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1.
Nat Commun ; 12(1): 7102, 2021 Dec 07.
Artigo em Inglês | MEDLINE | ID: mdl-34876589

RESUMO

Various stressors such as viral infection lead to the suppression of cap-dependent translation and the activation of the integrated stress response (ISR), since the stress-induced phosphorylated eukaryotic translation initiation factor 2 [eIF2(αP)] tightly binds to eIF2B to prevent it from exchanging guanine nucleotide molecules on its substrate, unphosphorylated eIF2. Sandfly fever Sicilian virus (SFSV) evades this cap-dependent translation suppression through the interaction between its nonstructural protein NSs and host eIF2B. However, its precise mechanism has remained unclear. Here, our cryo-electron microscopy (cryo-EM) analysis reveals that SFSV NSs binds to the α-subunit of eIF2B in a competitive manner with eIF2(αP). Together with SFSV NSs, eIF2B retains nucleotide exchange activity even in the presence of eIF2(αP), in line with the cryo-EM structures of the eIF2B•SFSV NSs•unphosphorylated eIF2 complex. A genome-wide ribosome profiling analysis clarified that SFSV NSs expressed in cultured human cells attenuates the ISR triggered by thapsigargin, an endoplasmic reticulum stress inducer. Furthermore, SFSV NSs introduced in rat hippocampal neurons and human induced-pluripotent stem (iPS) cell-derived motor neurons exhibits neuroprotective effects against the ISR-inducing stress. Since ISR inhibition is beneficial in various neurological disease models, SFSV NSs may be a promising therapeutic ISR inhibitor.

2.
Mol Cell ; 81(1): 88-103.e6, 2021 01 07.
Artigo em Inglês | MEDLINE | ID: mdl-33220178

RESUMO

The small molecule ISRIB antagonizes the activation of the integrated stress response (ISR) by phosphorylated translation initiation factor 2, eIF2(αP). ISRIB and eIF2(αP) bind distinct sites in their common target, eIF2B, a guanine nucleotide exchange factor for eIF2. We have found that ISRIB-mediated acceleration of eIF2B's nucleotide exchange activity in vitro is observed preferentially in the presence of eIF2(αP) and is attenuated by mutations that desensitize eIF2B to the inhibitory effect of eIF2(αP). ISRIB's efficacy as an ISR inhibitor in cells also depends on presence of eIF2(αP). Cryoelectron microscopy (cryo-EM) showed that engagement of both eIF2B regulatory sites by two eIF2(αP) molecules remodels both the ISRIB-binding pocket and the pockets that would engage eIF2α during active nucleotide exchange, thereby discouraging both binding events. In vitro, eIF2(αP) and ISRIB reciprocally opposed each other's binding to eIF2B. These findings point to antagonistic allostery in ISRIB action on eIF2B, culminating in inhibition of the ISR.


Assuntos
Acetamidas/química , Cicloexilaminas/química , Fator de Iniciação 2B em Eucariotos/química , Fator de Iniciação 2 em Eucariotos/química , Regulação Alostérica , Animais , Sítios de Ligação , Células CHO , Cricetulus , Microscopia Crioeletrônica , Fator de Iniciação 2 em Eucariotos/genética , Fator de Iniciação 2 em Eucariotos/metabolismo , Fator de Iniciação 2B em Eucariotos/genética , Fator de Iniciação 2B em Eucariotos/metabolismo , Células HeLa , Humanos , Fosforilação
3.
Plant Direct ; 5(12): e370, 2021 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-34988354

