Glutamatergic plasticity by synaptic delivery of GluR-B(long)-containing AMPA receptors

Activity-driven delivery of AMPA receptors is proposed to mediate glutamatergic synaptic plasticity, both during development and learning. In hippocampal CA1 principal neurons, such trafficking is primarily mediated by the abundant GluR-A subunit. We now report a study of GluR-B(long), a C-terminal splice variant of the GluR-B subunit. GluR-B(long) synaptic delivery is regulated by two forms of activity. Spontaneous synaptic activity-driven GluR-B(long) transport maintains one-third of the steady-state AMPA receptor-mediated responses, while GluR-B(long) delivery following the induction of LTP is responsible for approximately 50% of the resulting potentiation at the hippocampal CA3 to CA1 synapses at the time of GluR-B(long) peak expression-the second postnatal week. Trafficking of GluR-B(long)-containing receptors thus mediates a GluR-A-independent form of glutamatergic synaptic plasticity in the juvenile hippocampus.     

Catalytically Active Monomers of E. coli Glyceraldehyde-3-Phosphate Dehydrogenase

Monomeric forms of E. coli glyceraldehyde-3-phosphate dehydrogenase have been prepared using two different experimental approaches: (1) covalent immobilization of a tetramer on a solid support via a single subunit with subsequent dissociation of non-covalently bound subunits in the presence of urea, and (2) entrapment of monomeric species into reversed micelles of Aerosol OT in octane. Isolated monomers were shown to be catalytically active, exhibiting K _M values close to the parameters characteristic of the tetrameric forms. Like tetramers, isolated monomers did not use NADP7 as a coenzyme.     

EAST Organizes Drosophila Insulator Proteins in the Interchromosomal Nuclear Compartment and Modulates CP190 Binding to Chromatin

Recent data suggest that insulators organize chromatin architecture in the nucleus. The best studied Drosophila insulator proteins, dCTCF (a homolog of the vertebrate insulator protein CTCF) and Su(Hw), are DNA-binding zinc finger proteins. Different isoforms of the BTB-containing protein Mod(mdg4) interact with Su(Hw) and dCTCF. The CP190 protein is a cofactor for the dCTCF and Su(Hw) insulators. CP190 is required for the functional activity of insulator proteins and is involved in the aggregation of the insulator proteins into specific structures named nuclear speckles. Here, we have shown that the nuclear distribution of CP190 is dependent on the level of EAST protein, an essential component of the interchromatin compartment. EAST interacts with CP190 and Mod(mdg4)-67.2 proteins in vitro and in vivo. Over-expression of EAST in S2 cells leads to an extrusion of the CP190 from the insulator bodies containing Su(Hw), Mod(mdg4)-67.2, and dCTCF. In consistent with the role of the insulator bodies in assembly of protein complexes, EAST over-expression led to a striking decrease of the CP190 binding with the dCTCF and Su(Hw) dependent insulators and promoters. These results suggest that EAST is involved in the regulation of CP190 nuclear localization.     

Oxidative Damage of U937 Human Leukemic Cells Caused by Hydroxyl Radical Results in Singlet Oxygen Formation

The exposure of human cells to oxidative stress leads to the oxidation of biomolecules such as lipids, proteins and nuclei acids. In this study, the oxidation of lipids, proteins and DNA was studied after the addition of hydrogen peroxide and Fenton reagent to cell suspension containing human leukemic monocyte lymphoma cell line U937. EPR spin-trapping data showed that the addition of hydrogen peroxide to the cell suspension formed hydroxyl radical via Fenton reaction mediated by endogenous metals. The malondialdehyde HPLC analysis showed no lipid peroxidation after the addition of hydrogen peroxide, whereas the Fenton reagent caused significant lipid peroxidation. The formation of protein carbonyls monitored by dot blot immunoassay and the DNA fragmentation measured by comet assay occurred after the addition of both hydrogen peroxide and Fenton reagent. Oxidative damage of biomolecules leads to the formation of singlet oxygen as conformed by EPR spin-trapping spectroscopy and the green fluorescence of singlet oxygen sensor green detected by confocal laser scanning microscopy. It is proposed here that singlet oxygen is formed by the decomposition of high-energy intermediates such as dioxetane or tetroxide formed by oxidative damage of biomolecules.     

Systems‐wide analysis of BCR signalosomes and downstream phosphorylation and ubiquitylation

B‐cell receptor (BCR) signaling is essential for the development and function of B cells; however, the spectrum of proteins involved in BCR signaling is not fully known. Here we used quantitative mass spectrometry‐based proteomics to monitor the dynamics of BCR signaling complexes (signalosomes) and to investigate the dynamics of downstream phosphorylation and ubiquitylation signaling. We identify most of the previously known components of BCR signaling, as well as many proteins that have not yet been implicated in this system. BCR activation leads to rapid tyrosine phosphorylation and ubiquitylation of the receptor‐proximal signaling components, many of which are co‐regulated by both the modifications. We illustrate the power of multilayered proteomic analyses for discovering novel BCR signaling components by demonstrating that BCR‐induced phosphorylation of RAB7A at S72 prevents its association with effector proteins and with endo‐lysosomal compartments. In addition, we show that BCL10 is modified by LUBAC‐mediated linear ubiquitylation, and demonstrate an important function of LUBAC in BCR‐induced NF‐κB signaling. Our results offer a global and integrated view of BCR signaling, and the provided datasets can serve as a valuable resource for further understanding BCR signaling networks.     

Fate of the Molar Dental Lamina in the Monophyodont Mouse

The successional dental lamina (SDL) plays an essential role in the development of replacement teeth in diphyodont and polyphyodont animals. A morphologically similar structure, the rudimental successional dental lamina (RSDL), has been described in monophyodont (only one tooth generation) lizards on the lingual side of the developing functional tooth. This rudimentary lamina regresses, which has been proposed to play a role in preventing the formation of future generations of teeth. A similar rudimentary lingual structure has been reported associated with the first molar in the monophyodont mouse, and we show that this structure is common to all murine molars. Intriguingly, a lingual lamina is also observed on the non-replacing molars of other diphyodont mammals (pig and hedgehog), initially appearing very similar to the successional dental lamina on the replacing teeth. We have analyzed the morphological as well as ultrastructural changes that occur during the development and loss of this molar lamina in the mouse, from its initiation at late embryonic stages to its disappearance at postnatal stages. We show that loss appears to be driven by a reduction in cell proliferation, down-regulation of the progenitor marker Sox2, with only a small number of cells undergoing programmed cell death. The lingual lamina was associated with the dental stalk, a short epithelial connection between the tooth germ and the oral epithelium. The dental stalk remained in contact with the oral epithelium throughout tooth development up to eruption when connective tissue and numerous capillaries progressively invaded the dental stalk. The buccal side of the dental stalk underwent keratinisation and became part of the gingival epithelium, while most of the lingual cells underwent programmed cell death and the tissue directly above the erupting tooth was shed into the oral cavity.