracts amounted to 40-50%. Specific binding of [3H]glibenclamide to the solubilized receptors corresponded well to specific binding to microsomes. In microsomes and detergent extracts, the Mg2+ complexes of ATP, ADP, GTP, and GDP inhibited binding of [3H]glibenclamide. These effects were not observed in the absence of Mg2+. In detergent extracts, Mg-ATP (300 microM) reduced the number of high-affinity sites for [3H]-glibenclamide by 52% and increased the dissociation constant for [3H]glibenclamide by eightfold; Mg-ATP was half-maximally effective at 41 microM. Alkaline phosphatase accelerated the reversal of Mg-ATP-induced inhibition of [3H]glibenclamide binding. The data suggest similar control of the sulfonylurea receptor from brain and pancreatic islets by protein phosphorylation.     

MODBASE, a database of annotated comparative protein structure models

MODBASE (http://guitar.rockefeller.edu/modbase) is a relational database of annotated comparative protein structure models for all available protein sequences matched to at least one known protein structure. The models are calculated by MODPIPE, an automated modeling pipeline that relies on PSI-BLAST, IMPALA and MODELLER. MODBASE uses the MySQL relational database management system for flexible and efficient querying, and the MODVIEW Netscape plugin for viewing and manipulating multiple sequences and structures. It is updated regularly to reflect the growth of the protein sequence and structure databases, as well as improvements in the software for calculating the models. For ease of access, MODBASE is organized into different datasets. The largest dataset contains models for domains in 304 517 out of 539 171 unique protein sequences in the complete TrEMBL database (23 March 2001); only models based on significant alignments (PSI-BLAST E-value < 10^–4) and models assessed to have the correct fold are included. Other datasets include models for target selection and structure-based annotation by the New York Structural Genomics Research Consortium, models for prediction of genes in the Drosophila melanogaster genome, models for structure determination of several ribosomal particles and models calculated by the MODWEB comparative modeling web server.     

Exposure of Petunia Seedlings to Ethylene Decreased Apical Dominance by Reducing the Ratio of Auxin to Cytokinin

Seedlings of Petunia x hybrida ‘Orchid’ treated with the ethylene-releasing compound ethephon at 0.9, 1.7, and 3.5 mM evolved ethylene at a higher rate as the concentration of ethephon increased. Regardless of the concentration of ethephon applied, ethylene evolution peaked 6 to 8 h following application. Evidence that ethephon application decreased apical dominance included an increase in the number of new nodes on the main stem and a sustained increase in the length of new and existing lateral shoots compared to the control (no ethephon). Plants treated with 3.5 mM ethephon developed mild chlorosis, whereas a concentration of 1.7 mM ethephon decreased apical dominance without phytotoxic effects. The auxin/cytokinin ratio decreased in the apical shoot section as early as 1 h after ethephon treatment. In contrast, a decrease in the ratio in the subapical shoot section was not detected until 24 h after ethephon application. Reduction in auxin/cytokinin ratio was a result of a decrease in indole-3-acetic acid (IAA) and an increase of zeatin riboside (ZR), but not isopentenyladenosine (iPA). These results suggest that exposing ‘Orchid’ petunia seedlings to ethylene via ethephon lowers the auxin/cytokinin ratio, thereby promoting the outgrowth of lateral shoots.     

Electrostatic recognition in substrate binding to serine proteases

Serine proteases of the Chymotrypsin family are structurally very similar but have very different substrate preferences. This study investigates a set of 9 different proteases of this family comprising proteases that prefer substrates containing positively charged amino acids, negatively charged amino acids, and uncharged amino acids with varying degree of specificity. Here, we show that differences in electrostatic substrate preferences can be predicted reliably by electrostatic molecular interaction fields employing customized GRID probes. Thus, we are able to directly link protease structures to their electrostatic substrate preferences. Additionally, we present a new metric that measures similarities in substrate preferences focusing only on electrostatics. It efficiently compares these electrostatic substrate preferences between different proteases. This new metric can be interpreted as the electrostatic part of our previously developed substrate similarity metric. Consequently, we suggest, that substrate recognition in terms of electrostatics and shape complementarity are rather orthogonal aspects of substrate recognition. This is in line with a 2‐step mechanism of protein‐protein recognition suggested in the literature.     

An RNAi-Based Approach to Down-Regulate a Gene Family In Vivo

Genetic redundancy poses a major problem to the analysis of gene function. RNA interference allows the down-regulation of several genes simultaneously, offering the possibility to overcome genetic redundancy, something not easily achieved with traditional genetic approaches. Previously we have used a polycistronic miR155-based framework to knockdown expression of three genes of the early B cell factor family in cultured cells. Here we develop the system further by generating transgenic mice expressing the RNAi construct in vivo in an inducible manner. Expression of the transgene from the strong CAG promoter is compatible with a normal function of the basal miRNA/RNAi machinery, and the miR155 framework readily allows inducible expression from the Rosa26 locus as shown by Gfp. However, expression of the transgene in hematopoietic cells does not lead to changes in B cell development and neuronal expression does not affect cerebellar architecture as predicted from genetic deletion studies. Protein as well as mRNA levels generated from Ebf genes in hetero- and homozygous animals are comparable to wild-type levels. A likely explanation for the discrepancy in the effectiveness of the RNAi construct between cultured cells and transgenic animals lies in the efficiency of the sequences used, possibly together with the complexity of the transgene. Since new approaches allow to overcome efficiency problems of RNAi sequences, the data lay the foundation for future work on the simultaneous knockdown of several genes in vivo.     

The social position of male Barbary macaques (Macaca sylvanus) in a semifree-ranging population

A study was carried out on the social position of 12 subadult males of a semifreeranging Barbary macaque population during the non-mating season. The social position was measured in terms of spatial as well as interactive parameters. The subadult males had social contacts to members of nearly all other age-sex classes but showed clear preferences for same-sexed partners. Besides this differences were found between 5- and 6-year-old males with respect to their interaction profiles and the preference for special classes of interaction partners. The terms “peripheral-central” is discussed with reference to the social structures of macaque societies. The data of the present study indicate that the social position of subadult male Barbary macaques can not be described by one of these terms exclusively. The results are compared to other studies on Barbary macaques and other macaque species. It is concluded that in macaque societies subadult males are not obligatorily forced to live at the periphery or to abide. It is proposed not to postulate stiff social structures but to put more emphasis on the range of variation among macaque species.