RESUMO

The suppressor of gamma response 1 (SOG1), a NAM, ATAF1, 2, and CUC2 (NAC)-type transcription factor found in seed plants, is a master regulator of DNA damage responses (DDRs). Upon DNA damage, SOG1 regulates the expression of downstream DDR genes. To know the origin of the DDR network in land plants, we searched for a homolog(s) of SOG1 in a moss Physcomitrium (Physcomitrella) patens and identified PpSOG1a and PpSOG1b. To assess if either or both of them function(s) in DDR, we knocked out the PpSOG1s using CRISPR/Cas9-mediated gene editing and analyzed the responses to DNA-damaging treatments. The double-knockout (KO) sog1a sog1b plants showed resistance to γ-rays, bleomycin, and ultraviolet B (UVB) treatments similarly seen in Arabidopsis sog1 plants. Next, we irradiated wild-type (WT) and KO plants with γ-rays and analyzed the whole transcriptome to examine the effect on the expression of DDR genes. The results revealed that many P. patens genes involved in the checkpoint, DNA repair, replication, and cell cycle-related genes were upregulated after γ-irradiation, which was not seen in sog1a sog1b plant. These results suggest that PpSOG1a and PpSOG1b work redundantly on DDR response in P. patens; in addition, plant-specific DDR systems had been established before the emergence of vascular plants.

5.
Front Plant Sci ; 10: 1208, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31649692

RESUMO

Plant genomes sustain various forms of DNA damage that stall replication forks. Translesion synthesis (TLS) is one of the pathways to overcome stalled replication in which specific polymerases (TLS polymerase) perform bypass synthesis across DNA damage. This article gives a brief overview of plant TLS polymerases. In Arabidopsis, DNA polymerase (Pol) ζ, η, κ, θ, and λ and Reversionless1 (Rev1) are shown to be involved in the TLS. For example, AtPolη bypasses ultraviolet (UV)-induced cyclobutane pyrimidine dimers in vitro. Disruption of AtPolζ or AtPolη increases root stem cell death after UV irradiation. These results suggest that AtPolζ and ATPolη bypass UV-induced damage, prevent replication arrest, and allow damaged cells to survive and grow. In general, TLS polymerases have low fidelity and often induce mutations. Accordingly, disruption of AtPolζ or AtRev1 reduces somatic mutation frequency, whereas disruption of AtPolη elevates it, suggesting that plants have both mutagenic and less mutagenic TLS activities. The stalled replication fork can be resolved by a strand switch pathway involving a DNA helicase Rad5. Disruption of both AtPolζ and AtRAD5a shows synergistic or additive effects in the sensitivity to DNA-damaging agents. Moreover, AtPolζ or AtRev1 disruption elevates homologous recombination frequencies in somatic tissues. These results suggest that the Rad5-dependent pathway and TLS are parallel. Plants grown in the presence of heat shock protein 90 (HSP90) inhibitor showed lower mutation frequencies, suggesting that HSP90 regulates mutagenic TLS in plants. Hypersensitivities of TLS-deficient plants to γ-ray and/or crosslink damage suggest that plant TLS polymerases have multiple roles, as reported in other organisms.

6.
Science ; 364(6439): 495-499, 2019 05 03.
Artigo em Inglês | MEDLINE | ID: mdl-31048492

RESUMO

A core event in the integrated stress response, an adaptive pathway common to all eukaryotic cells in response to various stress stimuli, is the phosphorylation of eukaryotic translation initiation factor 2 (eIF2). Normally, unphosphorylated eIF2 transfers the methionylated initiator tRNA to the ribosome in a guanosine 5'-triphosphate-dependent manner. By contrast, phosphorylated eIF2 inhibits its specific guanine nucleotide exchange factor, eIF2B. To elucidate how the eIF2 phosphorylation status regulates the eIF2B activity, we determined cryo-electron microscopic and crystallographic structures of eIF2B in complex with unphosphorylated or phosphorylated eIF2. The unphosphorylated and phosphorylated forms of eIF2 bind to eIF2B in completely different manners: the nucleotide exchange-active and -inactive modes, respectively. These structures explain how phosphorylated eIF2 dominantly inhibits the nucleotide exchange activity of eIF2B.


