The binding motifs for Ac transposase are absolutely required for excision of Ds1 in maize

A reverse genetic system for studying excision of the transposable elementDs1 in maize plants has been established previously. In this system, theDs1 element, as part of the genome of maize streak virus (MSV), is introduced into maize plants via agroinfection. In the presence of theAc element, excision ofDs1 from the MSV genome results in the appearance of viral symptoms on the maize plants. Here, we used this system to study DNA sequences requiredin cis for excision ofDs1. TheDs1 element contains theAc transposase binding motif AAACGG in only one of its subterminal regions (defined here as the 5′ subterminal region). We showed that mutation of these motifs abolished completely the excision capacity ofDs1. This is the first direct demonstration that the transposase binding motifs are essential for excision. Mutagenesis with oligonucleotide insertions in the other (3′) subterminal region resulted in elements with either a reduced or an increased excision efficiency, indicating that this subterminal region also has an important function.     

A focal adhesion factor directly linking intracellularly motile Listeria monocytogenes and Listeria ivanovii to the actin-based cytoskeleton of mammalian cells.

The surface-bound ActA polypeptide of the intracellular bacterial pathogen Listeria monocytogenes is the sole listerial factor needed for recruitment of host actin filaments by intracellularly motile bacteria. Here we report that following Listeria infection the host vasodilator-stimulated phosphoprotein (VASP), a microfilament- and focal adhesion-associated substrate of both the cAMP- and cGMP-dependent protein kinases, accumulates on the surface of intracytoplasmic bacteria prior to the detection of F-actin 'clouds'. VASP remains associated with the surface of highly motile bacteria, where it is polarly located, juxtaposed between one extremity of the bacterial surface and the front of the actin comet tail. Since actin filament polymerization occurs only at the very front of the tail, VASP exhibits properties of a host protein required to promote actin polymerization. Purified VASP binds directly to the ActA polypeptide in vitro. A ligand-overlay blot using purified radiolabelled VASP enabled us to identify the ActA homologue of the related intracellular motile pathogen, Listeria ivanovii, as a protein with a molecular mass of approximately 150 kDa. VASP also associates with actin filaments recruited by another intracellularly motile bacterial pathogen, Shigella flexneri. Hence, by the simple expedient of expressing surface-bound attractor molecules, bacterial pathogens effectively harness cytoskeletal components to achieve intracellular movement.     

Feeding biology of two brachionid rotifers: Brachionus quadridentatus and Brachionus plicatilis

Brachionus quadridentatus and Brachionus plicatilis were examined in laboratory cultures to investigate their (1) food requirements and (2) ability to select particles on the basis of size. Growth experiments showed that the Chlorophyceans Nannochloris sp. and Chlorella vulgaris possess good nutritional value for these rotifers. However, B. quadridentatus declined in cultures with Microcystis firma, detritus of Enteromorpha sp. or Pseudomonas sp. (bacteria) as a sole source of food, thus indicating that these foods were inadequate. Utilization of latex microspheres by these two species indicate that they respond to particle size in different ways: B. quadridentatus had a higher selectivity index for 3 and 5 µm beads, while B. plicatilis had a higher selectivity index for 2 µm beads.     

