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Cytochrome c oxidase (CcO) reduces O2 to water, coupled with a proton-pumping process. The structure of the O2-reduction site of CcO contains two reducing equivalents, Fea32+ and CuB1+, and suggests that a peroxide-bound state (Fea33+–O−–O−–CuB2+) rather than an O2-bound state (Fea32+–O2) is the initial catalytic intermediate. Unexpectedly, however, resonance Raman spectroscopy results have shown that the initial intermediate is Fea32+–O2, whereas Fea33+–O−–O−–CuB2+ is undetectable. Based on X-ray structures of static noncatalytic CcO forms and mutation analyses for bovine CcO, a proton-pumping mechanism has been proposed. It involves a proton-conducting pathway (the H-pathway) comprising a tandem hydrogen-bond network and a water channel located between the N- and P-side surfaces. However, a system for unidirectional proton-transport has not been experimentally identified. Here, an essentially identical X-ray structure for the two catalytic intermediates (P and F) of bovine CcO was determined at 1.8 Å resolution. A 1.70 Å Fe–O distance of the ferryl center could best be described as Fea34+ = O2−, not as Fea34+–OH−. The distance suggests an ∼800-cm−1 Raman stretching band. We found an interstitial water molecule that could trigger a rapid proton-coupled electron transfer from tyrosine-OH to the slowly forming Fea33+–O−–O−–CuB2+ state, preventing its detection, consistent with the unexpected Raman results. The H-pathway structures of both intermediates indicated that during proton-pumping from the hydrogen-bond network to the P-side, a transmembrane helix closes the water channel connecting the N-side with the hydrogen-bond network, facilitating unidirectional proton-pumping during the P-to-F transition.

Optic atrophy 1 (OPA1) is a dynamin protein that mediates mitochondrial fusion at the inner membrane. OPA1 is also necessary for maintaining the cristae and thus essential for supporting cellular energetics. OPA1 exists as membrane-anchored long form (L-OPA1) and short form (S-OPA1) that lacks the transmembrane region and is generated by cleavage of L-OPA1. Mitochondrial dysfunction and cellular stresses activate the inner membrane–associated zinc metallopeptidase OMA1 that cleaves L-OPA1, causing S-OPA1 accumulation. The prevailing notion has been that L-OPA1 is the functional form, whereas S-OPA1 is an inactive cleavage product in mammals, and that stress-induced OPA1 cleavage causes mitochondrial fragmentation and sensitizes cells to death. However, S-OPA1 contains all functional domains of dynamin proteins, suggesting that it has a physiological role. Indeed, we recently demonstrated that S-OPA1 can maintain cristae and energetics through its GTPase activity, despite lacking fusion activity. Here, applying oxidant insult that induces OPA1 cleavage, we show that cells unable to generate S-OPA1 are more sensitive to this stress under obligatory respiratory conditions, leading to necrotic death. These findings indicate that L-OPA1 and S-OPA1 differ in maintaining mitochondrial function. Mechanistically, we found that cells that exclusively express L-OPA1 generate more superoxide and are more sensitive to Ca2+-induced mitochondrial permeability transition, suggesting that S-OPA1, and not L-OPA1, protects against cellular stress. Importantly, silencing of OMA1 expression increased oxidant-induced cell death, indicating that stress-induced OPA1 cleavage supports cell survival. Our findings suggest that S-OPA1 generation by OPA1 cleavage is a survival mechanism in stressed cells.

