scholarly journals 3.7 Å cryo-EM structure of the core Centromere Binding Factor 3 complex

2017 ◽  
Author(s):  
Wenjuan Zhang ◽  
Natalya Lukoynova ◽  
Shomon Miah ◽  
Cara K Vaughan
Keyword(s):  
Atomic Structure ◽  
The Other ◽  
In Vitro Assays ◽  
Core Complex ◽  
The Third ◽  
The Core ◽  
Binding Factor

SummaryThe Centromere Binding Factor 3 (CBF3) complex binds the third Centromere DNA Element in organisms with point centromeres, such as S. cerevisiae. It is the only essential centromere binding complex as it facilitates genetic specification of point centromeres. It is therefore the most fundamental complex of the kinetochore in these organisms and its association with centromere DNA allows association of all other kinetochore components. We have determined the atomic structure of the core complex of CBF3, comprising 3 of its 4 components, using cryo-EM. The architecture of the complex is 'U'-shaped, with a deep, strongly basic channel that is narrow at one end and wide at the other. Combining our structure and in vitro assays, we present a model for its association with centromere DNA.

scholarly journals Determination of functional domains in the C subunit of the CCAAT-binding factor (CBF) necessary for formation of a CBF-DNA complex: CBF-B interacts simultaneously with both the CBF-A and CBF-C subunits to form a heterotrimeric CBF molecule.

1996 ◽  
Vol 16 (8) ◽  
pp. 4003-4013 ◽  
Author(s):  
I S Kim ◽  
S Sinha ◽  
B de Crombrugghe ◽  
S N Maity
Keyword(s):  
Mutational Analysis ◽  
The Other ◽  
In Vitro Assays ◽  
Histone H2a ◽  
Interaction Domain ◽  
Histone Fold ◽  
Binding Factor ◽  
Dna Complex

The mammalian CCAAT-binding factor (CBF; also called NF-Y and CP1) is a heterotrimeric protein consisting of three subunits, CBF-A, CBF-B, and CBF-C, all of which are required for DNA binding and all of which are present in the CBF-DNA complex. In this study using cross-linking and immunoprecipitation methods, we first established that CBF-B interacts simultaneously with both subunits of the CBF-A-CBF-C heterodimer to form a heterotrimeric CBF molecule. We then performed a mutational analysis of CBF-C to define functional interactions with the other two CBF subunits and with DNA using several in vitro assays and an in vivo yeast two-hybrid system. Our experiments established that the evolutionarily conserved segment of CBF-C, which shows similarities with the histone-fold motif of histone H2A, was necessary for formation of the CBF-DNA complex. The domain of CBF-C which interacts with CBF-A included a large portion of this segment, one that corresponds to the segment of the histone-fold motif in H2A used for interaction with H2B. Two classes of interactions involved in formation of the CBF-A-CBF-C heterodimer were detected; one class, provided by residues in the middle of the interaction domain, was needed for formation of the CBF-A-CBF-C heterodimer. The other, provided by sequences flanking those of the first class was needed for stabilization of the heterodimer. Two separate domains were identified in the conserved segment of CBF-C for interaction with CBF-B; these were located on each side of the CBF-A interaction domain. Since our previous experiments identified a single CBF-B interaction domain in the histone-fold motif of CBF-A, we propose that a tridentate interaction domain in the CBF-A-CBF-C heterodimer interacts with the 21-amino-acid-long subunit interaction domain of CBF-B. Together with our previous mutational analysis of CBF-A (S. Sinha, I.-S. Kim, K.-Y. Sohn, B. de Crombrugghe, and S. N. Maity, Mol. Cell. Biol. 16:328-337, 1996), this study demonstrates that the histone fold-motifs of CBF-A and CBF-C interact with each other to form the CBF-A-CBF-C heterodimer and generate a hybrid surface which then interacts with CBF-B to form the heterotrimeric CBF molecule.

scholarly journals Comparison of Four SARS-CoV-2 Neutralization Assays

Vaccines ◽  
2020 ◽  
Vol 9 (1) ◽  
pp. 13
Author(s):  
Lydia Riepler ◽  
Annika Rössler ◽  
Albert Falch ◽  
André Volland ◽  
Wegene Borena ◽  
...  
Keyword(s):  
Vesicular Stomatitis Virus ◽  
Neutralizing Antibodies ◽  
The Other ◽  
In Vitro Assays ◽  
The Novel ◽  
Effective Protection ◽  
Vaccine Candidates ◽  
Vesicular Stomatitis ◽  
Novel Coronavirus