MUTATIONAL MELTDOWNS IN SEXUAL POPULATIONS

Although it is widely acknowledged that the gradual accumulation of mildly deleterious mutations is an important source of extinction for asexual populations, it is generally assumed that this process is of little relevance to sexual species. Here we present results, based on computer simulations and supported by analytical approximations, that indicate that mutation accumulation in small, random-mating monoecious populations can lead to mean extinction times less than a few hundred to a few thousand generations. Unlike the situation in obligate asexuals in which the mean time to extinction (t?_e) increases more slowly than linearly with the population carrying capacity (K), t?_e increases approximately exponentially with K in outcrossing sexual populations. The mean time to extinction for obligately selfing populations is shown to be equivalent to that for asexual populations of the same size, but with half the mutation rate and twice the mutational effect; this suggests that obligate selfing, like obligate asexuality, is inviable as a long-term reproductive strategy. Under all mating systems, the mean time to extinction increases relatively slowly with the logarithm of fecundity, and mutations with intermediate effects (similar to those observed empirically) cause the greatest risk of extinction. Because our analyses ignore sources of demographic and environmental stochasticity, which have synergistic effects that exacerbate the accumulation of deleterious mutations, our results should yield liberal upper bounds to the mean time to extinction caused by mutational degradation. Thus, deleterious mutation accumulation cannot be ruled out generally as a significant source of extinction vulnerability in small sexual populations or as a selective force influencing mating-system evolution.     

BBReader: A computer program for the combined use of the BioMagResBank and PDB databases

A computer program (BBReader) was developed which performs an inverse search in theBioMagResBank database. Given (cross) peak positions of a protein, the program searchesfor atoms with matching chemical shifts and suggests possible assignments for user-specifiedhomo- and heteronuclear one- to three-dimensional COSY- and NOESY-type experiments.It can handle 1H, 13C and 15N spectra. Distance information from PDB files can be utilizedfor filtering possible NOESY cross peak assignments.     

Cholinergic Control of Nerve Growth Factor in Adult Rats: Evidence from Cortical Cholinergic Deafferentation and Chronic Drug Treatment

Abstract: It is well documented that nerve growth factor (NGF) plays an important role in maintaining functions of cholinergic basal forebrain neurons. In the present study, we tested the hypothesis that cholinergic activity controls NGF levels in cholinoceptive neurons of the cerebral cortex and hippocampus. To address that question, we used both cholinergic deafferentation of cerebral cortex and hippocampus by cholinergic immunolesion with 192IgG-saporin and chronic pharmacological treatment of sham-treated and immunolesioned rats with the cholinergic agonist pilocarpine and the cholinergic antagonist scopolamine. We observed an increase in NGF protein levels in the cortex and hippocampus after cholinergic immunolesions and also after muscarinic receptor blockade by chronic intracerebroventricular scopolamine infusion in sham-treated rats after 2 weeks. There was no further increase in the accumulation of NGF after scopolamine treatment of immunolesioned rats. Chronic infusion of pilocarpine had no effect on cortical and hippocampal NGF protein levels in sham-treated rats. In rats with cholinergic immunolesions, however, pilocarpine did prevent the lesion-induced accumulation of NGF. There was no effect of cholinergic lesion and drug treatment on cortical or hippocampal NGF mRNA levels, consistent with the importance of NGF retrograde transport as opposed to its de novo synthesis. This study provides strong evidence for the hypothesis that there is cholinergic control of cortical and hippocampal NGF protein but not mRNA levels in adult rats.     

Larval Planktotrophy—A Primitive Trait in the Bilateria?

The concept of Gösta Jägersten of a primary biphasic metazoan life-cycle, consisting of a planktotrophic larva and a benthic adult, forms the basis for several theories on metazoan phylogeny. In this paper the assumed planktotrophic life-style of the larva is critically analyzed and reconsidered. It is shown, in particular for the Mollusca, that a biphasic life-cycle with a lecithotrophic larva is probably the plesiomorphic condition. Character distribution and structural data suggest a parallel evolution of the downstream collecting system used in planktotrophic larvae or filter-feeding adults of gastropods, bivalves and other spiralian or aschelminth taxa. In the basic metazoans (Parazoa, Placozoa, coelenterates) direct or lecithotrophic development dominates by far. For the acoelomate (Platyhelminthes, Gnathostomulida) and pseudocoelomate taxa direct development is probably the plesiomorphic condition. The structural similarities of the upstream collecting system in tentaculate and deuterostome phyla may also be explained by parallel events of heterochrony out of an ancestor with adult filter-feeding. The main conclusion of this survey is that larval planktotrophy is likely to be secondary and not a plesiomorphic condition among the Bilateria. Accordingly, theories which are based on the assumed plesiomorphy of larval planktotrophy of the Bilateria, need careful reevaluation.     

