Implications for the domain arrangement of axonin-1 derived from the mapping of its NgCAM binding site.

The neuronal cell adhesion molecule axonin-1 is composed of six immunoglobulin and four fibronectin type III domains. Axonin-1 promotes neurite outgrowth, when presented as a substratum for neurons in vitro, via a neuronal receptor that has been identified as the neuron-glia cell adhesion molecule, NgCAM, based on the blocking effect of polyclonal antibodies directed to NgCAM. Here we report the identification of axonin-1 domains involved in NgCAM binding. NgCAM-conjugated microspheres were tested for binding to COS cells expressing domain deletion mutants of axonin-1. In addition, monoclonal antibodies directed to axonin-1 were assessed for their ability to block the axonin-1-NgCAM interaction, and their epitopes were mapped using the domain deletion mutants. The results suggest that the four amino-terminal immunoglobulin domains of axonin-1 form a domain conglomerate which is necessary and sufficient for NgCAM binding. Surprisingly, NgCAM binding to membrane-bound axonin-1 was increased strongly by deletion of the fifth or sixth immunoglobulin domains of axonin-1. Based on these results and on negative staining electron microscopy, we propose a horseshoe-shaped domain arrangement of axonin-1 that obscures the NgCAM binding site. Neurite outgrowth studies with truncated forms of axonin-1 show that axonin-1 is a neurite outgrowth-promoting substratum in the absence of the NgCAM binding site.     

Dynein-mediated cargo transport in vivo. A switch controls travel distance

Cytoplasmic dynein is a microtubule-based motor with diverse cellular roles. Here, we use mutations in the dynein heavy chain gene to impair the motor's function, and employ biophysical measurements to demonstrate that cytoplasmic dynein is responsible for the minus end motion of bidirectionally moving lipid droplets in early Drosophila embryos. This analysis yields an estimate for the force that a single cytoplasmic dynein exerts in vivo (1.1 pN). It also allows us to quantitate dynein-mediated cargo motion in vivo, providing a framework for investigating how dynein's activity is controlled. We identify three distinct travel states whose general features also characterize plus end motion. These states are preserved in different developmental stages. We had previously provided evidence that for each travel direction, single droplets are moved by multiple motors of the same type (Welte et al. 1998). Droplet travel distances (runs) are much shorter than expected for multiple motors based on in vitro estimates of cytoplasmic dynein processivity. Therefore, we propose the existence of a process that ends runs before the motors fall off the microtubules. We find that this process acts with a constant probability per unit distance, and is typically coupled to a switch in travel direction. A process with similar properties governs plus end motion, and its regulation controls the net direction of transport.     

Statistical confidence in parentage analysis with incomplete sampling: how many loci and offspring are needed?

We have recently presented models to estimate parentage in breeding systems with multiple mating and incomplete sampling of the candidate parents. Here we provide formulas to calculate the statistical confidence and the optimal trade-off between the number of loci and offspring. These calculations allow an understanding of the statistical significance of the parentage estimates as well as the appropriate sampling regime required to obtain a desired level of confidence. We show that the trade-off generally depends on the parentage of the putative parents. When parentage is low, sampling effort should concentrate on increasing the number of loci. Otherwise, there are similar benefits from increasing the number of loci or offspring. We demonstrate these methods using genetic data from a nest of the bluegill sunfish (Lepomis macrochirus).     

The above-ground coarse wood productivity of 104 Neotropical forest plots

The net primary production of tropical forests and its partitioning between long‐lived carbon pools (wood) and shorter‐lived pools (leaves, fine roots) are of considerable importance in the global carbon cycle. However, these terms have only been studied at a handful of field sites, and with no consistent calculation methodology. Here we calculate above‐ground coarse wood carbon productivity for 104 forest plots in lowland New World humid tropical forests, using a consistent calculation methodology that incorporates corrections for spatial variations in tree‐size distributions and wood density, and for census interval length. Mean wood density is found to be lower in more productive forests. We estimate that above‐ground coarse wood productivity varies by more than a factor of three (between 1.5 and 5.5 Mg C ha^?1 a^?1) across the Neotropical plots, with a mean value of 3.1 Mg C ha^?1 a^?1. There appear to be no obvious relationships between wood productivity and rainfall, dry season length or sunshine, but there is some hint of increased productivity at lower temperatures. There is, however, also strong evidence for a positive relationship between wood productivity and soil fertility. Fertile soils tend to become more common towards the Andes and at slightly higher than average elevations, so the apparent temperature/productivity relationship is probably not a direct one. Coarse wood productivity accounts for only a fraction of overall tropical forest net primary productivity, but the available data indicate that it is approximately proportional to total above‐ground productivity. We speculate that the large variation in wood productivity is unlikely to directly imply an equivalent variation in gross primary production. Instead a shifting balance in carbon allocation between respiration, wood carbon and fine root production seems the more likely explanation.     

State of art and limitations in genetic engineering to induce stable chondrogenic phenotype

Current protocols for chondrocyte expansion and chondrogenic differentiation of stem cells fail to reduce phenotypic loss and to mitigate hypertrophic tendency. To this end, cell genetic manipulation is gaining pace as a means of generating cells with stable chondrocyte phenotype. Herein, we provide an overview of candidate genes that either induce cartilage regeneration or inhibit cartilage degeneration. We further discuss in vitro, ex vivo and in vivo viral transduction and non-viral transfection strategies for targeted cells (chondrocytes, mesenchymal stem cells, induced pluripotent stem cells and synovial cells), along with the most representative results obtained in pre-clinical models and in clinical trials. We highlight current challenges and associated risks that slowdown clinical acceptance and commercialisation of gene transfer technologies.     