Neutralizing antibodies are a major correlate of protection for many viruses including the novel coronavirus SARS-CoV-2. Thus, vaccine candidates should potently induce neutralizing antibodies to render effective protection from infection. A variety of in vitro assays for the detection of SARS-CoV-2 neutralizing antibodies has been described. However, validation of the different assays against each other is important to allow comparison of different studies. Here, we compared four different SARS-CoV-2 neutralization assays using the same set of patient samples. Two assays used replication competent SARS-CoV-2, a focus forming assay and a TCID50-based assay, while the other two assays used replication defective lentiviral or vesicular stomatitis virus (VSV)-based particles pseudotyped with SARS-CoV-2 spike. All assays were robust and produced highly reproducible neutralization titers. Titers of neutralizing antibodies correlated well between the different assays and with the titers of SARS-CoV-2 S-protein binding antibodies detected in an ELISA. Our study showed that commonly used SARS-CoV-2 neutralization assays are robust and that results obtained with different assays are comparable.

scholarly journals Waktu Inkubasi pada Derajat Distilasi Kitosan Enzim dan Efektifitas Penghambatannya terhadap Penyakit Antraknosa

2017 ◽  
Vol 12 (6) ◽  
pp. 209
Author(s):  
Yadi Suryadi ◽  
Tri Puji Priyatno ◽  
I Made Samudra ◽  
Dwi Ningsih Susilowati ◽  
Hermawati Nurzulaika ◽  
...  
Keyword(s):  
Spore Germination ◽  
Burkholderia Cepacia ◽  
Fungal Growth ◽  
The Other ◽  
In Vitro Assays ◽  
Incubation Condition ◽  
Coating Agent ◽  
Optimal Incubation

The use of chitosan as a coating agent of harvested fruits is an alternative method in controlling anthracnose disease (Colletotrichum sp.). This study aimed to obtain an optimal enzymatic chitosan (EC) that hydrolyzed using chitinase from Burkholderia cepacia isolate E76. The optimal incubation condition to produce EC was 2 h with the yield of 3.52 ± 0.38 g. The degree of deacetylation (DD) chitosan and  EC was 66.91% and 80.91%, respectively. Based on in vitro assays, EC 2% was the most effective in inhibiting the growth of Colletotrichum sp. (94.22%)  than chitosan, while the highest inhibition for chitosan 3% was 55.26%. Moreover, the EC 2% showed the highest inhibition of spore germination (74.12%). The in vivo assay revealed that EC 2% showed the highest inhibition on the fungal growth (88.88%), compared to the other concentrations. On the other hand, the EC 2% and 3% gave similar results on inhibition of Colletotrichum sp.of chili (55.55%). 

scholarly journals Assessing Democracy In Vitro, In Vivo, and In Actu and the Role of Democratic Theory Today

2019 ◽  
Vol 6 (2) ◽  
pp. 70-84
Author(s):  
Anastasia Deligiaouri ◽  
Jane Suiter
Keyword(s):  
Democratic Theory ◽  
Discourse Theory ◽  
Second Step ◽  
The Third ◽  
The Core ◽  
Ongoing Project

How can we define democracy today given the continuous changes that modern societies are undergoing? What is the role of a democratic theorist? This paper articulates a threefold argument in responding to these questions by analyzing the term of democracy in vitro, in vivo, and in actu. The first step is to secure a democratic minimum and the core principles of democracy. The second step involves studying democracy as an ongoing project and examining how the principles of this democratic minimum are encoded. In the third step we deploy the basic premises of discourse theory of Laclau and Mouffe when evaluating a specific discourse of democracy, as this approach encompasses both discursive and nondiscursive practices. Utilizing this three-level evaluative framework for democratic theory will allow us to not only articulate normative principles but also evaluate them according to their mode of implementation.

scholarly journals SAGA–CORE subunit Spt7 is required for correct Ubp8 localization, chromatin association and deubiquitinase activity