Assuntos
Fator de Iniciação 2B em Eucariotos/antagonistas & inibidores , Fator de Iniciação 2B em Eucariotos/química , Fator de Iniciação 2 em Eucariotos/química , Estresse Fisiológico , Motivos de Aminoácidos , Microscopia Crioeletrônica , Fator de Iniciação 2B em Eucariotos/metabolismo , Humanos , Fosforilação
7.
Mol Cell ; 74(6): 1205-1214.e8, 2019 06 20.
Artigo em Inglês | MEDLINE | ID: mdl-31080011

RESUMO

Translation initiation of hepatitis C virus (HCV) genomic RNA is induced by an internal ribosome entry site (IRES). Our cryoelectron microscopy (cryo-EM) analysis revealed that the HCV IRES binds to the solvent side of the 40S platform of the cap-dependently translating 80S ribosome. Furthermore, we obtained the cryo-EM structures of the HCV IRES capturing the 40S subunit of the IRES-dependently translating 80S ribosome. In the elucidated structures, the HCV IRES "body," consisting of domain III except for subdomain IIIb, binds to the 40S subunit, while the "long arm," consisting of domain II, remains flexible and does not impede the ongoing translation. Biochemical experiments revealed that the cap-dependently translating ribosome becomes a better substrate for the HCV IRES than the free ribosome. Therefore, the HCV IRES is likely to efficiently induce the translation initiation of its downstream mRNA with the captured translating ribosome as soon as the ongoing translation terminates.


Assuntos
Fatores de Iniciação em Eucariotos/química , Hepacivirus/genética , Iniciação Traducional da Cadeia Peptídica , RNA Viral/química , Subunidades Ribossômicas Maiores de Eucariotos/ultraestrutura , Subunidades Ribossômicas Menores de Eucariotos/ultraestrutura , Sítios de Ligação , Microscopia Crioeletrônica , Fatores de Iniciação em Eucariotos/genética , Fatores de Iniciação em Eucariotos/metabolismo , Células HEK293 , Hepacivirus/metabolismo , Interações Hospedeiro-Patógeno , Humanos , Sítios Internos de Entrada Ribossomal , Modelos Moleculares , Conformação de Ácido Nucleico , RNA Viral/genética , RNA Viral/metabolismo , Subunidades Ribossômicas Maiores de Eucariotos/genética , Subunidades Ribossômicas Maiores de Eucariotos/metabolismo , Subunidades Ribossômicas Menores de Eucariotos/genética , Subunidades Ribossômicas Menores de Eucariotos/metabolismo
8.
Pediatrics ; 143(3)2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30765462

RESUMO

A 14-year-old girl developed 4 episodes of anaphylaxis of unknown etiology, which required intramuscular adrenaline administration each time. She had eaten pizza and a cheeseburger immediately before the first 2 episodes, respectively, but had not eaten anything for several hours before the last 2 episodes. It turned out that she had eaten the same ice lolly 4 hours before the first 3 episodes and a Café au lait Swirkle (a half-frozen beverage) 4 hours before the last episode. We detected carboxymethylcellulose sodium as the only common ingredient in all anaphylactic episodes. Skin prick tests were positive for carboxymethylcellulose solution and carboxymethylcellulose-containing food products. We obtained a custom-made carboxymethylcellulose sodium-free ice lolly from the manufacturer and confirmed that it did not induce anaphylactic reactions by a challenge test. Carboxymethylcellulose, an anionic water-soluble polymer derived from native cellulose, is considered to be unabsorbable from the human gut and has been widely and increasingly used in pharmaceutical preparations, cosmetics, and food. This article is the first report of anaphylaxis caused by carboxymethylcellulose-containing foods, whereas anaphylaxis to carboxymethylcellulose has been rarely associated with carboxymethylcellulose-containing pharmaceuticals. Although the exact mechanisms underlying the induction of late-onset anaphylaxis by carboxymethylcellulose remain unclear, a small minority of cellulose-digesting microbial flora in the human colon and contamination of food products with carboxymethylcellulose of low molecular weight might be involved. The induction of recurrent anaphylaxis by various products should be a clue that prompts physicians to suspect food additives as a cause for anaphylaxis.


