scholarly journals Thermodynamics of protein destabilization in live cells

2015 ◽  
Vol 112 (40) ◽  
pp. 12402-12407 ◽  
Author(s):  
Jens Danielsson ◽  
Xin Mu ◽  
Lisa Lang ◽  
Huabing Wang ◽  
Andres Binolfi ◽  
...  
Keyword(s):  
Self Organization ◽  
Live Cells ◽  
Heat Capacity Change ◽  
Bacterial Cells ◽  
Crowding Effects ◽  
Capacity Change ◽  
The Individual ◽  
Cellular Components

Although protein folding and stability have been well explored under simplified conditions in vitro, it is yet unclear how these basic self-organization events are modulated by the crowded interior of live cells. To find out, we use here in-cell NMR to follow at atomic resolution the thermal unfolding of a β-barrel protein inside mammalian and bacterial cells. Challenging the view from in vitro crowding effects, we find that the cells destabilize the protein at 37 °C but with a conspicuous twist: While the melting temperature goes down the cold unfolding moves into the physiological regime, coupled to an augmented heat-capacity change. The effect seems induced by transient, sequence-specific, interactions with the cellular components, acting preferentially on the unfolded ensemble. This points to a model where the in vivo influence on protein behavior is case specific, determined by the individual protein’s interplay with the functionally optimized “interaction landscape” of the cellular interior.

scholarly journals Self-organization of keratin intermediate filaments into cross-linked networks

2009 ◽  
Vol 186 (3) ◽  
pp. 409-421 ◽  
Author(s):  
Chang-Hun Lee ◽  
Pierre A. Coulombe
Keyword(s):  
Epithelial Cells ◽  
Self Organization ◽  
Live Cells ◽  
Type I ◽  
Filamentous Actin ◽  
Basal Progenitor ◽  
Filament Bundles ◽  
10 Nm Filaments

Keratins, the largest subgroup of intermediate filament (IF) proteins, form a network of 10-nm filaments built from type I/II heterodimers in epithelial cells. A major function of keratin IFs is to protect epithelial cells from mechanical stress. Like filamentous actin, keratin IFs must be cross-linked in vitro to achieve the high level of mechanical resilience characteristic of live cells. Keratins 5 and 14 (K5 and K14), the main pairing occurring in the basal progenitor layer of epidermis and related epithelia, can readily self-organize into large filament bundles in vitro and in vivo. Here, we show that filament self-organization is mediated by multivalent interactions involving distinct regions in K5 and K14 proteins. Self-organization is determined independently of polymerization into 10-nm filaments, but involves specific type I–type II keratin complementarity. We propose that self-organization is a key determinant of the structural support function of keratin IFs in vivo.

scholarly journals Transcription-dependent confined diffusion of enzymes within subcellular spaces of the bacterial cytoplasm

BMC Biology ◽  
2021 ◽  
Vol 19 (1) ◽  
Author(s):  
Daniel A. O. Rotter ◽  
Christoph Heger ◽  
Luis M. Oviedo-Bocanegra ◽  
Peter L. Graumann
Keyword(s):  
Single Cells ◽  
Single Particle Tracking ◽  
Live Cells ◽  
Fast Diffusion ◽  
Bacterial Cells ◽  
Substrate Channeling ◽  
Active Transcription ◽  
Bacterial Cytoplasm

