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1.
PLoS One ; 16(9): e0256588, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34506539

RESUMO

Bovine leukemia virus (BLV) is the causative agent of enzootic bovine leukosis (EBL), a malignant B cell lymphoma. However, the mechanisms of BLV-associated lymphomagenesis remain poorly understood. Here, after deep sequencing, we performed comparative analyses of B cell microRNAs (miRNAs) in cattle infected with BLV and those without BLV. In BLV-infected cattle, BLV-derived miRNAs (blv-miRNAs) accounted for 38% of all miRNAs in B cells. Four of these blv-miRNAs (blv-miR-B1-5p, blv-miR-B2-5p, blv-miR-B4-3p, and blv-miR-B5-5p) had highly significant positive correlations with BLV proviral load (PVL). The read counts of 90 host-derived miRNAs (bta-miRNAs) were significantly down-regulated in BLV-infected cattle compared to those in uninfected cattle. Only bta-miR-375 had a positive correlation with PVL in BLV-infected cattle and was highly expressed in the B cell lymphoma tissue of EBL cattle. There were a few bta-miRNAs that correlated with BLV tax/rex gene expression; however, BLV AS1 expression had a significant negative correlation with many of the down-regulated bta-miRNAs that are important for tumor development and/or tumor suppression. These results suggest that BLV promotes lymphomagenesis via AS1 and blv-miRNAs, rather than tax/rex, by down-regulating the expression of bta-miRNAs that have a tumor-suppressing function, and this downregulation is linked to increased PVL.


Assuntos
Linfócitos B/metabolismo , Leucose Enzoótica Bovina/metabolismo , Vírus da Leucemia Bovina/isolamento & purificação , MicroRNAs/metabolismo , Animais , Linfócitos B/citologia , Bovinos , Provírus/isolamento & purificação , Carga Viral
2.
Vet Immunol Immunopathol ; 239: 110301, 2021 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-34340138

RESUMO

Bovine leukemia virus (BLV) proviral load is controlled by T-cell responses, which require vitamin A (VA) derived from food. However, whether dietary VA restriction for marbling impairs the T-cell responses that control BLV proviral load in beef cattle is unknown. We assessed T-cell subsets, interferon (IFN)-γ gene expression, and BLV proviral load in naturally BLV-infected Japanese Black cattle that were fed a diet with decreased VA levels. We found that the percentage of CD4+ T cells increased over time during dietary VA restriction. In addition, BLV proviral load was negatively correlated with the percentage of CD4+ T cells and with the level of IFN-γ gene expression. These observations suggest that dietary VA restriction for marbling enhances T-cell responses that control BLV proviral load and thus does not promote leukemogenesis in fattening beef cattle.

3.
Nat Commun ; 12(1): 3338, 2021 06 07.
Artigo em Inglês | MEDLINE | ID: mdl-34099686

RESUMO

The versatile nucleotide excision repair (NER) pathway initiates as the XPC-RAD23B-CETN2 complex first recognizes DNA lesions from the genomic DNA and recruits the general transcription factor complex, TFIIH, for subsequent lesion verification. Here, we present a cryo-EM structure of an NER initiation complex containing Rad4-Rad23-Rad33 (yeast homologue of XPC-RAD23B-CETN2) and 7-subunit coreTFIIH assembled on a carcinogen-DNA adduct lesion at 3.9-9.2 Å resolution. A ~30-bp DNA duplex could be mapped as it straddles between Rad4 and the Ssl2 (XPB) subunit of TFIIH on the 3' and 5' side of the lesion, respectively. The simultaneous binding with Rad4 and TFIIH was permitted by an unwinding of DNA at the lesion. Translocation coupled with torque generation by Ssl2 and Rad4 would extend the DNA unwinding at the lesion and deliver the damaged strand to Rad3 (XPD) in an open form suitable for subsequent lesion scanning and verification.


