Evolution of a multigene family of chorion proteins in silkmoths.

The evolution of the A family of chorion genes was examined by comparing new protein and DNA sequences from the silkmoths Antheraea pernyi and Bombyx mori with previously known sequences from Antheraea polyphemus. The comparisons indicated that the A family and its major subfamilies are ancient and revealed how parts of the genes corresponding to distinct regions of the protein structure have evolved, both by base substitutions and by segmental reduplications and deletions.     

Chordin knockdown enhances the osteogenic differentiation of human mesenchymal stem cells

Introduction

Bone morphogenetic proteins (BMPs) are critical growth factors in the osteogenic differentiation of progenitor cells during development in embryos and fracture repair in adults. Although recombinant BMPs are in use clinically, their clinical efficiency needs to be improved. The biological activities of BMPs are naturally regulated by extracellular binding proteins. The specific hypotheses tested in this study were as follows: the BMP inhibitor chordin is produced endogenously during the osteogenic differentiation of human mesenchymal stem cells (MSCs); and blockade of the activity of the BMP inhibitor increases the rate of osteogenic differentiation of human MSCs in vitro.

Methods

Human MSCs were derived from bone marrow from an iliac crest aspirate and from patients undergoing hip hemiarthroplasty. The MSCs were induced down the osteogenic pathway using standard osteogenic differentiation media, and expressions of BMP-2 and chordin were determined by gene expression analysis. During osteogenic differentiation, chordin knockdown was induced using RNA interference. Osteogenic differentiation was assessed by measuring the expression of alkaline phosphatase and calcium deposition. The differences in expression of osteogenic makers between groups were compared by analysis of variance, followed by Gabriel post hoc test.

Results

We demonstrate the expression of BMP-2 and chordin in human MSCs during osteogenic differentiation. Knockdown of chordin by RNA interference in vitro resulted in a significant increase in the expression of the osteogenic marker alkaline phosphatase and the deposition of extracellular mineral, in response to osteogenic stimulation.

Conclusion

We conclude that endogenously produced chordin constrains the osteogenic differentiation of human MSCs. The targeting of BMP inhibitors, such as chordin, may provide a novel strategy for enhancing bone regeneration.     

Evolution of chorion structural genes and regulatory mechanisms in two wild silkmoths: A preliminary analysis

Summary We report a preliminary analysis of structural and regulatory evolution of the A and B chorion gene families in two wild silkmoths,Antheraea pernyi andAntheraea polyphemus. Homospecific and heterospecific dot hybridizations were performed between previously characterizedA. polyphemus complementary DNA clones and total or stage-specific follicular mRNAs from the two species. The hybridization patterns indicated substantial interspecies changes in the abundance of corresponding mRNA sequences (heteroposic evolution) without substantial changes in their developmental specificities (heterochronic evolution). In addition, the proteins encoded in the two species by corresponding mRNAs were determined by hybrid-selected translation followed by electrophoretic analysis. The results suggested that the proteins evolve in size, presumably through internal deletions and duplications.     

Cloning, chromosomal organization and expression analysis of Neurl, the mouse homolog of Drosophila melanogaster neuralized gene

The Drosophila neuralized (neur) gene belongs to the neurogenic group of genes involved in regulating cell-cell interactions required for neural precursor development. neur mutant phenotypes include strong overcommitment to neural fates at the expense of epidermal fates. The human neuralized homolog (NEURL) has been recently determined and found to map to chromosome 10q25.1 within the region frequently deleted in malignant astrocytomas. Because of its potential importance in developmental processes, we analyzed the structure of the mouse homolog, Neurl, and its expression pattern in embryonic tissues. Neurl activity is detected from early developmental stages in several tissues and organs including neural tissues, limbs, the skeletal system, sense organs and internal organs undergoing epithelial-mesenchymal interactions. Neurl encodes a polypeptide associated with the plasma membrane but also detected in the cytoplasm. Similarly to the Drosophila gene, mammalian neuralized may code for an important regulatory factor.     