pncRNA-D is an irradiation-induced 602-nt long noncoding RNA transcribed from the promoter region of the cyclin D1 (CCND1) gene. CCND1 expression is predicted to be inhibited through an interplay between pncRNA-D and RNA-binding protein TLS/FUS. Because the pncRNA-D–TLS interaction is essential for pncRNA-D–stimulated CCND1 inhibition, here we studied the possible role of RNA modification in this interaction in HeLa cells. We found that osmotic stress induces pncRNA-D by recruiting RNA polymerase II to its promoter. pncRNA-D was highly m6A-methylated in control cells, but osmotic stress reduced the methylation and also arginine methylation of TLS in the nucleus. Knockdown of the m6A modification enzyme methyltransferase-like 3 (METTL3) prolonged the half-life of pncRNA-D, and among the known m6A recognition proteins, YTH domain-containing 1 (YTHDC1) was responsible for binding m6A of pncRNA-D. Knockdown of METTL3 or YTHDC1 also enhanced the interaction of pncRNA-D with TLS, and results from RNA pulldown assays implicated YTHDC1 in the inhibitory effect on the TLS–pncRNA-D interaction. CRISPR/Cas9-mediated deletion of candidate m6A site decreased the m6A level in pncRNA-D and altered its interaction with the RNA-binding proteins. Of note, a reduction in the m6A modification arrested the cell cycle at the G0/G1 phase, and pncRNA-D knockdown partially reversed this arrest. Moreover, pncRNA-D induction in HeLa cells significantly suppressed cell growth. Collectively, these findings suggest that m6A modification of the long noncoding RNA pncRNA-D plays a role in the regulation of CCND1 gene expression and cell cycle progression.

tRNAs universally carry a CCA nucleotide triplet at their 3'-ends. In eukaryotes, the CCA is added post-transcriptionally by the CCA-adding enzyme (CAE). The mitochondrion of the parasitic protozoan Trypanosoma brucei lacks tRNA genes and therefore imports all of its tRNAs from the cytosol. This has generated interest in the tRNA modifications and their distribution in this organism, including how CCA is added to tRNAs. Here, using a BLAST search for genes encoding putative CAE proteins in T. brucei, we identified a single ORF, Tb927.9.8780, as a potential candidate. Knockdown of this putative protein, termed TbCAE, resulted in the accumulation of truncated tRNAs, abolished translation, and inhibited both total and mitochondrial CCA-adding activities, indicating that TbCAE is located both in the cytosol and mitochondrion. However, mitochondrially localized tRNAs were much less affected by the TbCAE ablation than the other tRNAs. Complementation assays revealed that the N-terminal 10 amino acids of TbCAE are dispensable for its activity and mitochondrial localization and that deletion of 10 further amino acids abolishes both. A growth arrest caused by the TbCAE knockdown was rescued by the expression of the cytosolic isoform of yeast CAE, even though it was not imported into mitochondria. This finding indicated that the yeast enzyme complements the essential function of TbCAE by adding CCA to the primary tRNA transcripts. Of note, ablation of the mitochondrial TbCAE activity, which likely has a repair function, only marginally affected growth.

The ribosome biogenesis factor Las1 is an essential endoribonuclease that is well-conserved across eukaryotes and a newly established member of the higher eukaryotes and prokaryotes nucleotide-binding (HEPN) domain-containing nuclease family. HEPN nucleases participate in diverse RNA cleavage pathways and share a short HEPN nuclease motif (RφXXXH) important for RNA cleavage. Most HEPN nucleases participate in stress-activated RNA cleavage pathways; Las1 plays a fundamental role in processing pre-rRNA. Underscoring the significance of Las1 function in the cell, mutations in the human LAS1L (LAS1-like) gene have been associated with neurological dysfunction. Two juxtaposed HEPN nuclease motifs create Las1's composite nuclease active site, but the roles of the individual HEPN motif residues are poorly defined. Here using a combination of in vivo experiments in Saccharomyces cerevisiae and in vitro assays, we show that both HEPN nuclease motifs are required for Las1 nuclease activity and fidelity. Through in-depth sequence analysis and systematic mutagenesis, we determined the consensus HEPN motif in the Las1 subfamily and uncovered its canonical and specialized elements. Using reconstituted Las1 HEPN-HEPN' chimeras, we defined the molecular requirements for RNA cleavage. Intriguingly, both copies of the Las1 HEPN motif were important for nuclease function, revealing that both HEPN motifs participate in coordinating the RNA within the Las1 active site. We also established that conformational flexibility of the two HEPN domains is important for proper nuclease function. The results of our work reveal critical information about how dual HEPN domains come together to drive Las1-mediated RNA cleavage.

