Microsatellite analysis as a tool for discriminating an interfamily hybrid between olive flounder and starry flounder

An interspecific artificial hybrid was produced between two economically important aquaculture flatfish: olive flounder (Paralichthys olivaceus) and starry flounder (P. stellatus). This hybrid displays the rapid growth characteristic of the former and tolerance to low temperatures and low salinity of the latter, but the genetics of inheritance in this hybrid have not been elucidated. Polymorphic microsatellite markers developed for P. olivaceus and P. stellatus were tested to determine if these markers can be used for analysis of parentage and genetic inheritance. Multiplex PCR using two primer sets that were specific to each species produced PCR products of different sizes; these could be used for the identification of interspecific hybrids. Among the 192 primers derived from olive flounder, 25.5% of the primer sets successfully amplified genomic DNA from starry flounder, and 23% of the 56 primer sets originating from starry flounder amplified DNA from olive flounder. Analysis of genetic inheritance in the hybrid using seven of the 62 microsatellite markers common to both species demonstrated classic Mendelian inheritance of these markers in the hybrid progeny, with the exception of one locus identified as a null allele in the hybrid. These results demonstrate that cross-specific microsatellite markers can be used tools for parentage analysis of hybrid flatfish, for mapping quantitative trait loci, for marker-assisted selective breeding, and for studies of the evolution of fish.     

Comparative structural analyses of purified glycogen particles from rat liver,human skeletal muscle and commercial preparations

Glycogen is a cellular energy store that is crucial for whole body energy metabolism, metabolic regulation and exercise performance. To understand glycogen structure we have purified glycogen particles from rat liver and human skeletal muscle tissues and compared their biophysical properties with those found in commercial glycogen preparations. Ultrastructural analysis of commercial liver glycogens fails to reveal the classical α-rosette structure but small irregularly shaped particles. In contrast, commercial slipper limpet glycogen consists of β-particles with similar branching and chain lengths to purified rat liver glycogen together with a tendency to form small α-particles, and suggest it should be used as a source of glycogen for all future studies requiring a substitute for mammalian liver glycogen.     

Isolation of the protein tyrosine phosphatase 1B inhibitory metabolite from the marine-derived fungus Cosmospora sp. SF-5060

In the course of bioassay-guided study on the EtOAc extract of a culture broth of the marine-derived fungus Cosmospora sp. SF-5060, aquastatin A (1) was isolated as a protein tyrosine phosphatase 1B (PTP1B) inhibitory component produced by the fungus. The compound was isolated by various chromatographic methods, and the structure was determined mainly by analysis of NMR spectroscopic data. Compound 1 exhibited potent inhibitory activity against PTP1B with IC₅₀ value of 0.19 μM, and the kinetic analyses of PTP1B inhibition by compound 1 suggested that the compound is inhibiting PTP1B activity in a competitive manner. Aquastatin A (1) also showed modest but selective inhibitory activity toward PTP1B over other protein tyrosine phosphatases, such as TCPTP, SHP-2, LAR, and CD45. In addition, the result of hydrolyzing aquastatin A (1) suggested that the dihydroxypentadecyl benzoic acid moiety in the molecule is responsible for the inhibitory activity.     

Smad-dependent cooperative regulation of interleukin 2 receptor alpha chain gene expression by T cell receptor and transforming growth factor-beta

The interleukin 2 receptor alpha chain (IL-2Ralpha) is a component of high affinity IL-2 receptors and thus critically regulates T cell growth and other lymphoid functions. Five positive regulatory regions together control lineage-restricted and activation-dependent IL-2Ralpha induction in response to antigen and IL-2. We now show that TGF-beta cooperates with T cell receptor (TCR) signaling to increase IL-2Ralpha gene expression. Moreover, we identify a sixth positive regulatory region that regulates IL-2Ralpha expression in cells treated with anti-CD3 + anti-CD28 as well as TGF-beta and show that this region contains binding sites for Smad3, AP-1, and cAMP-responsive element-binding protein/ATF proteins. The importance of Smad complexes is indicated by impaired IL-2Ralpha induction by TGF-beta in CD4+ T cells from both Smad3-/- and Smad4-/- mice. Thus, we have identified a novel positive regulatory region in the IL-2Ralpha gene that mediates TGF-beta-dependent induction of the gene. These findings have implications related to IL-2Ralpha expression on activated T cells and regulatory T cells.     