Assuntos
Anafilaxia/induzido quimicamente , Anafilaxia/diagnóstico , Carboximetilcelulose Sódica/efeitos adversos , Carboximetilcelulose Sódica/análise , Aditivos Alimentares/efeitos adversos , Aditivos Alimentares/análise , Adolescente , Feminino , Humanos
9.
Mol Cell ; 73(4): 738-748.e9, 2019 02 21.
Artigo em Inglês | MEDLINE | ID: mdl-30595437

RESUMO

A class of translation inhibitors, exemplified by the natural product rocaglamide A (RocA), isolated from Aglaia genus plants, exhibits antitumor activity by clamping eukaryotic translation initiation factor 4A (eIF4A) onto polypurine sequences in mRNAs. This unusual inhibitory mechanism raises the question of how the drug imposes sequence selectivity onto a general translation factor. Here, we determined the crystal structure of the human eIF4A1⋅ATP analog⋅RocA⋅polypurine RNA complex. RocA targets the "bi-molecular cavity" formed characteristically by eIF4A1 and a sharply bent pair of consecutive purines in the RNA. Natural amino acid substitutions found in Aglaia eIF4As changed the cavity shape, leading to RocA resistance. This study provides an example of an RNA-sequence-selective interfacial inhibitor fitting into the space shaped cooperatively by protein and RNA with specific sequences.


Assuntos
Benzofuranos/metabolismo , Fator de Iniciação 4A em Eucariotos/metabolismo , Biossíntese de Proteínas , Inibidores da Síntese de Proteínas/metabolismo , RNA/metabolismo , Ribossomos/metabolismo , Adenilil Imidodifosfato/química , Adenilil Imidodifosfato/metabolismo , Aglaia/química , Aglaia/genética , Aglaia/metabolismo , Substituição de Aminoácidos , Benzofuranos/química , Benzofuranos/isolamento & purificação , Benzofuranos/farmacologia , Sítios de Ligação , Resistência a Medicamentos/genética , Fator de Iniciação 4A em Eucariotos/química , Fator de Iniciação 4A em Eucariotos/genética , Células HEK293 , Humanos , Modelos Moleculares , Estrutura Molecular , Mutação , Proteínas de Plantas/química , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Ligação Proteica , Biossíntese de Proteínas/efeitos dos fármacos , Biossíntese de Proteínas/genética , Domínios e Motivos de Interação entre Proteínas , Inibidores da Síntese de Proteínas/química , Inibidores da Síntese de Proteínas/isolamento & purificação , Inibidores da Síntese de Proteínas/farmacologia , RNA/química , Ribossomos/química , Ribossomos/efeitos dos fármacos , Ribossomos/genética , Relação Estrutura-Atividade
10.
Sci Rep ; 8(1): 16622, 2018 11 09.
Artigo em Inglês | MEDLINE | ID: mdl-30413746

RESUMO

When a spermatozoon shows chemotactic behavior, transient [Ca2+]i increases in the spermatozoon are induced by an attractant gradient. The [Ca2+]i increase triggers a series of stereotypic responses of flagellar waveforms that comprise turning and straight-swimming. However, the molecular mechanism of [Ca2+]i modulation controlled by the attractants is not well defined. Here, we examined receptive mechanisms for the sperm attractant, SAAF, in the ascidian, Ciona intestinalis, and identified a plasma membrane Ca2+-ATPase (PMCA) as a SAAF-binding protein. PMCA is localized in sperm flagella membranes and seems to interact with SAAF through basic amino acids located in the second and third extracellular loops. ATPase activity of PMCA was enhanced by SAAF, and PMCA inhibitors, 5(6)-Carboxyeosin diacetate and Caloxin 2A1, inhibited chemotactic behavior of the sperm. Furthermore, Caloxin 2A1 seemed to inhibit efflux of [Ca2+]i in the sperm, and SAAF seemed to competitively reduce the effect of Caloxin 2A1. On the other hand, chemotactic behavior of the sperm was disordered not only at low-Ca2+, but also at high-Ca2+ conditions. Thus, PMCA is a potent candidate for the SAAF receptor, and direct control of Ca2+ efflux via PMCA is a fundamental mechanism to mediate chemotactic behavior in the ascidian spermatozoa.