Abstract Background Knowledge on the localization and mobility of enzymes inside bacterial cells is scarce, but important for understanding spatial regulation of metabolism. The four central enzymes (Rib enzymes) of the riboflavin (RF) biosynthesis pathway in the Gram positive model bacterium Bacillus subtilis have been studied extensively in vitro, especially the heavy RF synthase, a large protein complex with a capsid structure formed by RibH and an encapsulated RibE homotrimer, which mediates substrate-channeling. However, little is known about the behavior and mobility of these enzymes in vivo. Results We have investigated the localization and diffusion of the Rib enzymes in the cytoplasm of B. subtilis. By characterizing the diffusion of Rib enzymes in live cells using single particle tracking (SPT) we provide evidence for confined diffusion at the cell poles and otherwise Brownian motion. A majority of RibH particles showed clear nucleoid occlusion and a high degree of confined motion, which is largely abolished after treatment with Rifampicin, revealing that confinement is dependent on active transcription. Contrarily, RibE is mostly diffusive within the cell, showing only 14% encapsulation by RibH nanocompartments. By localizing different diffusive populations within single cells, we find that fast diffusion occurs mostly across the nucleoids located in the cell centers, while the slower, confined subdiffusion occurs at the crowded cell poles. Conclusions Our results provide evidence for locally different motion of active enzymes within the bacterial cytoplasm, setting up metabolic compartmentalization mostly at the poles of cells.

scholarly journals Tracking of quiescence inLeishmaniaby quantifying the expression of GFP in the ribosomal DNA locus

2019 ◽  
Author(s):  
Marlene Jara ◽  
Ilse Maes ◽  
Hideo Imamura ◽  
Malgorzata A. Domagalska ◽  
Jean Claude Dujardin ◽  
...  
Keyword(s):  
Ribosomal Dna ◽  
Rdna Locus ◽  
Live Cells ◽  
Brdu Incorporation ◽  
Reference Laboratory ◽  
Latent Infections ◽  
The Individual ◽  
First Time

ABSTRACTUnder stressful conditions some microorganisms adopt a reversible non or slow proliferative quiescent stage that allows their survival. Although quiescence has been described broadly in bacteria, this phenotype has been only recently discovered inLeishmania. In the present work we developed a biosensor of quiescence that allows to monitor the physiological stage of the parasite at population and single cell levels. We inserted a GFP gene into the ribosomal DNA locus and followed the expression of this reporter gene, driven by the ribosomal promotor (rGFP expression). We showed that rGFP expression decreased significantly and rapidly during thein vitrotransition from extracellular promastigotes to intracellular amastigotes ofL. mexicanaanL. braziliensisand that the decrease in rGFP expression was coupledin vitrowith a decrease in replication as measured by BrdU incorporation. Quiescence was not only observed in reference laboratory strains, but also among clinical isolates. We found that quiescence was reversible as the parasites could rapidly resume their metabolically active and proliferative stage when they were put back in an optimal environment for growth. We demonstrated for the first time in live cells that amastigotes are a heterogeneous population in which shallow and deep quiescent stages may coexist. Finally, we showed that rGFP expression could be monitoredin vivoand that quiescent amastigotes could reside in tissues of animals with latent infections ofL. braziliensisorL. mexicana. We propose rGFP expression as a simple parameter to define quiescent cells and further characterize them.IMPORTANCEQuiescence is a physiological diversification that allows pathogens to overcome chemotherapy without the development of drug resistance and to be invisible to the immune system of their host. Quiescent pathogens can cause latent infections and (re-) emerge in an unpredictable time during the lifetime of the individual. The phenomenon was recently described inLeishmaniain which it could explain several clinical and sub-clinical features, like therapeutic failure, reactivation of the disease and asymptomatic infections. However, a simple biosensor of quiescence forLeishmaniais not yet available. We show for the first time that the integration of GFP within the rDNA locus and the subsequent quantification of its expression can be used as a biosensor to distinguish quiescent subpopulations among live amastigotes. Moreover, we show quiescence is quickly reversible bothin vivoandin vitro. We offer a tool that will allow the further molecular characterization of quiescent parasites.