An Arabidopsis F-box protein regulates tapetum degeneration and pollen maturation during anther development

The Arabidopsis anther has a bilateral symmetry with four lobes, each consisting of four distinct layers of somatic cells from the outer to inner side: epidermis, endothecium, middle layer and tapetum. The tapetum is a layer of cells comprising the inner surface of the pollen wall. It plays an important role in anther development by providing enzymes, materials and nutrients required for pollen maturation. Genes and molecular mechanisms underlying tapetum formation and pollen wall biosynthesis have been studied in Arabidopsis. However, tapetum degeneration and anther dehiscence have not been well characterized at the molecular level. Here, we report that an Arabidopsis gene, designated reduced male fertility (RMF), regulates degeneration of tapetum and middle layer during anther development. The Arabidopsis dominant mutant rmf-1D overexpressing the RMF gene exhibited pleiotropic phenotypes, including dwarfed growth with small, dark-green leaves and low male fertility. Tapetum development and subsequent degeneration were impaired in the mutant. Accordingly, pollen maturation was disturbed, reducing the male fertility. In contrast, tapetum degeneration was somewhat accelerated in the RMF RNAi plants. The RMF gene was expressed predominantly in the anther, particularly in the pollen grains. Notably, the RMF protein contains an F-box motif and is localized to the nucleus. It physically interacts with the Arabidopsis-Skp1-like1 protein via the F-box motif. These observations indicate that the RMF gene encodes an F-box protein functioning in tapetum degeneration during anther development.     

Methylglyoxal induces cellular damage by increasing argpyrimidine accumulation and oxidative DNA damage in human lens epithelial cells

Methylglyoxal (MGO) is a cytotoxic metabolite and modifies tissue proteins through the Maillard reaction, resulting in advanced glycation end products (AGEs), which can alter protein structure and functions. Several MGO-derived AGEs have been described, including argpyrimidine, a fluorescent product of the MGO reaction with arginine residues. Herein, we evaluated the cytotoxic role of MGO in human lens epithelial cell line (HLE-B3). HLE-B3 cells were exposed to 400 μM MGO in the present or absence of pyridoxamine for 24 h. We then examined the formation of argpyrimidine, apoptosis and oxidative stress in HLE-B3 cells. In MGO-treated HLE-B3 cells, the accumulation of argpyrimidine was markedly increased, and caspase-3 and 8-hydroxydeoxyguanosine (8-OHdG) were highly expressed, which paralleled apoptotic cell death. However, pyridoxamine (AGEs inhibitor) prevented the argpyrimidine formation and apoptosis of MGO-treated HLE-B3 cells. These results suggested that the accumulation of argpyrimidine and oxidative DNA damage caused by MGO are involved in apoptosis of HLE-B3 cells.     

An in vitro model for cell-cell interactions

Summary Heterotypic cell-cell interactions appear to be involved in the control of development and function in a wide variety of tissues. In the vasculature, endothelial cells and mural cells (smooth muscle cells or pericytes) make frequent contacts, suggesting a role for intercellular interactions in the regulation of vascular growth and function. We have previously grown endothelial cells and mural cells together in mixed cultures and found that heterocellular contact led to endothelial growth inhibition. However, this mixed culture system does not lend itself to the examination of the effects of contact on the phenotype of the individual cell types. We have therefore developed a co-culture system in which cells can be co-cultured across a porous membrane, permitting intercellular contact while maintaining pure cell populations. Co-culture of endothelial cells and smooth muscle cells across membranes with pore sizes of 0.02, 0.4, 0.6, and 0.8μm maintained the two cell types as homogeneous populations, whereas smooth muscle cells migrated across the membrane through pores of 2.0μm. Vascular cell co-culture across membranes with 0.8-μm pores resulted the inhibition of endothelial cell proliferation and the generation of conditioned media which inhibited endothelial cell growth. The arrangement of the cells in this co-culture system mimics thein vivo orientation of vascular cells in which mural cells are separated from the abluminal surface of the endothelium by a fenestrated internal elastic lamina or basement membrane. Because this co-culture system maintains separable populations of cells in contact or close proximity allowing for biochemical and molecular analyses of pure populations, it should prove useful for the study of cell-cell interactions in a variety of systems.     

Expression of dihydropyridine-sensitive brain calcium channels in the rat central nervous system.

We have localized dihydropyridine (DHP-sensitive calcium channels in rat brain by in situ hybridization and immunohistochemistry. The mRNA for the dihydropyridine-sensitive calcium channel alpha 1 subunit (DHPR-B) is prominently localized in neuronal cells in the olfactory bulb, dentate gyrus, hippocampus, arcuate nucleus, paraventricular nucleus, ventromedial nucleus, cerebral cortex, superior colliculus and the cerebellar Purkinje cell layer. Strong expression of DHPR-B mRNA was also found in the pituitary and pineal glands. DHP-sensitive calcium channel alpha 1 subunit distribution has also been examined immunohistochemically with polyclonal antibodies raised against synthetic peptides specific for the DHPR-B alpha 1 subunit protein. The results from immunohistochemistry were in good agreement with those from in situ hybridization. Thus, regional distribution and localization of DHPR-B mRNA and alpha 1 subunit protein in rat brain suggest that this type of DHP-sensitive brain calcium channel may play an important role in excitation-secretion coupling functions in the neuroendocrine system.     

Antidotes to anthrax lethal factor intoxication. Part 2: structural modifications leading to improved in vivo efficacy

New anthrax lethal factor inhibitors (LFIs) were designed based upon previously identified potent inhibitors 1a and 2. Combining the new core structures with modifications to the C2-side chain yielded analogs with improved efficacy in the rat lethal toxin model.