Assuntos
Cálcio/metabolismo , Membrana Celular/enzimologia , Quimiotaxia , Ciona intestinalis/fisiologia , Peptídeos/metabolismo , ATPases Transportadoras de Cálcio da Membrana Plasmática/metabolismo , Espermatozoides/fisiologia , Animais , Sinalização do Cálcio , Colestanóis/metabolismo , Peptídeos e Proteínas de Sinalização Intercelular , Masculino , ATPases Transportadoras de Cálcio da Membrana Plasmática/genética , Motilidade Espermática , Ésteres do Ácido Sulfúrico/metabolismo
11.
J Cell Biol ; 217(12): 4155-4163, 2018 12 03.
Artigo em Inglês | MEDLINE | ID: mdl-30275105

RESUMO

The Tau family microtubule-associated proteins (MAPs) promote microtubule stabilization and regulate microtubule-based motility. They share the C-terminal microtubule-binding domain, which includes three to five tubulin-binding repeats. Different numbers of repeats formed by alternative splicing have distinct effects on the activities of these proteins, and the distribution of these variants regulates fundamental physiological phenomena in cells. In this study, using cryo-EM, we visualized the MAP4 microtubule complex with the molecular motor kinesin-1. MAP4 bound to the C-terminal domains of tubulins along the protofilaments stabilizes the longitudinal contacts of the microtubule. The strongest bond of MAP4 was found around the intertubulin-dimer interface such that MAP4 coexists on the microtubule with kinesin-1 bound to the intratubulin-dimer interface as well. MAP4, consisting of five repeats, further folds and accumulates above the intertubulin-dimer interface, interfering with kinesin-1 movement. Therefore, these cryo-EM studies reveal new insight into the structural basis of microtubule stabilization and inhibition of kinesin motility by the Tau family MAPs.


Assuntos
Proteínas Associadas aos Microtúbulos , Microtúbulos , Humanos , /metabolismo , Proteínas Associadas aos Microtúbulos/química , Proteínas Associadas aos Microtúbulos/metabolismo , Microtúbulos/química , Microtúbulos/metabolismo , Microtúbulos/ultraestrutura
12.
Genes (Basel) ; 9(2)2018 Feb 07.
Artigo em Inglês | MEDLINE | ID: mdl-29414843

RESUMO

The purpose of this study was to investigate whether the moss Physcomitrella patens cells are more resistant to ionizing radiation than animal cells. Protoplasts derived from P. patens protonemata were irradiated with γ-rays of 50-1000 gray (Gy). Clonogenicity of the protoplasts decreased in a γ-ray dose-dependent manner. The dose that decreased clonogenicity by half (LD50) was 277 Gy, which indicated that the moss protoplasts were 200-times more radioresistant than human cells. To investigate the mechanism of radioresistance in P. patens, we irradiated protoplasts on ice and initial double-strand break (DSB) yields were measured using the pulsed-field gel electrophoresis assay. Induced DSBs linearly increased dependent on the γ-ray dose and the DSB yield per Gb DNA per Gy was 2.2. The DSB yield in P. patens was half to one-third of those reported in mammals and yeasts, indicating that DSBs are difficult to induce in P. patens. The DSB yield per cell per LD50 dose in P. patens was 311, which is three- to six-times higher than those in mammals and yeasts, implying that P. patens is hyperresistant to DSBs. Physcomitrella patens is indicated to possess unique mechanisms to inhibit DSB induction and provide resistance to high numbers of DSBs.