Developmental regulation of presence of binding sites for neoglycoproteins and endogenous lectins in various embryonic stages of human lung,liver and heart

Protein-carbohydrate interactions are supposed to play key roles in the mechanisms of cell adhesion, biosignalling and intracellular routing, warranting the analysis of the developmental course of expression of epitopes of this system. Thus, a panel of carrier-immobilized carbohydrate ligands was used as probes, namely lactose,N-acetylgalactosamine,N-acetylglucosamine, mannose, fucose and maltose. Additionally, an antibody to an endogenous -galactoside-binding lectin (anti-galectin-1), the biotinylated lectin and two further human lectins, namely the macrophage migration inhibitory factor-binding sarcolectin and serum amyloid P component (SAP) that displays selectivity for sulphated sugars and mannose-6-phosphate, were included. They enabled us to assess the extent of the presence of respective binding sites in fixed sections from human lungs (pulmonary epithelial cells), livers (hepatocytes) and hearts (myocard cells) of 10–50 weeks gestation. Invariably, specific binding was detected in the three organ types, at least in certain stages. In most of the cases, the intensity of staining exhibited developmental regulation. The apparent patterns reveal similarities between the different cell types, as seen with immobilizedN-acetylglucosamine as well as with labelled galectin-1 and sarcolectin. However, drastic differences among such patterns with nearly opposite developmental courses do also occur, as detected for carrier-attached mannose and maltose residues. These results point to a potential importance for the detected glycohistochemical features in human development and substantiate the possibility of differential regulation of the presence of binding sites for distinct sugars within a certain organ and between the individual cell types of the monitored organs.     

One of three nuclear localization signals of maize Activator (Ac) transposase overlaps the DNA-binding domain

The nuclear localization sequences (NLSs) of the Ac transposase (TPase) protein have been characterized by indirect immunofluorescence detection of TPase deletion derivatives and TPase/β-glucuronidase (GUS) fusion proteins in transiently transfected Petunia cells. The TPase contains three NLSs near its amino-terminal end, NLS(44–62), NLS(159–178) and NLS(174–206), each of which is sufficient to redirect GUS to the nucleus. Deletion of the N-terminal 102 TPase residues including NLS(44–62) results in strongly reduced nuclear import of the truncated TPase. NLS(44–62) and NLS(159–178) are bipartite NLSs, whereas the structure of NLS(174–206) does not allow a classification into one of the three major NLS categories. NLS(174–206) overlaps with the basic DNA-binding domain of TPase. A substitution of two amino acids in this segment (HiS₁₉₁→Arg and Arg₁₉₃→His) results in a total loss of DNA-binding activity, but retains reduced NLS activity. Accordingly, the two functions can be separated. In addition, we show that a NLS-deficient 71 kDa TPase derivative is co-imported into the nucleus in the presence of wildtype TPase.     

Histidine decarboxylase: Isolation and molecular characteristics

High levels of histidine decarboxylase activity were measured in rat basophilic leukemia cells grown in ascitic form in 4 week old WKY/N rats. The potent inhibition of this enzyme by brocresine and -methylhistidine but not by -methyl DOPA identified it as a specific histidine decarboxylase. Gel filtration and polyacrylamide gel electrophoresis revealed a molecular weight of 125,000 for the native enzyme, similar to that of fetal rat liver histidine decarboxylase. Using rat basophilic leukemia cells as starting material, histidine decarboxylase was purified extensively in a seven step procedure. Electrophoresis under denaturing conditions revealed that histidine decarboxylase is a dimeric protein consisting of two identical subunits with a molecular weight of 62,000. The results indicate that rat basophilic leukemia cells provide a new and rich source for the purification of histidine decarboxylase.