Site-specific arrest of RNA polymerases (RNAPs) is fundamental to several technologies that assess RNA structure and function. Current in vitro transcription “roadblocking” approaches inhibit transcription elongation by blocking RNAP with a protein bound to the DNA template. One limitation of protein-mediated transcription roadblocking is that it requires inclusion of a protein factor extrinsic to the minimal in vitro transcription reaction. In this work, we developed a chemical approach for halting transcription by Escherichia coli RNAP. We first established a sequence-independent method for site-specific incorporation of chemical lesions into dsDNA templates by sequential PCR and translesion synthesis. We then show that interrupting the transcribed DNA strand with an internal desthiobiotin-triethylene glycol modification or 1,N6-etheno-2'-deoxyadenosine base efficiently and stably halts Escherichia coli RNAP transcription. By encoding an intrinsic stall site within the template DNA, our chemical transcription roadblocking approach enables display of nascent RNA molecules from RNAP in a minimal in vitro transcription reaction.

The arrangement of functionally-related genes in operons is a fundamental element of how genetic information is organized in prokaryotes. This organization ensures coordinated gene expression by co-transcription. Often, however, alternative genetic responses to specific stress conditions demand the discoordination of operon expression. During cold temperature stress, accumulation of the gene encoding the sole Asp–Glu–Ala–Asp (DEAD)-box RNA helicase in Synechocystis sp. PCC 6803, crhR (slr0083), increases 15-fold. Here, we show that crhR is expressed from a dicistronic operon with the methylthiotransferase rimO/miaB (slr0082) gene, followed by rapid processing of the operon transcript into two monocistronic mRNAs. This cleavage event is required for and results in destabilization of the rimO transcript. Results from secondary structure modeling and analysis of RNase E cleavage of the rimO–crhR transcript in vitro suggested that CrhR plays a role in enhancing the rate of the processing in an auto-regulatory manner. Moreover, two putative small RNAs are generated from additional processing, degradation, or both of the rimO transcript. These results suggest a role for the bacterial RNA helicase CrhR in RNase E-dependent mRNA processing in Synechocystis and expand the known range of organisms possessing small RNAs derived from processing of mRNA transcripts.

Kainate and α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors are two major, closely related receptor subtypes in the glutamate ion channel family. Excessive activities of these receptors have been implicated in a number of central nervous system diseases. Designing potent and selective antagonists of these receptors, especially of kainate receptors, is useful for developing potential treatment strategies for these neurological diseases. Here, we report on two RNA aptamers designed to individually inhibit kainate and AMPA receptors. To improve the biostability of these aptamers, we also chemically modified these aptamers by substituting their 2'-OH group with 2'-fluorine. These 2'-fluoro aptamers, FB9s-b and FB9s-r, were markedly resistant to RNase-catalyzed degradation, with a half-life of ∼5 days in rat cerebrospinal fluid or serum-containing medium. Furthermore, FB9s-r blocked AMPA receptor activity. Aptamer FB9s-b selectively inhibited GluK1 and GluK2 kainate receptor subunits, and also GluK1/GluK5 and GluK2/GluK5 heteromeric kainate receptors with equal potency. This inhibitory profile makes FB9s-b a powerful template for developing tool molecules and drug candidates for treatment of neurological diseases involving excessive activities of the GluK1 and GluK2 subunits.

K. D. Cherednichenko, Yu. Yu. Ershova, A. V. Kiselev and S. N. Naboko
Trans. Moscow Math. Soc. 80 (2020), 251-294.
Abstract, references and article information

A. M. Savchuk and I. V. Sadovnichaya
Trans. Moscow Math. Soc. 80 (2020), 235-250.
Abstract, references and article information

I. V. Astashova, D. A. Lashin and A. V. Filinovskii
Trans. Moscow Math. Soc. 80 (2020), 221-234.
Abstract, references and article information

A. A. Vladimirov
Trans. Moscow Math. Soc. 80 (2020), 211-219.
Abstract, references and article information

I. A. Sheipak and T. A. Garmanova
Trans. Moscow Math. Soc. 80 (2020), 189-210.
Abstract, references and article information