2020 ◽  
Vol 13 (1) ◽  
Author(s):  
Carme Nuño-Cabanes ◽  
Varinia García-Molinero ◽  
Manuel Martín-Expósito ◽  
María-Eugenia Gas ◽  
Paula Oliete-Calvo ◽  
...  
Keyword(s):  
Eukaryotic Cells ◽  
In Vitro Assays ◽  
Saga Complex ◽  
Wild Type ◽  
Core Complex ◽  
Independent Manner ◽  
Tetrameric Structure ◽  
Core Components

Abstract Background Histone H2B deubiquitination is performed by numerous deubiquitinases in eukaryotic cells including Ubp8, the catalytic subunit of the tetrameric deubiquitination module (DUBm: Ubp8; Sus1; Sgf11; Sgf73) of the Spt-Ada-Gcn5 acetyltransferase (SAGA). Ubp8 is linked to the rest of SAGA through Sgf73 and is activated by the adaptors Sus1 and Sgf11. It is unknown if DUBm/Ubp8 might also work in a SAGA-independent manner. Results Here we report that a tetrameric DUBm is assembled independently of the SAGA–CORE components SPT7, ADA1 and SPT20. In the absence of SPT7, i.e., independent of the SAGA complex, Ubp8 and Sus1 are poorly recruited to SAGA-dependent genes and to chromatin. Notably, cells lacking Spt7 or Ada1, but not Spt20, show lower levels of nuclear Ubp8 than wild-type cells, suggesting a possible role for SAGA–CORE subunits in Ubp8 localization. Last, deletion of SPT7 leads to defects in Ubp8 deubiquitinase activity in in vivo and in vitro assays. Conclusions Collectively, our studies show that the DUBm tetrameric structure can form without a complete intact SAGA–CORE complex and that it includes full-length Sgf73. However, subunits of this SAGA–CORE influence DUBm association with chromatin, its localization and its activity.

Investigation of Aminomethyl Indole Derivatives as Hyaluronidase Inhibitors

2010 ◽  
Vol 65 (7-8) ◽  
pp. 445-450 ◽  
Author(s):  
Süreyya Ölgen ◽  
Andre Kaessler ◽  
Zühal Zühal Kılıç-Kurt ◽  
Joachim Jose
Keyword(s):  
Bacterial Infections ◽  
The Other ◽  
In Vitro Assays ◽  
Indole Derivatives ◽  
Malignant Tumours ◽  
Lipophilic Compounds ◽  
Hyaluronidase Activity ◽  
Therapeutic Value

Hyaluronidase inhibitors are of potential therapeutic value for the treatment of a variety of diseases, such as cancer, arthrosis, or bacterial infections. Potent and selective hyaluronidase inhibitors are not known so far, and current approaches to the development of hyaluronidase inhibitors are limited. Elevated levels of hyaluronan (HA) are connected with most malignant tumours. Therefore, the search for drugs modulating the hyaluronidase activity became very important. In the present study, a new series of aminomethyl indole derivatives (AMIDs) were tested for inhibition of bovine testes hyaluronidase (BTH). In vitro assays were performed using stains-all at pH 7 and Morgan-Elson reaction at pH 3.5. Among the AMIDs, 3-[(4-methylpiperazin-1-yl)methyl]-5-phenyl-1H-indole (9) was found to be active with 23% inhibition at 50 μM and pH 7. All the other inhibitors showed less activity at pH 3.5 and pH 7. These activity results demonstrated that compounds with phenyl substitution at position 5 have higher activity. The results confirmed that more lipophilic compounds have better inhibition against the hyaluronidase enzyme.

scholarly journals IOP1 Protein Is an External Component of the Human Cytosolic Iron-Sulfur Cluster Assembly (CIA) Machinery and Functions in the MMS19 Protein-dependent CIA Pathway

2013 ◽  
Vol 288 (23) ◽  
pp. 16680-16689 ◽  
Author(s):  
Mineaki Seki ◽  
Yukiko Takeda ◽  
Kazuhiro Iwai ◽  
Kiyoji Tanaka
Keyword(s):  
Cluster Assembly ◽  
Core Complex ◽  
Core Component ◽  
Iron Sulfur Cluster ◽  
Sulfur Cluster ◽  
The Core ◽  
Iron Sulfur ◽  
External Component