13.
Biochim Biophys Acta Biomembr ; 1860(5): 981-990, 2018 May.
Artigo em Inglês | MEDLINE | ID: mdl-29317195

RESUMO

The voltage sensor domain (VSD) is a protein domain that confers sensitivity to membrane potential in voltage-gated ion channels as well as the voltage-sensing phosphatase. Although VSDs have long been considered to function as regulatory units acting on adjacent effectors, recent studies have revealed the existence of direct ion permeation paths in some mutated VSDs and in the voltage-gated proton channel. In this study, we show that calcium currents are evoked upon membrane hyperpolarization in cells expressing a VSD derived from an ascidian voltage-gated ion channel superfamily. Unlike the previously reported omega-pore in the Shaker K+ channel and rNav1.4, mutations are not required. From electrophysiological experiments in heterologous expression systems, we found that the conductance is directly mediated by the VSD itself and is carried by both monovalent and divalent cations. This is the first report of divalent cation permeation through a VSD-like structure.


Assuntos
Canais de Cálcio , Cátions Bivalentes/metabolismo , Ativação do Canal Iônico , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Animais , Canais de Cálcio/química , Canais de Cálcio/genética , Canais de Cálcio/metabolismo , Ciona intestinalis/genética , Ciona intestinalis/metabolismo , Condutividade Elétrica , Feminino , Células HEK293 , Humanos , Ativação do Canal Iônico/genética , Potenciais da Membrana/genética , Permeabilidade , Domínios Proteicos/genética , Xenopus
14.
J Radiat Res ; 58(6): 772-781, 2017 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-28637346

RESUMO

Ion beams have been used as an effective tool in mutation breeding for the creation of crops with novel characteristics. Recent analyses have revealed that ion beams induce large chromosomal alterations, in addition to small mutations comprising base changes or frameshifts. In an effort to understand the potential capability of ion beams, we analyzed an Arabidopsis mutant possessing an abnormal genetic trait. The Arabidopsis mutant uvh3-2 is hypersensitive to UVB radiation when photoreactivation is unavailable. uvh3-2 plants grow normally and produce seeds by self-pollination. SSLP and CAPS analyses of F2 plants showed abnormal recombination frequency on chromosomes 2 and 3. PCR-based analysis and sequencing revealed that one-third of chromosome 3 was translocated to chromosome 2 in uvh3-2. FISH analysis using a 180 bp centromeric repeat and 45S ribosomal DNA (rDNA) as probes showed that the 45S rDNA signal was positioned away from that of the 180 bp centromeric repeat in uvh3-2, suggesting the insertion of a large chromosome fragment into the chromosome with 45S rDNA clusters. F1 plants derived from a cross between uvh3-2 and wild-type showed reduced fertility. PCR-based analysis of F2 plants suggested that reproductive cells carrying normal chromosome 2 and uvh3-2-derived chromosome 3 are unable to survive and therefore produce zygote. These results showed that ion beams could induce marked genomic alterations, and could possibly lead to the generation of novel plant species and crop strains.


Assuntos
Arabidopsis/genética , Aberrações Cromossômicas , Cromossomos de Plantas/genética , Mutação/genética , Arabidopsis/efeitos da radiação , Pareamento de Bases/genética , Segregação de Cromossomos/efeitos da radiação , Cruzamentos Genéticos , DNA Ribossômico/genética , Fertilidade/genética , Fertilidade/efeitos da radiação , Íons , Reação em Cadeia da Polimerase , Recombinação Genética/genética , Raios Ultravioleta
15.
J Vis Exp ; (119)2017 01 04.
Artigo em Inglês | MEDLINE | ID: mdl-28117786