Assuntos
Microscopia Crioeletrônica , Dano ao DNA , Reparo do DNA , Proteínas de Ligação a DNA/química , DNA/química , Proteínas de Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/metabolismo , Fator de Transcrição TFIIH/química , Adutos de DNA/metabolismo , DNA Helicases/química , DNA Helicases/genética , Proteínas de Ligação a DNA/genética , Proteínas de Saccharomyces cerevisiae/genética , Fator de Transcrição TFIIH/genética
4.
Nat Commun ; 12(1): 3487, 2021 06 09.
Artigo em Inglês | MEDLINE | ID: mdl-34108468

RESUMO

Fusicoccadiene synthase from Phomopsis amygdali (PaFS) is a unique bifunctional terpenoid synthase that catalyzes the first two steps in the biosynthesis of the diterpene glycoside Fusicoccin A, a mediator of 14-3-3 protein interactions. The prenyltransferase domain of PaFS generates geranylgeranyl diphosphate, which the cyclase domain then utilizes to generate fusicoccadiene, the tricyclic hydrocarbon skeleton of Fusicoccin A. Here, we use cryo-electron microscopy to show that the structure of full-length PaFS consists of a central octameric core of prenyltransferase domains, with the eight cyclase domains radiating outward via flexible linker segments in variable splayed-out positions. Cryo-electron microscopy and chemical crosslinking experiments additionally show that compact conformations can be achieved in which cyclase domains are more closely associated with the prenyltransferase core. This structural analysis provides a framework for understanding substrate channeling, since most of the geranylgeranyl diphosphate generated by the prenyltransferase domains remains on the enzyme for cyclization to form fusicoccadiene.


Assuntos
Alquil e Aril Transferases/química , Diterpenos/metabolismo , Proteínas Fúngicas/química , Alquil e Aril Transferases/metabolismo , Ascomicetos/química , Ascomicetos/enzimologia , Catálise , Domínio Catalítico , Microscopia Crioeletrônica , Ciclização , Dimetilaliltranstransferase/química , Dimetilaliltranstransferase/metabolismo , Proteínas Fúngicas/metabolismo , Glicosídeos/biossíntese , Liases/química , Liases/metabolismo , Enzimas Multifuncionais , Fosfatos de Poli-Isoprenil/metabolismo , Conformação Proteica
5.
Sci Adv ; 7(15)2021 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-33827808

RESUMO

During transcription initiation, the general transcription factor TFIIH marks RNA polymerase II by phosphorylating Ser5 of the carboxyl-terminal domain (CTD) of Rpb1, which is followed by extensive modifications coupled to transcription elongation, mRNA processing, and histone dynamics. We have determined a 3.5-Å resolution cryo-electron microscopy (cryo-EM) structure of the TFIIH kinase module (TFIIK in yeast), which is composed of Kin28, Ccl1, and Tfb3, yeast homologs of CDK7, cyclin H, and MAT1, respectively. The carboxyl-terminal region of Tfb3 was lying at the edge of catalytic cleft of Kin28, where a conserved Tfb3 helix served to stabilize the activation loop in its active conformation. By combining the structure of TFIIK with the previous cryo-EM structure of the preinitiation complex, we extend the previously proposed model of the CTD path to the active site of TFIIK.

6.
Sci Adv ; 7(3)2021 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-33523904

RESUMO

The Cdk8 kinase module (CKM) in Mediator, comprising Med13, Med12, CycC, and Cdk8, regulates RNA polymerase II transcription through kinase-dependent and -independent functions. Numerous pathogenic mutations causative for neurodevelopmental disorders and cancer congregate in CKM subunits. However, the structure of the intact CKM and the mechanism by which Cdk8 is non-canonically activated and functionally affected by oncogenic CKM alterations are poorly understood. Here, we report a cryo-electron microscopy structure of Saccharomyces cerevisiae CKM that redefines prior CKM structural models and explains the mechanism of Med12-dependent Cdk8 activation. Med12 interacts extensively with CycC and activates Cdk8 by stabilizing its activation (T-)loop through conserved Med12 residues recurrently mutated in human tumors. Unexpectedly, Med13 has a characteristic Argonaute-like bi-lobal architecture. These findings not only provide a structural basis for understanding CKM function and pathological dysfunction, but also further impute a previously unknown regulatory mechanism of Mediator in transcriptional modulation through its Med13 Argonaute-like features.