The emerging link between iron metabolism and genome integrity is increasingly clear. Recent studies have revealed that MMS19 and cytosolic iron-sulfur cluster assembly (CIA) factors form a complex and have central roles in CIA pathway. However, the composition of the CIA complex, particularly the involvement of the Fe-S protein IOP1, is still unclear. The roles of each component are also largely unknown. Here, we show that MMS19, MIP18, and CIAO1 form a tight “core” complex and that IOP1 is an “external” component of this complex. Although IOP1 and the core complex form a complex both in vivo and in vitro, IOP1 behaves differently in vivo. A deficiency in any core component leads to down-regulation of all of the components. In contrast, IOP1 knockdown does not affect the level of any core component. In MMS19-overproducing cells, other core components are also up-regulated, but the protein level of IOP1 remains unchanged. IOP1 behaves like a target protein in the CIA reaction, like other Fe-S helicases, and the core complex may participate in the maturation process of IOP1. Alternatively, the core complex may catch and hold IOP1 when it becomes mature to prevent its degradation. In any case, IOP1 functions in the MMS19-dependent CIA pathway. We also reveal that MMS19 interacts with target proteins. MIP18 has a role to bridge MMS19 and CIAO1. CIAO1 also binds IOP1. Based on our in vivo and in vitro data, new models of the CIA machinery are proposed.

scholarly journals A Dynamic Scaffold of Pre-snoRNP Factors Facilitates Human Box C/D snoRNP Assembly

2007 ◽  
Vol 27 (19) ◽  
pp. 6782-6793 ◽  
Author(s):  
Kenneth Scott McKeegan ◽  
Charles Maurice Debieux ◽  
Séverine Boulon ◽  
Edouard Bertrand ◽  
Nicholas James Watkins
Keyword(s):  
Protein Interactions ◽  
Core Protein ◽  
The Novel ◽  
Protein Protein Interactions ◽  
Core Complex ◽  
The Core ◽  
Snornp Biogenesis ◽  
The Individual ◽  
Vitro Protein

ABSTRACT The box C/D small nucleolar RNPs (snoRNPs) are essential for the processing and modification of rRNA. The core box C/D proteins are restructured during human U3 box C/D snoRNP biogenesis; however, the molecular basis of this is unclear. Here we show that the U8 snoRNP is also restructured, suggesting that this may occur with all box C/D snoRNPs. We have characterized four novel human biogenesis factors (BCD1, NOP17, NUFIP, and TAF9) which, along with the ATPases TIP48 and TIP49, are likely to be involved in the formation of the pre-snoRNP. We have analyzed the in vitro protein-protein interactions between the assembly factors and core box C/D proteins. Surprisingly, this revealed few interactions between the individual core box C/D proteins. However, the novel biogenesis factors and TIP48 and TIP49 interacted with one or more of the core box C/D proteins, implying that they mediate the assembly of the pre-snoRNP. Consistent with this, we show that NUFIP bridges interactions between the core box C/D proteins in a partially reconstituted pre-snoRNP. Restructuring of the core complex probably reflects the conversion of the pre-snoRNP, where core protein-protein interactions are maintained by the bridging biogenesis factors, to the mature snoRNP.

scholarly journals Mycotic Keratitis Due to Curvularia senegalensis and In Vitro Antifungal Susceptibilities ofCurvularia spp.

1999 ◽  
Vol 37 (12) ◽  
pp. 4170-4173 ◽  
Author(s):  
Josep Guarro ◽  
Tiyomi Akiti ◽  
Roberto Almada- Horta ◽  
L. A. Morizot Leite-Filho ◽  
Josepa Gené ◽  
...  
Keyword(s):  
Amphotericin B ◽  
Rare Species ◽  
Antifungal Agents ◽  
The Other ◽  
Corneal Transplant ◽  
Mycotic Keratitis ◽  
The Third ◽  
Fungal Hyphae ◽  
Highly Effective

A case of mycotic keratitis due to Curvularia senegalensis is reported. This case represents the third known reported infection caused by this rare species. Fungal hyphae were detected in corneal scrapings, and repeated cultures were positive for this fungi. The patient was presumed cured after a corneal transplant and treatment with itraconazole, but the infection recurred and the patient is waiting for a keratoplasty. The in vitro antifungal susceptibilities of the case strain and another 24 strains belonging to seven species of Curvularia were tested for six antifungal agents. With the exception of flucytosine, and occasionally fluconazole, the other drugs assayed (amphotericin B, miconazole, itraconazole, and ketoconazole) were highly effective in vitro.

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