RESUMO

In most mammals, auditory ossicles in the middle ear, including the malleus, incus and stapes, are the smallest bones. In mice, a bony structure called the auditory bulla houses the ossicles, whereas the auditory capsule encloses the inner ear, namely the cochlea and semicircular canals. Murine ossicles are essential for hearing and thus of great interest to researchers in the field of otolaryngology, but their metabolism, development, and evolution are highly relevant to other fields. Altered bone metabolism can affect hearing function in adult mice, and various gene-deficient mice show changes in morphogenesis of auditory ossicles in utero. Although murine auditory ossicles are tiny, their manipulation is feasible if one understands their anatomical orientation and 3D structure. Here, we describe how to dissect the auditory bulla and capsule of postnatal mice and then isolate individual ossicles by removing part of the bulla. We also discuss how to embed the bulla and capsule in different orientations to generate paraffin or frozen sections suitable for preparation of longitudinal, horizontal, or frontal sections of the malleus. Finally, we enumerate anatomical differences between mouse and human auditory ossicles. These methods would be useful in analyzing pathological, developmental and evolutionary aspects of auditory ossicles and the middle ear in mice.


Assuntos
Ossículos da Orelha/patologia , Animais , Ossículos da Orelha/diagnóstico por imagem , Bigorna/patologia , Martelo/patologia , Camundongos , Estribo/patologia , Microtomografia por Raio-X
17.
Biol Reprod ; 92(1): 8, 2015 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-25395676

RESUMO

Seminal vesicle secretion 2 (SVS2) is a protein secreted by the mouse seminal vesicle. We previously demonstrated that SVS2 regulates fertilization in mice; SVS2 is attached to a ganglioside GM1 on the plasma membrane of the sperm head and inhibits sperm capacitation in in vitro fertilization as a decapacitation factor. Furthermore, male mice lacking SVS2 display prominently reduced fertility in vivo, which indicates that SVS2 protects spermatozoa from some spermicidal attack in the uterus. In this study, we tried to investigate the mechanisms by which SVS2 controls in vivo sperm capacitation. SVS2-deficient males that mated with wild-type partners resulted in decreased cholesterol levels on ejaculated sperm in the uterine cavity. SVS2 prevented cholesterol efflux from the sperm plasma membrane and incorporated liberated cholesterol in the sperm plasma membrane, thereby reversibly preventing the induction of sperm capacitation by bovine serum albumin and methyl-beta-cyclodextrin in vitro. SVS2 enters the uterus and the uterotubal junction, arresting sperm capacitation in this area. Therefore, our results show that SVS2 keeps sterols on the sperm plasma membrane and plays a key role in unlocking sperm capacitation in vivo.


Assuntos
Proteínas Secretadas pela Vesícula Seminal/farmacologia , Capacitação Espermática/efeitos dos fármacos , Espermatozoides/efeitos dos fármacos , Esteróis/metabolismo , Animais , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Citoproteção/efeitos dos fármacos , Tubas Uterinas/efeitos dos fármacos , Tubas Uterinas/fisiologia , Feminino , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Proteínas Recombinantes/farmacologia , Proteínas Secretadas pela Vesícula Seminal/fisiologia , Espermatozoides/metabolismo
18.
Brain Dev ; 37(2): 200-5, 2015 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-24768169

RESUMO

AIM: Congenital cytomegalovirus (CMV) infection can cause a variety of neurological deficits of delayed onset in infants who are asymptomatic at birth. The aim of this study was to investigate the prevalence of congenital CMV infection among children with autism spectrum disorder (ASD) in Nagasaki, Japan. METHODS: Twenty-nine children with ASD who were born in Nagasaki and had no other major neurological deficits were recruited. Two of the patients were excluded due to significant perinatal events. The remaining 27 children were investigated retrospectively for congenital CMV infection by analyzing dried blood spot samples or dried umbilical cords for CMV DNA using real-time PCR. RESULTS: CMV DNA was detected in two (7.4%) of the 27 children. Neither of the patients had perinatal histories suggestive of congenital CMV disease or other neurological deficits, including hearing impairment and epilepsy. The severity of their autistic disorders varied considerably. CONCLUSIONS: The rate of congenital CMV infection in this study (two of 27 children with ASD), which was significantly (p=0.004) higher than the incidence of congenital CMV infection in Nagasaki (0.31%, 10/3230 live births), suggests the involvement of congenital CMV infection in a portion of children with ASD, although definite diagnosis was not obtained due to limited clinical data of the study subjects.