7.
Nat Commun ; 12(1): 929, 2021 02 10.
Artigo em Inglês | MEDLINE | ID: mdl-33568648

RESUMO

Respiratory electron transport complexes are organized as individual entities or combined as large supercomplexes (SC). Gram-negative bacteria deploy a mitochondrial-like cytochrome (cyt) bc1 (Complex III, CIII2), and may have specific cbb3-type cyt c oxidases (Complex IV, CIV) instead of the canonical aa3-type CIV. Electron transfer between these complexes is mediated by soluble (c2) and membrane-anchored (cy) cyts. Here, we report the structure of an engineered bc1-cbb3 type SC (CIII2CIV, 5.2 Å resolution) and three conformers of native CIII2 (3.3 Å resolution). The SC is active in vivo and in vitro, contains all catalytic subunits and cofactors, and two extra transmembrane helices attributed to cyt cy and the assembly factor CcoH. The cyt cy is integral to SC, its cyt domain is mobile and it conveys electrons to CIV differently than cyt c2. The successful production of a native-like functional SC and determination of its structure illustrate the characteristics of membrane-confined and membrane-external respiratory electron transport pathways in Gram-negative bacteria.


Assuntos
Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Complexo III da Cadeia de Transporte de Elétrons/química , Complexo III da Cadeia de Transporte de Elétrons/metabolismo , Complexo IV da Cadeia de Transporte de Elétrons/química , Complexo IV da Cadeia de Transporte de Elétrons/metabolismo , Rhodobacter capsulatus/enzimologia , Proteínas de Bactérias/genética , Domínio Catalítico , Coenzimas/química , Coenzimas/metabolismo , Microscopia Crioeletrônica , Transporte de Elétrons , Complexo III da Cadeia de Transporte de Elétrons/genética , Complexo IV da Cadeia de Transporte de Elétrons/genética , Engenharia Genética , Rhodobacter capsulatus/química , Rhodobacter capsulatus/genética , Rhodobacter capsulatus/metabolismo
8.
Can J Vet Res ; 85(1): 72-76, 2021 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-33390657

RESUMO

Changes in immune factors expressed by milk somatic cells from Holstein cows with hypocalcemia after calving were investigated in this study. Fourteen multiparous Holstein cows after their 3rd or 4th calving in one farm were used. The cows were divided into 2 groups: 7 cows needing treatment due to onset of hypocalcemia (hypocalcemia group; age = 5.53 ± 0.27 years, parity = 3.14 ± 0.14) and 7 cows without health problems (control group; age = 5.88 ± 0.31 years, parity = 3.57 ± 0.26). Milk samples were collected aseptically using a cannula and mRNA of immune factors expressed by milk somatic cells were analyzed. Milk samples (50 mL) were collected from the right rear mammary gland of cows before milking at day 1 and weeks 1, 2, 4, and 8 after calving. All milk samples showed a negative reaction to the California Mastitis Test. Levels of relative interleukin (IL)-6 and cathelicidin in the hypocalcemia group were lower than those in the control group in weeks 1 to 8. A significant difference in relative IL-6 levels was found in week 4 (P < 0.05). These results suggest that levels of IL-6 expressed by milk somatic cells may be affected by hypocalcemia in dairy cows.


Assuntos
Doenças dos Bovinos/imunologia , Regulação da Expressão Gênica/imunologia , Hipocalcemia/veterinária , Fatores Imunológicos/metabolismo , Leite/citologia , RNA Mensageiro/metabolismo , Animais , Bovinos , Doenças dos Bovinos/genética , Feminino , Hipocalcemia/genética , Hipocalcemia/imunologia , Período Pós-Parto , RNA Mensageiro/genética
9.
Anim Sci J ; 91(1): e13495, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-33372705

RESUMO

The effectiveness of on-farm continuous flow high-temperature short-time (HTST) pasteurization (i.e., 72°C for 15 s) for the inactivation of bovine leukemia virus (BLV) in milk was investigated with a sheep bioassay. Four sheep that had been inoculated with completely pasteurized milk containing approximately 3.4 × 107 BLV-infected peripheral blood mononuclear cells (PBMC) and treated by either HTST pasteurization or laboratory-scale low-temperature long-time (LTLT) pasteurization (i.e., 60°C for 30 min), remained negative for BLV for at least 17 weeks after inoculation. In contrast, all sheep inoculated with unpasteurized or inadequately pasteurized milk containing the same number of BLV-infected PBMC were tested positive for BLV and anti-BLV antibodies within 3 weeks after inoculation. These results suggest that on-farm continuous flow HTST pasteurization was equivalent value with inactivated BLV on the LTLT procedure and can effectively inactivate BLV in the milk. Therefore, on-farm HTST pasteurization of the pooled colostrum or milk used in automated feeding systems is likely to protect group-housed preweaned calves from BLV infection, thereby improving animal health on dairy farms.


