Characterization of the proteasome from the extremely halophilic archaeonHaloarcula marismortui

A 20S proteasome, comprising two subunits α and β, was purified from the extreme halophilic archaeonHaloarcula marismortui, which grows only in saturated salt conditions. The three-dimensional reconstruction of theH. marismortuiproteasome (Hm proteasome), obtained from negatively stained electron micrographs, is virtually identical to the structure of a thermophilic proteasome filtered to the same resolution. The stability of the Hm proteasome was found to be less salt-dependent than that of other halophilic enzymes previously described. The proteolytic activity of the Hm proteasome was investigated using the malate dehydrogenase fromH. marismortui(HmMalDH) as a model substrate. The HmMalDH denatures when the salt concentration is decreased below 2 M. Under these conditions, the proteasome efficiently cleaves HmMalDH during its denaturation process, but the fully denatured HmMalDH is poorly degraded. These in vitro experiments show that, at low salt concentrations, the 20S proteasome from halophilic archaea eliminates a misfolded protein.

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In Vitro Selection and Characterization of Hepatitis C Virus Serine Protease Variants Resistant to an Active-Site Peptide Inhibitor

Journal of Virology ◽

10.1128/jvi.77.6.3669-3679.2003 ◽

2003 ◽

Vol 77 (6) ◽

pp. 3669-3679 ◽

Cited By ~ 85

Author(s):

Caterina Trozzi ◽

Linda Bartholomew ◽

Alessandra Ceccacci ◽

Gabriella Biasiol ◽

Laura Pacini ◽

...

Keyword(s):

Hepatitis C Virus ◽

Hepatitis C ◽

Serine Protease ◽

In Vitro Selection ◽

Three Dimensional ◽

Host Cells ◽

Dimensional Structure ◽

In Vitro Systems

ABSTRACT The hepatitis C virus (HCV) serine protease is necessary for viral replication and represents a valid target for developing new therapies for HCV infection. Potent and selective inhibitors of this enzyme have been identified and shown to inhibit HCV replication in tissue culture. The optimization of these inhibitors for clinical development would greatly benefit from in vitro systems for the identification and the study of resistant variants. We report the use HCV subgenomic replicons to isolate and characterize mutants resistant to a protease inhibitor. Taking advantage of the replicons' ability to transduce resistance to neomycin, we selected replicons with decreased sensitivity to the inhibitor by culturing the host cells in the presence of the inhibitor and neomycin. The selected replicons replicated to the same extent as those in parental cells. Sequence analysis followed by transfection of replicons containing isolated mutations revealed that resistance was mediated by amino acid substitutions in the protease. These results were confirmed by in vitro experiments with mutant enzymes and by modeling the inhibitor in the three-dimensional structure of the protease.

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X-ray topographic determination of the granular structure in a graphite mosaic crystal: a three-dimensional reconstruction

Journal of Applied Crystallography ◽

10.1107/s0021889800005975 ◽

2000 ◽

Vol 33 (4) ◽

pp. 1023-1030 ◽

Cited By ~ 13

Author(s):

M. Ohler ◽

M. Sanchez del Rio ◽

A. Tuffanelli ◽

M. Gambaccini ◽

A. Taibi ◽

...

Keyword(s):

Structural Parameters ◽

Pyrolytic Graphite ◽

Three Dimensional ◽

X Ray ◽

Dimensional Reconstruction ◽

Mosaic Crystals ◽

Spatial Locations ◽

Crystalline Domains

Section topographs recorded at different spatial locations and at different rocking angles of a highly oriented pyrolytic graphite (HOPG) crystal allow three-dimensional maps of the local angular-dependent scattering power to be obtained. This is performed with a direct reconstruction from the intensity distribution on such topographs. The maps allow the extraction of information on local structural parameters such as size, form and internal mosaic spread of crystalline domains. This data analysis leads to a new method for the characterization of mosaic crystals. Perspectives and limits of applicability of this method are discussed.

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Dysbindin deficiency Alters Cardiac BLOC-1 Complex and Myozap Levels in Mice

Cells ◽

10.3390/cells9112390 ◽

2020 ◽

Vol 9 (11) ◽

pp. 2390

Author(s):

Ankush Borlepawar ◽

Nesrin Schmiedel ◽

Matthias Eden ◽

Lynn Christen ◽

Alexandra Rosskopf ◽

...

