Cellular processes underlying maturation of P19 neurons: Changes in protein folding regimen and cytoskeleton organization

Embryonal carcinoma P19 cells provide an ideal model to study molecular programs along differentiation. Upon induction by retinoic acid (RA), the cells undergo a program of differentiation that generates functioning neurons within 60 h. RA induced cells that were plated as sparse (1000 cells/mm(2)) or dense (4000 cells/mm(2)) cultures showed a marked difference in the culture morphology with the dense cultures exhibiting rapid maturation and accelerated neurite outgrowth. The protein expression levels of the sparse and dense cultures were compared 48 h following RA. Cell extracts were separated by 1-DE and 2-DE and differential expression (>four-fold) proteins were identified by MS. Here, we focus on 20 proteins associated with cytoskeletal regulation and stress-dependent protein refolding. The first group includes drebrin, cofilin, alpha-internexin, vimentin, and nestin. Among the proteins in the second group are subunits of the TCP-1, and several chaperones of the Hsp70 and Hsp90 families. We show that coordinated remodeling of the cytoskeleton and modulations in chaperone activity underlie the change in neurite extension rate. Furthermore, a proteomics-based analysis applied on P19 neurons demonstrated pathways underlying neuronal outgrowth, suggesting that a malfunction of such pathways leads to neuropathological conditions.     

Jane: a new tool for the cophylogeny reconstruction problem

Background  

This paper describes the theory and implementation of a new software tool, called Jane, for the study of historical associations. This problem arises in parasitology (associations of hosts and parasites), molecular systematics (associations of orderings and genes), and biogeography (associations of regions and orderings). The underlying problem is that of reconciling pairs of trees subject to biologically plausible events and costs associated with these events. Existing software tools for this problem have strengths and limitations, and the new Jane tool described here provides functionality that complements existing tools.     

Comparison of neurolin (ALCAM) and neurolin-like cell adhesion molecule (NLCAM) expression in zebrafish

Many immunoglobulin (Ig)-superfamily cell adhesion molecules influence skeletal muscle formation. In Drosophila, dumbfounded (duf/kirre), irreC, sticks and stones and hibris encode related Ig-family proteins expressed in subsets of neurons and muscle precursor cells. The family mediates cell migration, axon guidance and fusion of myoblasts. Despite the importance of these genes in invertebrate myogenesis, no obvious functional parallels are known in vertebrate myogenesis. Here we investigate the gene expression pattern and phylogenetic and protein-structural relationships of the duf-related molecules neurolin and neurolin-like cell adhesion molecule (NLCAM), members of the activated leukocyte cell adhesion molecule (ALCAM) sub-family of Ig-molecules. These proteins are among the closest to Duf/Kirre by sequence. During zebrafish development, neurolin is expressed in subsets of somite and muscle cells, heart and numerous sites of neuronal maturation. The new ALCAM-family member, NLCAM, appears to have arisen by duplication of neurolin/ALCAM. NLCAM is expressed widely during gastrulation, particularly in the nascent neural plate, but later becomes predominantly expressed in sites of muscle and nerve maturation and in the fin fold. The expression of each gene is often in groups of cells in similar parts of the embryo; for example, in the region of Rohon Beard neurons, trigeminal ganglion and fusing fast and migrating slow muscle fibres. However, expression can also be distinct and dynamic; for example, muscle pioneer fibres express neurolin but not NLCAM at high level. Both molecules are expressed in subsets of muscle precursors at times prior to fusion.     

Comprehensive metabolic profiling and phenotyping of interspecific introgression lines for tomato improvement

Tomato represents an important source of fiber and nutrients in the human diet and is a central model for the study of fruit biology. To identify components of fruit metabolic composition, here we have phenotyped tomato introgression lines (ILs) containing chromosome segments of a wild species in the genetic background of a cultivated variety. Using this high-diversity population, we identify 889 quantitative fruit metabolic loci and 326 loci that modify yield-associated traits. The mapping analysis indicates that at least 50% of the metabolic loci are associated with quantitative trait loci (QTLs) that modify whole-plant yield-associated traits. We generate a cartographic network based on correlation analysis that reveals whole-plant phenotype associated and independent metabolic associations, including links with metabolites of nutritional and organoleptic importance. The results of our genomic survey illustrate the power of genome-wide metabolic profiling and detailed morphological analysis for uncovering traits with potential for crop breeding.     