Assuntos
Ração Animal/virologia , Indústria de Laticínios/métodos , Leucose Enzoótica Bovina/prevenção & controle , Leucose Enzoótica Bovina/virologia , Fazendas , Vírus da Leucemia Bovina/fisiologia , Leite/virologia , Pasteurização/métodos , Temperatura , Inativação de Vírus , Animais , Bovinos , Ovinos , Fatores de Tempo
10.
Arch Virol ; 165(12): 2961-2966, 2020 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-33037940

RESUMO

Bovine leukemia virus (BLV) is the causative agent of enzootic bovine leukosis (EBL). We used microchip electrophoresis in combination with automatic image analysis to develop a novel high-throughput PCR-RFLP to type the gene sequences that encode BLV Tax 233. This method revealed that 233L-Tax is more prevalent than 233P-Tax in cattle in Japan. The proportion infected with BLV carrying the gene encoding 233L-Tax was significantly higher in Holstein cattle than in Japanese Black cattle. Holsteins infected with BLV encoding 233L-Tax had higher proviral loads than did Holsteins infected with BLV encoding 233P-Tax and Japanese Blacks infected with BLV encoding 233L-Tax or 233P-Tax. The novel method developed in this study will be a useful tool for identifying cattle harboring BLV with a higher risk of EBL and viral transmission.


Assuntos
Eletroforese em Microchip/instrumentação , Produtos do Gene tax/genética , Vírus da Leucemia Bovina/genética , Reação em Cadeia da Polimerase/métodos , Polimorfismo de Fragmento de Restrição , Animais , Bovinos , Leucose Enzoótica Bovina/virologia , Japão , Carga Viral
11.
Sci Adv ; 6(23)2020 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-32917641

RESUMO

Actin-related protein (Arp) 2/3 complex nucleates branched actin networks that drive cell motility. It consists of seven proteins, including two actin-related subunits (Arp2 and Arp3). Two nucleation-promoting factors (NPFs) bind Arp2/3 complex during activation, but the order, specific interactions, and contribution of each NPF to activation are unresolved. Here, we report the cryo-electron microscopy structure of recombinantly expressed human Arp2/3 complex with two WASP family NPFs bound and address the mechanism of activation. A cross-linking assay that captures the transition of the Arps into the activated filament-like conformation shows that actin binding to NPFs favors this transition. Actin-NPF binding to Arp2 precedes binding to Arp3 and is sufficient to promote the filament-like conformation but not activation. Structure-guided mutagenesis of the NPF-binding sites reveals their distinct roles in activation and shows that, contrary to budding yeast Arp2/3 complex, NPF-mediated delivery of actin at the barbed end of both Arps is required for activation of human Arp2/3 complex.

12.
PLoS One ; 15(9): e0239072, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32915894

RESUMO

We aimed to evaluate choroid structural changes using swept-source optical coherence tomography (SS-OCT) following hemodialysis initiation in diabetic and nondiabetic patients with end-stage kidney disease (ESKD). In this multicenter, prospective, cross-sectional study, diabetic (DM group; 30 eyes; 16 patients) and nondiabetic patients (NDM group; 30 eyes; 15 patients) with ESKD were evaluated after hemodialysis initiation. SS-OCT findings were analyzed using a manual delineation technique and binarization method before the first and last hemodialysis sessions, conducted approximately 2 weeks apart. Subfoveal choroidal thickness changes and mean large choroidal vessel layer thickness were significantly greater in the DM group (-13.3% ± 2.5% and -14.5% ± 5.2%, respectively) than the NDM group (-9.5% ± 3.1% and -9.2% ± 3.4%, respectively; p = 0.049 and p = 0.02, respectively). Binarized SS-OCT analysis revealed that the mean subfoveal choroidal area was significantly larger in the DM group (-21.9% ± 6.5%) than the NDM group (-17.2% ± 5.9%; p = 0.032). The change ratio in mean luminal area values was significantly greater in the DM group (-27.7% ± 8.7%) than the NDM group (-17.7% ± 5.8%; p = 0.007). The DM group exhibited substantial changes in the choroidal layer, possibly reflecting choroidal vascular disorders caused by diabetes.