Multi-mode light microscopy of microtubule assembly dynamics and chromosome movement in vivo and In vitro

1996 ◽  
Vol 54 ◽  
pp. 730-731
Author(s):  
E. D. Salmon ◽  
J. C. Waters ◽  
C. Waterman-Storer
Keyword(s):  
Imaging System ◽  
Ccd Camera ◽  
Transmitted Light ◽  
Microtubule Assembly ◽  
Live Cells ◽  
Optical Efficiency ◽  
Multiple Band ◽  
Multi Mode

We have developed a multi-mode digital imaging system which acquires images with a cooled CCD camera (Figure 1). A multiple band pass dichromatic mirror and robotically controlled filter wheels provide wavelength selection for epi-fluorescence. Shutters select illumination either by epi-fluorescence or by transmitted light for phase contrast or DIC. Many of our experiments involve investigations of spindle assembly dynamics and chromosome movements in live cells or unfixed reconstituted preparations in vitro in which photodamage and phototoxicity are major concerns. As a consequence, a major factor in the design was optical efficiency: achieving the highest image quality with the least number of illumination photons. This principle applies to both epi-fluorescence and transmitted light imaging modes. In living cells and extracts, microtubules are visualized using X-rhodamine labeled tubulin. Photoactivation of C2CF-fluorescein labeled tubulin is used to locally mark microtubules in studies of microtubule dynamics and translocation. Chromosomes are labeled with DAPI or Hoechst DNA intercalating dyes.

scholarly journals Preclinical Testing of Radiopharmaceuticals for the Detection and Characterization of Osteomyelitis: Experiences from a Porcine Model

Molecules ◽  
2021 ◽  
Vol 26 (14) ◽  
pp. 4221
Author(s):  
Aage Kristian Olsen Alstrup ◽  
Svend Borup Jensen ◽  
Ole Lerberg Nielsen ◽  
Lars Jødal ◽  
Pia Afzelius
Keyword(s):  
Animal Model ◽  
Animal Models ◽  
Porcine Model ◽  
Animal Experiments ◽  
Radioactive Tracers ◽  
The Individual ◽  
Selection Of

The development of new and better radioactive tracers capable of detecting and characterizing osteomyelitis is an ongoing process, mainly because available tracers lack selectivity towards osteomyelitis. An integrated part of developing new tracers is the performance of in vivo tests using appropriate animal models. The available animal models for osteomyelitis are also far from ideal. Therefore, developing improved animal osteomyelitis models is as important as developing new radioactive tracers. We recently published a review on radioactive tracers. In this review, we only present and discuss osteomyelitis models. Three ethical aspects (3R) are essential when exposing experimental animals to infections. Thus, we should perform experiments in vitro rather than in vivo (Replacement), use as few animals as possible (Reduction), and impose as little pain on the animal as possible (Refinement). The gain for humans should by far exceed the disadvantages for the individual experimental animal. To this end, the translational value of animal experiments is crucial. We therefore need a robust and well-characterized animal model to evaluate new osteomyelitis tracers to be sure that unpredicted variation in the animal model does not lead to a misinterpretation of the tracer behavior. In this review, we focus on how the development of radioactive tracers relies heavily on the selection of a reliable animal model, and we base the discussions on our own experience with a porcine model.

scholarly journals Insights in Behavior of Variably Formulated Alginate-Based Microcapsules for Cell Transplantation

2015 ◽  
Vol 2015 ◽  
pp. 1-11 ◽  
Author(s):  
Pia Montanucci ◽  
Silvia Terenzi ◽  
Claudio Santi ◽  
Ilaria Pennoni ◽  
Vittorio Bini ◽  
...  
Keyword(s):  
Divalent Cations ◽  
Chemical Properties ◽  
Live Cells ◽  
High Performing ◽  
Physical Chemical ◽  
Degenerative Disorders ◽  
Selection Of ◽  
Better Than

Alginate-based microencapsulation of live cells may offer the opportunity to treat chronic and degenerative disorders. So far, a thorough assessment of physical-chemical behavior of alginate-based microbeads remains cloudy. A disputed issue is which divalent cation to choose for a high performing alginate gelling process. Having selected, in our system, high mannuronic (M) enriched alginates, we studied different gelling cations and their combinations to determine their eventual influence on physical-chemical properties of the final microcapsules preparation,in vitroandin vivo. We have shown that used of ultrapure alginate allows for high biocompatibility of the formed microcapsules, regardless of gelation agents, while use of different gelling cations is associated with corresponding variable effects on the capsules’ basic architecture, as originally reported in this work. However, only the final application which the capsules are destined to will ultimately guide the selection of the ideal, specific gelling divalent cations, since in principle there are no capsules that are better than others.