Effects of experience on palatability hierarchies of novel plants in the polyphagous grasshopperSchistocerca americana

Summary We studied the influence of diet composition and breadth on the subsequent acceptability of three novel plants to sixth instarSchistocerca americana. Rearing diets of equal breadth differing in composition, and diets differing in breadth, significantly altered first meal length on some but not all of the test plants. These effects on palatability altered and at times reversed the palatability hierarchy of insects reared on different diets. The effects of rearing insects on broad diets were not produced by exposure to the plant odors alone, but apparently required contact with a diversity of plants while feeding. Switching diets for 24 h prior to testing did not alter preferences induced by rearing diets. The relationship of these patterns to induced preferences in other insects, and some possible mechanisms for generating induced preferences, are discussed.     

The AtRbx1 protein is part of plant SCF complexes,and its down-regulation causes severe growth and developmental defects

Recently in yeast and animal cells, one particular class of ubiquitin ligase (E3), called the SCF, was demonstrated to regulate diverse processes including cell cycle and development. In plants SCF-dependent proteolysis is also involved in different developmental and hormonal regulations. To further investigate the function of SCF, we characterized at the molecular level the Arabidopsis RING-H2 finger protein AtRbx1. We demonstrated that the plant gene is able to functionally complement a yeast knockout mutant strain and showed that AtRbx1 protein interacts physically with at least two members of the Arabidopsis cullin family (AtCul1 and AtCul4). AtRbx1 also associates with AtCul1 and the Arabidopsis SKP1-related proteins in planta, indicating that it is part of plant SCF complexes. AtRbx1 mRNAs accumulate in various tissues of the plant, but at higher levels in tissues containing actively dividing cells. Finally to study the function of the gene in planta, we either overexpressed AtRbx1 or reduced its expression by a dsRNA strategy. Down-regulation of AtRbx1 impaired seedling growth and development, indicating that the gene is essential in plants. Furthermore, the AtRbx1-silenced plants showed a reduced level of AtCul1 protein, but accumulated higher level of cyclin D3.     

Comparative analysis of the lipopolysaccharides of a rough and a smooth strain of Pseudomonas syringae pv. phaseolicola

The lipopolysaccharides (LPS) of a rough (R) and a smooth (S) strain of Pseudomonas syringae pv. phaseolicola were analysed. The S-LPS revealed markedly more rhamnose and fucose, but less glucose, than the R-LPS. The presence of 3-O-methyl-rhamnose (acofriose) in the S-LPS was confirmed by cochromatography with authentic acofriose. SDS polyacrylamide gel electrophoresis of the S-LPS demonstrated a cluster of regularly spaced high molecular weight fractions, which was almost lacking in the R-LPS. The main fatty acids of the lipid A of both LPS species were 3-OH-10:0,3-OH-12:0,2-OH-12:0, and 12:0. Two N-linked diesters were demonstrated: 3-O(12:0)-12:0 and 3-O(2-OH-12:0)-12:0. S-LPS was subjected to mild hydrolysis and the degraded polysaccharide separated into three fractions by gel permeation chromatography on a Fractogel TSK HW-50 column. Fraction I, representing nearly only the O-specific side chain, consisted of rhamnose and fucose in a molar ratio of 4:1, with 4% of the rhamnose being 3-O-methylated (acofriose). Fraction II, representing mostly core material, was composed of glucose, rhamnose, heptose, glucosamine, galactosamine, alanine, and a still unidentified amino compound, in an approximate molar ratio of 3:1:1:1:1:1:1, and KDO. Fraction III consisted of released monomers and salts. The LPS was highly phosphorylated (3.28% phosphorus in the core fraction). The thus characterized composition of the LPS O-chain seems to be unique for the pathovar phaseolicola of P. syringae, although many similarities exist to other pathovars as well as to other bacterial species.Abbreviations LPS lipopolysacchairdes - GC/MS combined gas liquid chromatography-mass spectrometry - HVE high voltage electrophoresis - KDO 2-keto-3-deoxyoctonic acid - PAGE polyacrylamide gel electrophoresis - SDS sodium dodecylsulfate P.s. pv. phaseolicola is termed P. phaseolicola in the text     

Sequence Heterogeneity in Closed Simian Virus 40 Deoxyribonucleic Acid

The heteroduplex molecules formed by self-annealing of denatured, singly nicked simian virus 40 (SV40) deoxyribonucleic acid (DNA) prepared from closed viral DNA were examined by formamide-protein film electron microscopy to test the DNA for sequence homogeneity. Sequence inhomogeneity appears in the heteroduplexes as single-strand loops. These result from sequence deletion or from sequence substitution, if regions greater than 50 nucleotides are involved. The undenatured DNA from viruses passaged twice at multiplicities of infection much less than 1 plaque-forming unit (PFU) per cell appeared to be homogeneous in size. The heteroduplexes formed by this DNA indicated that approximately 2% of the molecules carried deletions, but that substitutions were below the level of detection. In contrast, undenatured DNA from viruses grown by passaging undiluted lysates seven times or by infection with stock virus at a multiplicity of infection of 5 PFU per cell contained a large frequency of molecules shorter than the full length. The heteroduplex samples indicated that 12 and 7% of the undenatured material contained base substitutions, and 13 and 11% contained deletions. The deletions and substitutions appear to occur in separate molecules. Length measurements on heteroduplexes displaying the loop characteristic of substitutions have established that these molecules are from true sequence substitutions, and not from adjacent or overlapping deletions. More than 80% of the molecules carrying substitutions are shorter than the native SV40 length. On the average, the substituted sequence is about 20% of the length of SV40, but it replaces a sequence about 30% of the native length. The substituted sequences may be host cell nuclear DNA, possibly arising from integration of SV40 into the chromosome followed by excision of the SV40 DNA together with chromosomal DNA.