Assuntos
Doenças da Coroide/diagnóstico , Corioide/diagnóstico por imagem , Complicações do Diabetes/diagnóstico , Falência Renal Crônica/complicações , Diálise Renal , Idoso , Idoso de 80 Anos ou mais , Corioide/irrigação sanguínea , Corioide/patologia , Doenças da Coroide/etiologia , Doenças da Coroide/patologia , Estudos Transversais , Complicações do Diabetes/patologia , Feminino , Humanos , Falência Renal Crônica/terapia , Masculino , Pessoa de Meia-Idade , Estudos Prospectivos , Tomografia de Coerência Óptica
13.
Avian Dis ; 64(1): 80-84, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-32267128

RESUMO

After accumulating data through a nationwide survey, we characterized the recent prevalences and geographic distributions of various genotypes of infectious bronchitis virus (IBV) on layer farms in Japan. Reverse transcription PCR analysis of fecal samples revealed the presence of the IBV nucleoprotein (N) gene on approximately 30% of the farms surveyed. N-gene detection rates were higher in the Chugoku and Kyushu regions than in the remaining surveyed regions. Phylogenetic analysis of S1 gene sequences revealed that JP-I, JP-II, JP-III, and Massachusetts genotypes were particularly prevalent, with JP-I isolated throughout the country. Additionally, JP-II was the genotype detected most frequently in Chugoku, and JP-III was the most frequent in Kyushu. Unlike the previous results obtained in 1998 through 2003, the European-prevalent 4/91 genotype was no longer circulating in Japan. Moreover, the number of prefectures where multiple genotypes were detected simultaneously increased during that time.


Assuntos
Galinhas , Infecções por Coronavirus/veterinária , Vírus da Bronquite Infecciosa/isolamento & purificação , Doenças das Aves Domésticas/epidemiologia , Animais , Infecções por Coronavirus/epidemiologia , Infecções por Coronavirus/virologia , Genótipo , Vírus da Bronquite Infecciosa/genética , Japão/epidemiologia , Doenças das Aves Domésticas/virologia
14.
Ann Vasc Dis ; 13(4): 441-443, 2020 Dec 25.
Artigo em Inglês | MEDLINE | ID: mdl-33391567

RESUMO

This report describes a successful case of transcatheter arterial embolization for a critical vascular injury during lumbar disk surgery that resulted in a large retroperitoneal hematoma in a 72-year-old woman. A 4-Fr long sheath was inserted via the right popliteal artery in the prone position. Pelvic angiography revealed a pseudoaneurysm in the right internal iliac artery, which was managed with coil embolization. The patient underwent laparotomy because of abdominal compartment syndrome and was discharged in good condition after rehabilitation. The transpopliteal endovascular approach in the prone position may thus provide the best chance to treat this rare but critical condition.

15.
Proc Natl Acad Sci U S A ; 116(45): 22573-22582, 2019 11 05.
Artigo em Inglês | MEDLINE | ID: mdl-31591205

RESUMO

After synthesis of a short nascent RNA, RNA polymerase II (pol II) dissociates general transcription factors (GTFs; TFIIA, TFIIB, TBP, TFIIE, TFIIF, and TFIIH) and escapes the promoter, but many of the mechanistic details of this process remain unclear. Here we developed an in vitro transcription system from the yeast Saccharomyces cerevisiae that allows conversion of the preinitiation complex (PIC) to bona fide initially transcribing complex (ITC), elongation complex (EC), and reinitiation complex (EC+ITC). By biochemically isolating postinitiation complexes stalled at different template positions, we have determined the timing of promoter escape and the composition of protein complexes associated with different lengths of RNA. Almost all of the postinitiation complexes retained the GTFs when pol II was stalled at position +27 relative to the transcription start site, whereas most complexes had completed promoter escape when stalled at +49. This indicates that GTFs remain associated with pol II much longer than previously expected. Nevertheless, the long-persisting transcription complex containing RNA and all of the GTFs is unstable and is susceptible to extensive backtracking of pol II. Addition of the capping enzyme and/or Spt4/5 significantly increased the frequency of promoter escape as well as assembly of a follow-on PIC at the promoter for reinitiation. These data indicate that elongation factors play an important role in promoter escape and that ejection of TFIIB from the RNA exit tunnel of pol II by the growing nascent RNA is not sufficient to complete promoter escape.