Assuntos
Transtornos Globais do Desenvolvimento Infantil/complicações , Infecções por Citomegalovirus/congênito , Infecções por Citomegalovirus/diagnóstico , Citomegalovirus/genética , Criança , Transtornos Globais do Desenvolvimento Infantil/epidemiologia , Pré-Escolar , Citomegalovirus/patogenicidade , Infecções por Citomegalovirus/complicações , Infecções por Citomegalovirus/epidemiologia , Epilepsia/etiologia , Feminino , Perda Auditiva/etiologia , Humanos , Lactente , Masculino , Estudos Retrospectivos , Índice de Gravidade de Doença
19.
ACS Appl Mater Interfaces ; 6(10): 7695-704, 2014 May 28.
Artigo em Inglês | MEDLINE | ID: mdl-24712291

RESUMO

The hydrothermal treatment of a titanium plate in a mixed aqueous solution of hydrogen peroxide and aqueous phosphoric acid under different conditions results in the formation of various titanium phosphate thin films. The films have various crystal structures such as Ti2O3(H2PO4)2·2H2O, α-titanium phosphate (Ti(HPO4)2·H2O), π-titanium phosphate (Ti2O(PO4)2·H2O), or low-crystallinity titanium phosphate and different morphologies that have not been previously reported such as nanobelts, microflowers, nanosheets, nanorods, or nanoplates. The present study also suggests the mechanisms behind the formation of these thin films. The crystal structure and morphology of the titanium phosphate thin films depend strongly on the concentration of the aqueous hydrogen peroxide solution, the amount of phosphoric acid, and the reaction temperature. In particular, hydrogen peroxide plays an important role in the formation of the titanium phosphate thin films. Moreover, controllable wettability of the titanium phosphate thin films, including superhydrophilicity and superhydrophobicity, is reported. Superhydrophobic surfaces with controllable adhesion to water droplets are obtained on π-titanium phosphate nanorod thin films modified with alkylamine molecules. The adhesion force between a water droplet and the thin film depends on the alkyl chain length of the alkylamine and the duration of ultraviolet irradiation utilized for photocatalytic degradation.

20.
J Radiat Res ; 54(6): 1050-6, 2013 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-23728320

RESUMO

To assess the mutational effects of radiation on vigorously proliferating plant tissue, the mutation spectrum was analyzed with Arabidopsis seedlings using the plasmid-rescue method. Transgenic plants containing the Escherichia coli rpsL gene were irradiated with γ-rays and carbon ion beams (320-MeV (12)C(6+)), and mutations in the rpsL gene were analyzed. Mutant frequency increased significantly following irradiation by γ-rays, but not by 320-MeV (12)C(6+). Mutation spectra showed that both radiations increased the frequency of frameshifts and other mutations, including deletions and insertions, but only γ-rays increased the frequency of total base substitutions. These results suggest that the type of DNA lesions which cause base substitutions were less often induced by 320-MeV (12)C(6+) than by γ-rays in Arabidopsis seedlings. Furthermore, γ-rays never increased the frequencies of G:C to T:A or A:T to C:G transversions, which are caused by oxidized guanine; 320-MeV (12)C(6+), however, produced a slight increase in both transversions. Instead, γ-rays produced a significant increase in the frequency of G:C to A:T transitions. These results suggest that 8-oxoguanine has little effect on mutagenesis in Arabidopsis cells.


Assuntos
Arabidopsis/genética , Arabidopsis/efeitos da radiação , Dano ao DNA/genética , Íons Pesados , Mutação/genética , Mutação/efeitos da radiação , Plântula/genética , Plântula/efeitos da radiação , Carbono , Relação Dose-Resposta à Radiação , Proteínas de Escherichia coli , Plantas Geneticamente Modificadas/genética , Plantas Geneticamente Modificadas/efeitos da radiação , Doses de Radiação , Raios X
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