Unexpected differences between D- and L- tyrosine lead to chiral enhancement in racemic mixtures.

We report here an unexpected difference in the solubilities of D- and L-tyrosine in water, which could be discerned by their rate of crystallization and the resulting concentrations of their saturated solutions. A supersaturated solution of 10 mM L-tyrosine at 20 degrees C crystallized much more slowly than that of D-tyrosine under the same conditions, and the saturated solution of L-tyrosine was more concentrated than that of D-tyrosine. Supersaturated solutions of 10 mM DL-tyrosine in water formed precipitates of predominantly D-tyrosine and DL-tyrosine, resulting in an excess of L-tyrosine in the saturated solution. The experimental setups were monitored independently by UV-absorption, radioactivity tracing, optical rotation and X-ray diffraction. The process of nucleation and crystallization of D- and L-tyrosine is characterized by an exceptionally high cooperativity. It is possible that minute energy differences between D- and L-tyrosine, originating from parity violation or other non-conservative chiral discriminatory rules, could account for the observations. The physical process that initiated chiral selection in biological systems remains a challenging problem in understanding the origin of life, and it is possible that chiral compounds were concentrated from supersaturated racemic mixtures by preferential crystallization.     

Comparative study of calf thymus and wheat germ RNA polymerase II: stability of initiation complexes and elongation rates.

Pure wheat germ RNA polymerase II but not calf thymus RNA polymerase II forms relatively stable binary complexes (half life time of 30 minutes at 0°C) with superhelical SV 40 DNA. On the contrary, the addition of a specific dinucleotide and a single ribotriphosphate permits the formation of highly stable complexes between both enzymes and SV 40 DNA. The elongation of RNA chains with preinitiated wheat germ enzyme only is stimulated by sarkosyl. These observations suggest that the wheat germ enzyme, as compared to that isolated from calf thymus, may contain a protein factor, a more native structure or both that permit efficient initiation and elongation of RNA chains on double stranded DNA.     

Divergent mating systems and parental conflict as a barrier to hybridization in flowering plants

Parental conflicts can lead to antagonistic coevolution of the sexes and of parental genomes. Within a population, the resulting antagonistic effects should balance, but crosses between populations can reveal conflict. Parental conflict is less intense in self-pollinating plants than in outcrossers because outcrossing plants are pollinated by multiple pollen donors unrelated to the seed parent, while a self-pollinating plant is primarily pollinated by one individual (itself). Therefore, in crosses between plants with differing mating systems, outcrossing parents are expected to "overpower" selfing parents. We call this the weak inbreeder/strong outbreeder (WISO) hypothesis. Prezygotically, such overpowering can alter pollination success, and we argue that our hypothesis explains a common pattern of unilateral incompatibility, in which pollen from self-incompatible populations fertilizes ovules of self-compatible individuals but the reciprocal cross fails. A postzygotic manifestation of overpowering is aberrant seed development due to parent-of-origin effects such as genomic imprinting. We evaluate evidence for the WISO hypothesis by reviewing published accounts of crosses between plants of different mating systems. Many, but not all, of such reports support our hypothesis. Since parental conflicts can perturb fertilization and development, such conflicts may strengthen reproductive barriers between populations, contributing to speciation.     

Anatomy of a homeoprotein revealed by the analysis of human MODY3 mutations

Hepatocyte nuclear factor 1alpha (HNF1alpha) is an atypical dimeric homeodomain-containing protein that is expressed in liver, intestine, stomach, kidney, and pancreas. Mutations in the HNF1alpha gene are associated with an autosomal dominant form of non-insulin-dependent diabetes mellitus called maturity-onset diabetes of the young (MODY3). More than 80 different mutations have been identified so far, many of which involve highly conserved amino acid residues among vertebrate HNF1alpha. In the present work, we investigated the molecular mechanisms by which MODY3 mutations could affect HNF1alpha function. For this purpose, we analyzed the properties of 10 mutants resulting in amino acid substitutions or protein truncation. Some mutants have a reduced protein stability, whereas others are either defective in the DNA binding or impaired in their intrinsic trans-activation potential. Three mutants, characterized by a complete loss of trans-activation, behave as dominant negatives when transfected with the wild-type protein. These data define a clear causative relationship between MODY3 mutations and functional defects in HNF1alpha trans-activation. In addition, our analysis sheds new light on the structure of a homeoprotein playing a key role in pancreatic beta cell function.