Assuntos
Regiões Promotoras Genéticas , RNA Polimerase II/metabolismo , Saccharomyces cerevisiae/genética , Transcrição Genética , RNA Polimerase II/genética , RNA Fúngico/genética , RNA Fúngico/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Fatores de Elongação da Transcrição/genética , Fatores de Elongação da Transcrição/metabolismo
16.
J Vet Res ; 63(3): 369-373, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31572817

RESUMO

Introduction: The characteristics of immune factors in somatic cells from lactating dairy cows and their association with commensal bacteria in normal milk have not been clarified. This study investigated the relationship between the pathogenic bacteria in milk and somatic cell immune factors in healthy lactating cows. Material and Methods: In total 44 healthy Holstein cows were studied on one farm. Milk samples were collected aseptically using a cannula and these samples were cultured for detection of bacteria and analysis of mRNA of immune factors expressed by somatic cells. Cows were divided into two groups based on the microbial status of their milk samples: 12 cows showed bacteria in cultures (positive group), and the other 32 cows did not (negative group). Results: The mRNA levels of IL-6, lactotransferrin, and cathelicidin expressed by somatic cells after milking decreased significantly compared to those before milking in both groups (P < 0.05). There were significantly lower mRNA levels of IL-6 and cathelicidin in the positive group compared to those in the negative group before milking. Conclusion: These results suggest that mRNA levels of IL-6 and cathelicidin expressed by the somatic cells may be affected by the presence of bacteria in healthy lactating dairy cows.

17.
Curr Biol ; 29(16): 2625-2639.e5, 2019 08 19.
Artigo em Inglês | MEDLINE | ID: mdl-31353180

RESUMO

Centromeric nucleosomes are at the interface of the chromosome and the kinetochore that connects to spindle microtubules in mitosis. The core centromeric nucleosome complex (CCNC) harbors the histone H3 variant, CENP-A, and its binding proteins, CENP-C (through its central domain; CD) and CENP-N (through its N-terminal domain; NT). CENP-C can engage nucleosomes through two domains: the CD and the CENP-C motif (CM). CENP-CCD is part of the CCNC by virtue of its high specificity for CENP-A nucleosomes and ability to stabilize CENP-A at the centromere. CENP-CCM is thought to engage a neighboring nucleosome, either one containing conventional H3 or CENP-A, and a crystal structure of a nucleosome complex containing two copies of CENP-CCM was reported. Recent structures containing a single copy of CENP-NNT bound to the CENP-A nucleosome in the absence of CENP-C were reported. Here, we find that one copy of CENP-N is lost for every two copies of CENP-C on centromeric chromatin just prior to kinetochore formation. We present the structures of symmetric and asymmetric forms of the CCNC that vary in CENP-N stoichiometry. Our structures explain how the central domain of CENP-C achieves its high specificity for CENP-A nucleosomes and how CENP-C and CENP-N sandwich the histone H4 tail. The natural centromeric DNA path in our structures corresponds to symmetric surfaces for CCNC assembly, deviating from what is observed in prior structures using artificial sequences. At mitosis, we propose that CCNC asymmetry accommodates its asymmetric connections at the chromosome/kinetochore interface. VIDEO ABSTRACT.


Assuntos
Centrômero/ultraestrutura , Mitose/fisiologia , Nucleossomos/ultraestrutura , Proteínas Cromossômicas não Histona/ultraestrutura , Microscopia Crioeletrônica , Humanos
18.
Methods ; 159-160: 82-89, 2019 04 15.
Artigo em Inglês | MEDLINE | ID: mdl-30905750

RESUMO

Transcription initiation can be reconstituted from highly purified general transcription factors (GTFs), RNA polymerase II (pol II), and promoter DNA. However, earlier biochemical reconstitution systems had a serious technical limitation, namely very poor initiation efficiency. Due to the poor efficiency of the reaction and trace amounts of proteins involved in the pre-initiation complex (PIC) assembly, detection of transcription and PIC formation was only possible by the synthesis of a radiolabeled transcript and by immunoblotting for PIC components on templates. Here we describe a transcription system that is capable of initiating transcription with >90% efficiency of template usage using homogeneous, active yeast components including TFIIA, TFIIB, TBP, TFIIE, TFIIF, TFIIH, Sub1, and pol II. The abundant specifically assembled PICs on promoter DNA can be separated from free general transcription factors (GTFs) and pol II by density gradient sedimentation, irrespective of the length of promoter DNA. The system is robust, and can be modified to accommodate many other transcription factors, and the resulting complexes can be analyzed by SDS-PAGE followed by Coomassie Blue staining. This technical advance now paves the way to conduct definitive biochemical and structural studies of the complete process of pol II initiation from the PIC, through promoter escape, and finally to productive elongation.


