Efficacy of topiramate for intractable childhood generalized epilepsy with epileptic spasms: With special reference to electroencephalographic changes

AbstractPurposeEpileptic spasms (ES) beyond infancy are a highly refractory type of seizures that require the development of an effective treatment. We therefore studied the efficacy and safety of topiramate (TPM), which is a drug that is indicated to be effective for intractable childhood epilepsy, for ES.MethodsOut of 58 children with ES, we enrolled 33 patients treated with TPM at ≤12years of age. The administration of TPM was limited to cases of epilepsies that were resistant to any other potent treatment. We retrospectively investigated the efficacy of TPM for seizures and changes in electroencephalogram (EEG) findings.ResultsThe median age at the start of TPM treatment was 5years, 8months. All patients had ES and 28 also had tonic seizures. As for the efficacy of TPM for all seizures, five patients became seizure-free and two had a ≥50% reduction in seizures. Seizure aggravation was observed in six patients. Of 29 patients whose EEG findings were compared before and during TPM treatment, nine showed EEG improvement with reduced epileptic discharges. Adverse effects were observed in 13 patients and included somnolence, anorexia, and irritability. In general, TPM was well tolerated.ConclusionsTPM can be effective at suppressing very intractable ES in a proportion of patients who do not respond to any other treatment. The efficacy of TPM may be predictable based on EEG changes observed early in the course of treatment. TPM is promising for the treatment of extremely intractable childhood epilepsy and it has largely tolerable adverse effects

Immunohistochemical Demonstration of Membrane-bound Prostaglandin E2 Synthase-1 in Papillary Thyroid Carcinoma

Microsomal prostaglandin E2 synthase-1 (mPGES-1) is an inducible enzyme that catalyzes the conversion of prostaglandin (PG) H2 to PGE2 in downstream of cyclooxygenase-2 (COX-2). Recent studies have obtained in vitro evidence that PGE2 participates in carcinogenesis, angiogenesis, and induction of matrix metalloproteinase-9 (MMP-9), which plays a crucial role in cancer invasion. However, implications for mPGES-1 in thyroid carcinomas remain to be determined. To address this issue, we performed an immunohistochemical analysis for mPGES-1, COX-2 and MMP-9 in 20 papillary thyroid carcinoma (PTC) patients. mPGES-1 immunoreactivity was localized in the cytoplasm of carcinoma cells in 19 cases, with an intensity that tended to be distinct at the interface between the tumor and the surrounding non-neoplastic tissue. Staining was more intense in regions with papillary arrangement, while it was less intense in regions with trabecular or solid arrangement. In many cases, immunohistochemical localization of COX-2 and MMP-9 resemble that of mPGES-1. Taken together, our results suggest the involvement of mPGES-1 in proliferation and differentiation of PTC as well as local invasion of PTC

Disulfide bridge formation between SecY and a translocating polypeptide localizes the translocation pore to the center of SecY

During their biosynthesis, many proteins pass through the membrane via a hydrophilic channel formed by the heterotrimeric Sec61/SecY complex. Whether this channel forms at the interface of multiple copies of Sec61/SecY or is intrinsic to a monomeric complex, as suggested by the recently solved X-ray structure of the Methanococcus jannaschii SecY complex, is a matter of contention. By introducing a single cysteine at various positions in Escherichia coli SecY and testing its ability to form a disulfide bond with a single cysteine in a translocating chain, we provide evidence that translocating polypeptides pass through the center of the SecY complex. The strongest cross-links were observed with residues that would form a constriction in an hourglass-shaped pore. This suggests that the channel makes only limited contact with a translocating polypeptide, thus minimizing the energy required for translocation

SETDB1, HP1 and SUV39 promote repositioning of 53BP1 to extend resection during homologous recombination in G2 cells

Recent studies have shown that homologous recombination (HR) requires chromatin repression as well as relaxation at DNA double strand breaks (DSBs). HP1 and SUV39H1/2 are repressive factors essential for HR. Here, we identify SETDB1 as an additional compacting factor promoting HR. Depletion of HP1, SUV39, SETDB1 or BRCA1 confer identical phenotypes. The repressive factors, like BRCA1, are dispensable for the initiation of resection but promote the extension step causing diminished RPA or RAD51 foci and HR in irradiated G2 cells. Depletion of the compacting factors does not inhibit BRCA1 recruitment but at 8 h post IR, BRCA1 foci are smaller and aberrantly positioned compared to control cells. BRCA1 promotes 53BP1 repositioning to the periphery of enlarged foci and formation of a devoid core with BRCA1 becoming enlarged and localised internally to 53BP1. Depletion of the compacting factors precludes these changes at irradiation-induced foci. Thus, the repressive factors are required for BRCA1 function in promoting the repositioning of 53BP1 during HR. Additionally, depletion of these repressive factors in undamaged cells causes diminished sister chromatid association at centromeric sequences. We propose a model for how these findings may be functionally linked

Disulfide bridge formation between SecY and a translocating polypeptide localizes the translocation pore to the center of SecY

During their biosynthesis, many proteins pass through the membrane via a hydrophilic channel formed by the heterotrimeric Sec61/SecY complex. Whether this channel forms at the interface of multiple copies of Sec61/SecY or is intrinsic to a monomeric complex, as suggested by the recently solved X-ray structure of the Methanococcus jannaschii SecY complex, is a matter of contention. By introducing a single cysteine at various positions in Escherichia coli SecY and testing its ability to form a disulfide bond with a single cysteine in a translocating chain, we provide evidence that translocating polypeptides pass through the center of the SecY complex. The strongest cross-links were observed with residues that would form a constriction in an hourglass-shaped pore. This suggests that the channel makes only limited contact with a translocating polypeptide, thus minimizing the energy required for translocation