V. V. Vlasov and N. A. Rautian
Trans. Moscow Math. Soc. 80 (2020), 169-188.
Abstract, references and article information

N. F. Valeev, Ya. T. Sultanaev and É. A. Nazirova
Trans. Moscow Math. Soc. 80 (2020), 153-167.
Abstract, references and article information

K. A. Mirzoev and T. A. Safonova
Trans. Moscow Math. Soc. 80 (2020), 133-151.
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V. A. Sadovnichii, Ya. T. Sultanaev and A. M. Akhtyamov
Trans. Moscow Math. Soc. 80 (2020), 123-131.
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Trans. Moscow Math. Soc. 80 (2020), 113-122.
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A. G. Sergeev and Kh. A. Khachatryan
Trans. Moscow Math. Soc. 80 (2020), 95-111.
Abstract, references and article information

V. V. Ryzhikov
Trans. Moscow Math. Soc. 80 (2020), 83-94.
Abstract, references and article information

S. Yu. Orevkov
Trans. Moscow Math. Soc. 80 (2020), 73-81.
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S. A. Kashchenko
Trans. Moscow Math. Soc. 80 (2020), 53-71.
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S. A. Nazarov
Trans. Moscow Math. Soc. 80 (2020), 1-51.
Abstract, references and article information

Prótese auditiva ancorada em osso não emite um som como o aparelho convencional. Seu diferencial é vibrar o osso da cabeça.

The post SUS vai oferecer aparelho de última geração para tipo de perda auditiva até então sem tratamento appeared first on Saúde Próspera.

Todo mundo quer ter um sorriso bonito. Justamente por isso, a ideia de perder um ou mais dentes deixa qualquer pessoa desesperada.

The post Saiba como ter um sorriso perfeito appeared first on Saúde Próspera.

Exame poderá detectar a doença na fase inicial. Pesquisadores da Faculdade de Medicina Osteopática da Universidade de Rowan, nos Estados Unidos, afirmam que estão perto de desenvolver um exame de sangue para detectar Alzheimer com precisão, o que dar...

The post Teste de sangue para detectar Alzheimer está próximo da realidade appeared first on Saúde Próspera.

Unha encravada: quem teve jamais quer repetir a experiência. Os que nunca passaram pela situação, tremem com a ideia de sentir aquela dor de que só ouvem sobre nos relatos.

The post Unha encravada: Como evitar e desencravar appeared first on Saúde Próspera.

Aos primeiros sinais de visão embaçada, as hipóteses mais frequentes sempre são miopia, astigmatismo, hipermetropia. Mas esses sintomas podem indicar outra doença ocular chamada ceratocone - uma deformidade progressiva da córnea, que assume o formato...

The post Visão embaçada e distorcida nem sempre é miopia: fique atento aos sinais do ceratocone appeared first on Saúde Próspera.

Tem tomado seus cuidados com a pele? Neste artigo vamos ensinar a fazer limpeza de pele caseira. Conhecer  os benefícios para limpeza de pele. Conheça o passo a passo a…

The post Cuidados com a pele – Aprenda a fazer limpeza de pele caseira appeared first on Saúde Próspera.

Com que roupa eu vou? Todos os dias nós, mulheres, enfrentamos o dilema de decidir o que vestir. Veja algumas dicas para lhe ajudar a escolher qual roupa usar… A…

The post Com que roupa eu vou? Dicas de qual roupa usar appeared first on Saúde Próspera.

A cirurgia plástica é a área da medicina responsável pela reparação dos defeitos estéticos. Os problemas estéticos podem ser congênitos, ou seja, que já estão presentes no nascimento ou adquiridos…

The post Cirurgia Plástica não tem idade appeared first on Saúde Próspera.

O que é Candidíase? Candidíase, é uma infecção sistêmica causada pelo fungos da Candida albicans. A Candida albicans é um tipo de fungo (levedura) que vive em harmonia no organismo,…

The post Candidíase – Conheça as causas, sintomas e tratamentos appeared first on Saúde Próspera.

A. Knightly and C. Li
Memoirs of the AMS 224 (2012).
Abstract, references and article information

Publication Date: Apr 10 2020 The SEC will withdraw the existing approval order and the fund will become subject to the requirements of Rule 6c-11 and Nasdaq Rule 5704....