Assuntos
Regiões Promotoras Genéticas , RNA Polimerase II/metabolismo , Fatores de Transcrição/metabolismo , Iniciação da Transcrição Genética , Complexos Multiproteicos , Proteínas de Saccharomyces cerevisiae/metabolismo , Fator de Transcrição TFIIA/metabolismo , Fator de Transcrição TFIIB/metabolismo , Fatores de Transcrição TFII/metabolismo , Leveduras/enzimologia , Leveduras/genética , Leveduras/metabolismo
19.
Vet Microbiol ; 229: 81-89, 2019 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-30642602

RESUMO

Five mutations involved in changing of susceptibility to lincosamides and/or macrolides were investigated in field isolates of Mycoplasma californicum in Japan, and reconfirmed in laboratory-derived mutants. In addition, a quick and easy detection method for these mutations was established. Guanine at position 748 (Escherichia coli numbering) of the 23S rRNA gene (rrl) was shown to be involved with decreased susceptibility to 16-membered macrolides, and adenines at positions 2059 and 2062 of rrl were involved with decreased susceptibility to both lincosamides and macrolides. Both guanine at position 2576, and change from cytosine to thymine at position 2611 of rrl were found to be involved with decreased susceptibility to lincosamides, and the latter mutation also increased the susceptibility to erythromycin. These mutations were easily induced by several to approximately 30 passages in a medium containing the respective antimicrobial, but they did not return after their initial appearance. The melting curve analysis using hybridization probes revealed the existence of these mutations by the change in the melting curve shape and/or decrease in the melting peak temperature. The detection limit in milk samples with a somatic cell count up to 716 × 103 cell/mL was 133 cfu/mL, but an excessive increase in the cell count in milk or storage of the milk sample at chilling or freezing temperature decreased the sensitivity. This method requires only a few hours, so field veterinarians can make a same-day determination of susceptibility to macrolides and lincosamides, which are first-line antibiotics for bovine mycoplasmal mastitis.


Assuntos
Antibacterianos/farmacologia , Farmacorresistência Bacteriana/genética , Lincosamidas/farmacologia , Macrolídeos/farmacologia , Mycoplasma/efeitos dos fármacos , Mycoplasma/genética , Animais , Bovinos , DNA Bacteriano/genética , Feminino , Japão/epidemiologia , Mastite Bovina/epidemiologia , Mastite Bovina/microbiologia , Leite/microbiologia , Mutação , Técnicas de Amplificação de Ácido Nucleico/veterinária
20.
J Shoulder Elbow Surg ; 28(1): 149-157, 2019 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-30241983

RESUMO

BACKGROUND: Although past studies using video analysis indicated that the arm tackle and head-in-front shoulder tackle are possible risks for shoulder dislocation, the underlying mechanisms of tackling-related shoulder dislocation have not been sufficiently investigated. This study aimed to analyze the kinematic aspects of these tackling motions in 1-on-1 tackles in an experimental setting using a 3-dimensional motion-capture system. METHODS: A total of 65 one-on-one tackles were recorded using a marker-based, automatic, digitizing motion-capture system. A documented tackle was classified into 1 of 3 types, which was decided based on the first point of contact on the ball carrier and the head position at the time of impact: shoulder tackle (reference tackle), arm tackle, and head-in-front tackle. The orientations of the head, trunk, and shoulder at impact were calculated and statistically compared with each other. RESULTS: The distribution of tackles recorded in this study was as follows: 38 shoulder, 23 arm, and 4 head-in-front tackles. In comparison with the shoulder tackle as a reference, shoulder abduction on the side of impact was higher in both the arm and head-in-front tackles, while shoulder external rotation was lower in the head-in-front tackles. In the latter type of tackle, significant decreases in neck extension and ipsilateral neck rotation were also indicated. CONCLUSION: The kinematics in both the arm tackle and the head-in-front tackle is significantly different from that in the shoulder tackle and may represent a distinct risk factor for shoulder dislocation.


Assuntos
Fenômenos Biomecânicos/fisiologia , Simulação por Computador , Futebol Americano/lesões , Imageamento Tridimensional , Humanos , Masculino , Luxação do Ombro/fisiopatologia , Adulto Jovem
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