α- and β-Tubulin Lattice of the Axonemal Microtubule Doublet and Binding Proteins Revealed by Single Particle Cryo-Electron Microscopy and Tomography

SummaryMicrotubule doublet (MTD) is the main skeleton of cilia/flagella. Many proteins, such as dyneins and radial spokes, bind to MTD, and generate or regulate force. While the structure of the reconstituted microtubule has been solved at atomic resolution, nature of the axonemal MTD is still unclear. There are a few hypotheses of the lattice arrangement of its α- and β-tubulins, but it has not been described how dyneins and radial spokes bind to MTD. In this study, we analyzed the three-dimensional structure of Tetrahymena MTD at ∼19 Å resolution by single particle cryo-electron microscopy. To identify α- and β-tubulins, we combined image analysis of MTD with specific kinesin decoration. This work reveals that α- and β-tubulins form a B-lattice arrangement in the entire MTD with a seam at the outer junction. We revealed the unique way in which inner arm dyneins, radial spokes, and proteins inside MTD bind and bridge protofilaments

The Signal Sequence Coding Region Promotes Nuclear Export of mRNA

In eukaryotic cells, most mRNAs are exported from the nucleus by the transcription export (TREX) complex, which is loaded onto mRNAs after their splicing and capping. We have studied in mammalian cells the nuclear export of mRNAs that code for secretory proteins, which are targeted to the endoplasmic reticulum membrane by hydrophobic signal sequences. The mRNAs were injected into the nucleus or synthesized from injected or transfected DNA, and their export was followed by fluorescent in situ hybridization. We made the surprising observation that the signal sequence coding region (SSCR) can serve as a nuclear export signal of an mRNA that lacks an intron or functional cap. Even the export of an intron-containing natural mRNA was enhanced by its SSCR. Like conventional export, the SSCR-dependent pathway required the factor TAP, but depletion of the TREX components had only moderate effects. The SSCR export signal appears to be characterized in vertebrates by a low content of adenines, as demonstrated by genome-wide sequence analysis and by the inhibitory effect of silent adenine mutations in SSCRs. The discovery of an SSCR-mediated pathway explains the previously noted amino acid bias in signal sequences and suggests a link between nuclear export and membrane targeting of mRNAs

GM-CSF Regulates Alveolar Macrophage Differentiation and Innate Immunity in the Lung through PU.1

AbstractGM-CSF gene targeted (GM−/−) mice are susceptible to respiratory infections and develop alveolar proteinosis due to defects in innate immune function and surfactant catabolism in alveolar macrophages (AMs), respectively. Reduced cell adhesion, phagocytosis, pathogen killing, mannose- and Toll-like receptor expression, and LPS- or peptidoglycan-stimulated TNFα release were observed in AMs from GM−/− mice. The transcription factor PU.1 was markedly reduced in AMs of GM−/− mice in vivo and was restored by selective expression of GM-CSF in the lungs of SPC-GM/GM−/− transgenic mice. Retrovirus-mediated expression of PU.1 in AMs from GM−/− mice rescued host defense functions and surfactant catabolism by AMs. We conclude that PU.1 mediates GM-CSF-dependent effects on terminal differentiation of AMs regulating innate immune functions and surfactant catabolism by AMs

Co-operation of BRCA1 and POH1 relieves the barriers posed by 53BP1 and RAP80 to resection

In G2 phase cells, DNA double-strand break repair switches from DNA non-homologous end-joining to homologous recombination. This switch demands the promotion of resection. We examine the changes in 53BP1 and RAP80 ionizing radiation induced foci (IRIF) in G2 phase, as these are factors that restrict resection. We observed a 2-fold increase in the volume of 53BP1 foci by 8 h, which is not seen in G1 cells. Additionally, an IRIF core devoid of 53BP1 arises where RPA foci form, with BRCA1 IRIF forming between 53BP1 and replication protein A (RPA). Ubiquitin chains assessed using a-FK2 antibodies are similarly repositioned. Repositioning of all these components requires BRCA1’s BRCT but not the ring finger domain. 53BP1, RAP80 and ubiquitin chains are enlarged following POH1 depletion by small interfering RNA, but a devoid core does not form and RPA foci formation is impaired. Co-depletion of POH1 and RAP80, BRCC36 or ABRAXAS allows establishment of the 53BP1 and ubiquitin chain-devoid core. Thus, the barriers posed by 53BP1 and RAP80 are relieved by BRCA1 and POH1, respectively. Analysis of combined depletions shows that these represent distinct but interfacing barriers to promote loss of ubiquitin chains in the IRIF core, which is required for subsequent resection. We propose a model whereby BRCA1 impacts on 53BP1 to allow access of POH1 to RAP80. POH1-dependent removal of RAP80 within the IRIF core enables degradation of ubiquitin chains, which promotes loss of 53BP1. Thus, POH1 represents a novel component regulating the switch from nonhomologous end-joining to homologous recombination

Conformational diversity of dynactin

Dynactin is a principal regulator of the minus-end directed microtubule motor dynein. The sidearm of dynactin is essential for binding to microtubules and regulation of dynein activity. Although our understanding of the structure of the dynactin backbone (Arp1 rod) has greatly improved recently, structural details of the sidearm subcomplex remain elusive. Here, we report the flexible nature and diverse conformations of dynactin sidearm observed by electron microscopy. Using nanogold labeling and deletion mutant analysis, we determined the domain organization of the largest subunit p150 and discovered that its coiled-coil (CC1), dynein-binding domain, adopted either a folded or an extended form. Furthermore, the entire sidearm exhibited several characteristic forms, and the equilibrium among them depended on salt concentrations. These conformational diversities of the dynactin complex provide clues to understanding how it binds to microtubules and regulates dynein