Human estrogen receptor alpha gene is a target of Runx2 transcription factor in osteoblasts

Several studies into the mechanisms involved in control of osteoblast-specific gene expression have identified Runx2 and ERalpha (estrogen receptor alpha) as essential regulators of osteoblast differentiation. Recently, interactions between Runx2 and ERalpha have been described. Here, we investigate the role of Runx2 on the regulation of ERalpha expression by determining its interaction with the F promoter, one of the multiple promoters of the human ERalpha gene and the only one active in bone. We found that, in this promoter, three Runx2-like sites are present. By electrophoretic mobility shift assay in combination with supershift and ChIP experiments, we demonstrated that Runx2 preferentially binds one of the Runx2 motifs of the F promoter. To understand whether or not they are involved in influencing F promoter activity, different promoter-reporter deletion and mutation constructs were transiently transfected into human osteoblastic cells. Comparison of luciferase activities allowed the identification of a prevalent negative role of a sequence context, within the -117,877/-117,426 region, which may be under the control of Runx2 (a) site. Finally, silencing and overexpression of endogenous Runx2 provided evidence that Runx2 has a more complex role than initially expected. In fact, Runx2 (a) and Runx2 (b) sites carried out opposite roles which are conditioned by Runx2 levels in bone cells. Therefore, the resulting F promoter activity may be tightly regulated by a dynamic interplay between these two Runx2 sites, with a predominance of negative effect of the Runx2 (a) site.     

Leishmania amazonensis: Anionic currents expressed in oocytes upon microinjection of mRNA from the parasite

Transport mechanisms involved in pH homeostasis are relevant for the survival of Leishmania parasites. The presence of chloride conductive pathways in Leishmania has been anticipated since anion channel inhibitors limit the proton extrusion mediated by the H+ATPase, which is the major regulator of intracellular pH in amastigotes. In this study, we used Xenopus laevis oocytes as a heterologous expression system in which to study the expression of ion channels upon microinjection of polyA mRNA from Leishmania amazonensis. After injection of polyA mRNA into the oocytes, we measured three different types of currents. We discuss the possible origin of each, and propose that Type 3 currents could be the result of the heterologous expression of proteins from Leishmania since they show different pharmacological and biophysical properties as compared to endogenous oocyte currents.     

In vivo glyco-engineered antibody with improved lytic potential produced by an innovative non-mammalian expression system

Recent studies have demonstrated that the reduction of the core fucosylation on N-glycans of human IgGs is responsible for a clearly enhanced antibody-dependent cellular cytotoxicity (ADCC). This finding might give access to improved active therapeutic antibodies. Here, the expression of the tumor antigen-specific antibody IGN311 was performed in a glyco-optimized strain of the moss Physcomitrella patens. Removal of plant specific N-glycan structures in this plant expression host was achieved by targeted knockout of corresponding genes and included quantitative elimination of core fucosylation. Antibodies transiently expressed and secreted by such genetically modified moss protoplasts assembled correctly, showed an unaltered antigen-binding affinity and, in extensive tests, revealed an up to 40-fold enhanced ADCC. Thus, the glyco-engineered moss-based transient expression platform combines a rapid technology with the subsequent analysis of glycooptimized therapeutics with regard to advanced properties.     

Developmental characteristics of AMPA receptors in chick lumbar motoneurons

Ca2+ fluxes through ionotropic glutamate receptors regulate a variety of developmental processes, including neurite outgrowth and naturally occurring cell death. In the CNS, NMDA receptors were originally thought to be the sole source of Ca2+ influx through glutamate receptors; however, AMPA receptors also allow a significant influx of Ca2+ ions. The Ca2+ permeability of AMPA receptors is regulated by the insertion of one or more edited GluR2 subunits. In this study, we tested the possibility that changes in GluR2 expression regulate the Ca2+ permeability of AMPA receptors during a critical period of neuronal development in chick lumbar motoneurons. GluR2 expression is absent between embryonic day (E) 5 and E7, but increases significantly by E8 in the chick ventral spinal cord. Increased GluR2 protein expression is correlated with parallel changes in GluR2 mRNA in the motoneuron pool. Electrophysiological recordings of kainate-evoked currents indicate a significant reduction in the Ca2(+)-permeability of AMPA receptors between E6 and E11. Kainate-evoked currents were sensitive to the AMPA receptor blocker GYKI 52466. Application of AMPA or kainate generates a significant increase in the intracellular Ca2+ concentration in E6 spinal motoneurons, but generates a small response in older neurons. Changes in the Ca(2+)-permeability of AMPA receptors are not mediated by age-dependent changes in the editing pattern of GluR2 subunits. These findings raise the possibility that Ca2+ influx through Ca(2+)-permeable AMPA receptors plays an important role during early embryonic development in chick spinal motoneurons.     

Erwinia carotovora ssp. carotovora Infection Induced ＂Defense Lignin＂ Accumulation and Lignin Biosynthetic Gene Expression in Chinese Cabbage （Brassica rapa L. ssp. pekinensis）

Erwinia carotovora subsp, carotovora （Ecc） infects and causes soft rot disease in hundreds of crop species including vegetables, flowers and fruits. Lignin biosynthesis has been implicated in defensive reactions to injury and pathogen infection in plants. In this work, variations of lignin content and gene expression in the molecular interaction between Chinese cabbage and Ecc were investigated. H2O2 accumulation and peroxidase activity were detected by 3, 3＇- Dimethoxybenzidine staining at mocked and Ecc-inoculated sites of Chinese cabbage leafstalks. Klason lignin content in inoculated plants increased by about 7.84%, 40.37%, and 43.13% more than that of the mocked site at 12, 24 and 72 h after inoculation, respectively. Gas chromatography detected more p-coumaryl （H） and less coniferyl （G） and sinapyl （S） monolignins in leafstalks of Chinese cabbage. All three monomers increased in Ecc-infected leafstalks, and the Ecc-induced ＂defense lignin＂ were composed of more G and H monolignins, and less S monolignin. After searching the expressed sequence tags （EST） data of Chinese cabbage, 12 genes putatively encoding enzymes involved in lignin biosynthesis were selected to study their expression. All of these genes could be induced by mock inoculation and Ecc infection, while the gene expression lasted for several more hours in the infected samples than in mocked and untreated plants. Our results indicated that ＂defense lignin＂ was different from the developmental lignin in composition; G and S monolignins were significantly induced in plants in response to the soft rot Ecc; thus, lignin biosynthesis was differentially regulated and played a role in plant response to the soft rot Ecc.     

Expression of two types of acetylcholinesterase gene from the silkworm, Bombyx mori, in insect cells

Complementary DNAs encoding two types of acetylcholinesterase （ACHE） were isolated from the silkworm, Bombyx mori. The type 1 （Bmacel） and type 2 （Bmace2） ORFs are 2052 and 1917 bp in length, respectively. Both the complete ORFs of the Bmaces and C- terminal truncated forms were recombined into the Bacmid baculovirus vector under the control of the polyhedrin promoter and expressed in Trichoplusia ni （Tn-5B 1-4） cells. The resulting products exhibited ACHE activity and glycosylation of the expressed proteins. An inhibition assay indicated that the ace2-type enzyme was more sensitive than the acel-type enzyme to inhibition by eserine and paraoxon.