Keyword(s):

Direct Interaction ◽

Cardiomyocyte Hypertrophy ◽

Loss Of Function ◽

Interaction Partners ◽

Lysosome Related Organelles ◽

The Stability ◽

Schizophrenia Susceptibility

Dysbindin, a schizophrenia susceptibility marker and an essential constituent of BLOC-1 (biogenesis of lysosome-related organelles complex-1), has recently been associated with cardiomyocyte hypertrophy through the activation of Myozap-RhoA-mediated SRF signaling. We employed sandy mice (Dtnbp1_KO), which completely lack Dysbindin protein because of a spontaneous deletion of introns 5–7 of the Dtnbp1 gene, for pathophysiological characterization of the heart. Unlike in vitro, the loss-of-function of Dysbindin did not attenuate cardiac hypertrophy, either in response to transverse aortic constriction stress or upon phenylephrine treatment. Interestingly, however, the levels of hypertrophy-inducing interaction partner Myozap as well as the BLOC-1 partners of Dysbindin like Muted and Pallidin were dramatically reduced in Dtnbp1_KO mouse hearts. Taken together, our data suggest that Dysbindin’s role in cardiomyocyte hypertrophy is redundant in vivo, yet essential to maintain the stability of its direct interaction partners like Myozap, Pallidin and Muted.

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Characterization of covalent protein modification by triclosan in vivo and in vitro via three-dimensional liquid chromatography-mass spectrometry: New insight into its adverse effects

Environment International ◽

10.1016/j.envint.2019.105423 ◽

2020 ◽

Vol 136 ◽

pp. 105423 ◽

Cited By ~ 1

Author(s):

Meixian Liu ◽

Na Li ◽

Yida Zhang ◽

Zhiyuan Zheng ◽

Yue Zhuo ◽

...

Keyword(s):

Mass Spectrometry ◽

Liquid Chromatography ◽

Adverse Effects ◽

Protein Modification ◽

Three Dimensional ◽

Liquid Chromatography Mass Spectrometry ◽

Insight Into

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Structural Characterization of the Cell Division Cycle in Strigomonas culicis, an Endosymbiont-Bearing Trypanosomatid

Microscopy and Microanalysis ◽

10.1017/s1431927613013925 ◽

2014 ◽

Vol 20 (1) ◽

pp. 228-237 ◽

Cited By ~ 6

Author(s):

Felipe Lopes Brum ◽

Carolina Moura Costa Catta-Preta ◽

Wanderley de Souza ◽

Sergio Schenkman ◽

Maria Carolina Elias ◽

...

Keyword(s):

Cell Cycle ◽

Host Cell ◽

Light And Electron Microscopy ◽

Three Dimensional ◽

Symbiotic Bacterium ◽

Single Copy ◽

Cell Structures ◽

Dimensional Reconstruction ◽

Protozoan Cell

AbstractStrigomonas culicis (previously referred to as Blastocrithidia culicis) is a monoxenic trypanosomatid harboring a symbiotic bacterium, which maintains an obligatory relationship with the host protozoan. Investigations of the cell cycle in symbiont harboring trypanosomatids suggest that the bacterium divides in coordination with other host cell structures, particularly the nucleus. In this study we used light and electron microscopy followed by three-dimensional reconstruction to characterize the symbiont division during the cell cycle of S. culicis. We observed that during this process, the symbiotic bacterium presents different forms and is found at different positions in relationship to the host cell structures. At the G1/S phase of the protozoan cell cycle, the endosymbiont exhibits a constricted form that appears to elongate, resulting in the bacterium division, which occurs before kinetoplast and nucleus segregation. During cytokinesis, the symbionts are positioned close to each nucleus to ensure that each daughter cell will inherit a single copy of the bacterium. These observations indicated that the association of the bacterium with the protozoan nucleus coordinates the cell cycle in both organisms.

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Chapter 17 Electron Microscopy of Collagen Fibril Structure In Vitro and In Vivo Including Three-Dimensional Reconstruction

Methods in Cell Biology - Introduction to Electron Microscopy for Biologists ◽

10.1016/s0091-679x(08)00417-2 ◽

2008 ◽

pp. 319-345 ◽

Cited By ~ 29

Author(s):

Tobias Starborg ◽

Yinhui Lu ◽

Karl E. Kadler ◽

David F. Holmes

Keyword(s):

Electron Microscopy ◽

Collagen Fibril ◽

Three Dimensional ◽

Three Dimensional Reconstruction ◽

Fibril Structure ◽

Dimensional Reconstruction

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Applicability and Limitations in the Characterization of Poly-Dispersed Engineered Nanomaterials in Cell Media by Dynamic Light Scattering (DLS)

Materials ◽

10.3390/ma12233833 ◽

2019 ◽

Vol 12 (23) ◽

pp. 3833 ◽

Cited By ~ 2

Author(s):

Arianna Marucco ◽

Elisabetta Aldieri ◽

Riccardo Leinardi ◽

Enrico Bergamaschi ◽

Chiara Riganti ◽

...