Isolation and characterization of plasma membrane-associated cortical granules from sea urchin eggs

Cortical granules, which are specialized secretory organelles found in ova of many organisms, have been isolated from the eggs of the sea urchins Arbacia punctulata and Strongylocentrtus pupuratus by a simple, rapid procedure. Electron micropscope examination of cortical granules prepared by this procedure reveals that they are tightly attached to large segments of the plasma membrane and its associated vitelline layer. Further evidence that he cortical granules were associated with these cell surface layers was obtained by (125)I-labeling techniques. The cortical granule preparations were found to be rich in proteoesterase, which was purified 32-fold over that detected in a crude homogenate. Similarly, the specific radioactivity of a (125)I-labeled, surface glycoprotein was increased 40-fold. These facts, coupled with electron microscope observations, indicate the isolation procedure yields a preparation in which both the cortical granules and the plasma membrane-vitelline layer are purified to the same extent. Gel electrophoresis of the membrane-associated cortical granule preparation reveals the presence of at least eight polypeptides. The major polypeptide, which is a glycotprotein of apparent mol wt of 100,000, contains most of the radioactivity introduced by (125)I-labeling of the intact eggs. Lysis of the cortical granules is observed under hypotonic conditions, or under isotonic conditions if Ca(2+) ion is present. When lysis is under isotonic conditions is induced by addition of Ca(2+) ion, the electron-dense contents of the granules remain insoluble. In contrast, hypotonic lysis results in release of the contents of the granule in a soluble form. However, in both cases the (125)I-labeled glycoprotein remains insoluble, presumably because it is a component of either the plasma membrane or the vitelline layer. All these findings indicate that, using this purified preparation, it should be possible to carry out in vitro studies to better define some of the initial, surface-related events observed in vivo upon fertilization.     

Isolation and sources of 'blown pack' spoilage clostridia in beef abattoirs

Aims:  (i) To evaluate methods for isolation and molecular detection of blown pack spoilage (BPS) clostridia and (ii) to survey beef abattoirs for sources and distributions of Clostridium estertheticum and Cl. gasigenes .
Methods and Results:  Molecular detection and conventional isolation methods were used to detect and recover BPS associated clostridia ( Cl. estertheticum and Cl. gasigenes ), from four commercial Irish beef abattoirs and their environments, during a one year study. DNA-based methods detected 218 Cl. estertheticum and 300 Cl. gasigenes , from 1680 samples, whereas culture-methods only yielded 17 Cl. estertheticum and 176 Cl. gasigenes isolates. BPS Clostridia were frequently detected in beef abattoirs and their environments, especially at areas prior to hide removal. The study noted a higher percentage of positive samples during the month of May (38·6%).
Conclusions:  (i) DNA-based techniques are the most reliable ways to determine the presence of these organisms in various samples and (ii) hides and faeces are the main reservoirs of BPS clostridia in the abattoirs.
Significance and Impact of the Study:  This paper provides useful information to detect BPS organisms, as well as to develop a science-based control strategy of the problem.     

Chromosomal mapping of glutamate dehydrogenase gene sequences to mouse chromosomes 7 and 14

Glutamate dehydrogenase (GLUD) plays an important role in mammalian neuronal transmission. In human, GLUD is encoded by a small gene family. To determine whether defects in Glud genes are associated with known neurological mutations in the mouse and to contribute to the comparative mapping of homologous genes in man and mouse, the chromosomal location of genes reactive with a mouse brain GLUD cDNA were determined. Genomic Southern analysis of a well-characterized panel of Chinese hamster x mouse somatic cell hybrids identified two GLUD-reactive loci, one residing on mouse Chromosome 14 and the other on Chromosome 7. Progeny of an intersubspecies backcross were used to map one of these genes, Glud, proximal to Np-1 on Chromosome 14, but no restriction fragment polymorphisms could be identified for the second locus, Glud-2.