Keyword(s):

Light Scattering ◽

Dynamic Light Scattering ◽

Culture Media ◽

Raw 264.7 ◽

Pre Treatment ◽

The Stability ◽

Definition Of ◽

Cellular Tests

The dispersion protocol used to administer nanomaterials (NMs) in in vitro cellular tests might affect their toxicity. For this reason, several dispersion procedures have been proposed to harmonize the toxicological methods, allowing for the comparison of the data that were obtained by different laboratories. At the same time, several techniques and methods are available to monitor the identity of the NMs in the cell media. However, while the characterization of suspensions of engineered NMs having narrow size distribution may be easily performed, the description of aggregated NMs forming polydispersions is still challenging. In the present study, sub-micrometric/nanometric TiO2, SiO2, and CeO2 were dispersed in cell media by using two different dispersion protocols, with and without albumin (0.5%) and with different sonication procedures. Dynamic Light Scattering (DLS) was used to characterize NMs in stock solutions and culture media. Pitfalls that affect DLS measurements were identified and, guidance on a critical analysis of the results provided. The NMs were then tested for their cytotoxicity (LDH leakage) toward murine macrophages (RAW 264.7) and PMA-activated human monocytes (THP-1). As markers of pro-inflammatory response, nitric oxide (NO) and cytokine IL-1β production were measured on RAW 264.7 and THP-1 cells, respectively. The pre-treatment with albumin added to a strong sonication treatment increases the stability and homogeneity of the suspensions of nanometric samples, but not of the submicrometric-samples. Nevertheless, while TiO2 and CeO2 were non-cytotoxic in any conditions, differences in cytotoxicity, NO, and IL-1β releases were found for the SiO2, depending upon the protocol. Overall, the results suggest that there is no one-fits-all method valid for all NMs, since each class of NMs respond differently. The definition of validated procedures and parameters for the selection of the most appropriate method of dispersion for each class of NM appears to be a more efficacious strategy for the harmonization of the dispersion protocols.

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A Direct and Quantitative Three-Dimensional Reconstruction of the Internal Structure of Disordered Mesoporous Carbon with Tailored Pore Size

Microscopy and Microanalysis ◽

10.1017/s1431927613000238 ◽

2013 ◽

Vol 19 (3) ◽

pp. 745-750 ◽

Cited By ~ 9

Author(s):

Juan Balach ◽

Flavio Soldera ◽

Diego F. Acevedo ◽

Frank Mücklich ◽

César A. Barbero

Keyword(s):

Pore Size ◽

Focused Ion Beam ◽

Ion Beam ◽

Three Dimensional ◽

Quantitative Characterization ◽

Resorcinol Formaldehyde ◽

Dual Beam ◽

Dimensional Reconstruction ◽

A New Technique

AbstractA new technique that allows direct three-dimensional (3D) investigations of mesopores in carbon materials and quantitative characterization of their physical properties is reported. Focused ion beam nanotomography (FIB-nt) is performed by a serial sectioning procedure with a dual beam FIB-scanning electron microscopy instrument. Mesoporous carbons (MPCs) with tailored mesopore size are produced by carbonization of resorcinol-formaldehyde gels in the presence of a cationic surfactant as a pore stabilizer. A visual 3D morphology representation of disordered porous carbon is shown. Pore size distribution of MPCs is determined by the FIB-nt technique and nitrogen sorption isotherm methods to compare both results. The obtained MPCs exhibit pore sizes of 4.7, 7.2, and 18.3 nm, and a specific surface area of ca. 560 m2/g.

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Synthesis and Characterization of Novel Copolymeric Resveratrol Conjugates

Journal of Chemistry ◽

10.1155/2015/245625 ◽

2015 ◽

Vol 2015 ◽

pp. 1-6 ◽

Cited By ~ 4

Author(s):

Yan-Jing Ng ◽

Heather A. E. Benson ◽

David H. Brown ◽

Yan Chen

Keyword(s):

Micelle Formation ◽

Rat Plasma ◽

The Body ◽

Poly Ethylene Glycol ◽

Poly Ethylene ◽

The Stability ◽

Chain Lengths ◽

Cardioprotective Drug

Resveratrol (RSV), naturally found in plants, is known to have health benefits and has been proposed as a potential anticancer and cardioprotective drug. However, due to its molecular structure, it undergoes rapid metabolism in the body resulting in low bioavailability. Novel polymeric methoxy-poly(ethylene glycol)-block-poly(ε-caprolactone) (mPEG-PCL) RSV conjugates with varying PCL chain lengths have been synthesised and formulated into micelles and/or nanoparticles for preliminaryin vitrostability studies. RSV conjugated with mPEG2000-PCL9500 was found to have improved solubility and stability of RSV as compared to RSV alone. The length of the PCL chain was found to affect the micelle formation, hence the stability in physiological buffers and rat plasma.

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