scholarly journals Graphene and Reproduction: A Love-Hate Relationship

Nanomaterials ◽  
2021 ◽  
Vol 11 (2) ◽  
pp. 547
Author(s):  
Marina Ramal-Sanchez ◽  
Antonella Fontana ◽  
Luca Valbonetti ◽  
Alessandra Ordinelli ◽  
Nicola Bernabò ◽  
...  
Keyword(s):  
Graphene Oxide ◽  
Reproductive Function ◽  
Scientometric Analysis ◽  
Potential Toxicity ◽  
Good Tool ◽  
Vitro Fertilization ◽  
Number Of Publications ◽  
The Individual

Since its discovery, graphene and its multiple derivatives have been extensively used in many fields and with different applications, even in biomedicine. Numerous efforts have been made to elucidate the potential toxicity derived from their use, giving rise to an adequate number of publications with varied results. On this basis, the study of the reproductive function constitutes a good tool to evaluate not only the toxic effects derived from the use of these materials directly on the individual, but also the potential toxicity passed on to the offspring. By providing a detailed scientometric analysis, the present review provides an updated overview gathering all the research studies focused on the use of graphene and graphene-based materials in the reproductive field, highlighting the consequences and effects reported to date from experiments performed in vivo and in vitro and in different animal species (from Archea to mammals). Special attention is given to the oxidized form of graphene, graphene oxide, which has been recently investigated for its ability to increase the in vitro fertilization outcomes. Thus, the potential use of graphene oxide against infertility is hypothesized here, probably by engineering the spermatozoa and thus manipulating them in a safer and more efficient way.

scholarly journals ANTI-INFLAMMATORY ACTIVITY OF CURCUMIN AND CAPSAICIN AUGMENTED IN COMBINATION

2017 ◽  
Vol 9 (6) ◽  
pp. 145
Author(s):  
Thriveni Vasanth Kumar ◽  
Manjunatha H. ◽  
Rajesh Kp
Keyword(s):  
Acetic Acid ◽  
Protective Effect ◽  
Vascular Permeability ◽  
Diclofenac Sodium ◽  
Significant Inhibition ◽  
Inflammatory Activity ◽  
Anti Inflammatory ◽  
The Individual

Objective: Dietary curcumin and capsaicin are well known for their health beneficial potencies. The current study was done to assess the anti-inflammatory activity of curcumin, capsaicin and their combination by employing in vitro and in vivo models.Methods: We investigated the protective effect of curcumin, capsaicin and their combination using in vitro heat induced human red blood cell (HRBC) membrane stabilisation, in vivo 3% agar induced leukocyte mobilisation and acetic acid induced vascular permeability assay.Results: Curcumin, capsaicin and their combination exhibited concentration dependent protective effect against heat-induced HRBC membrane destabilisation, while combined curcumin and capsaicin restored 87.0±0.64 % membrane stability and it is found to be better than curcumin, capsaicin and diclofenac sodium (75.0±0.25. 72±0.9 and 80.0±0.31 %) protective effect. In agar suspension induced leukocyte mobilization assay, the combined curcumin and capsaicin had shown 39.5±1.58 % of inhibition compared to individual curcumin and capsaicin, which showed moderate inhibition of 16.0±3.14 and 21.6±2.17 % respectively. Besides, the combined curcumin and capsaicin had shown highly significant inhibition of acetic acid-induced vascular permeability in rats (62.0±3.14 %), whereas individual curcumin and capsaicin showed moderate inhibition of vascular permeability with 36.0±2.41 and 43.0±1.92 % respectively.Conclusion: This study demonstrates the significant anti-inflammatory property of combined curcumin and capsaicin at half of the individual concentration of curcumin and capsaicin.

scholarly journals SIMILARITY OF THE KINETICS OF INVERTASE ACTION IN VIVO AND IN VITRO. II

1932 ◽  
Vol 16 (2) ◽  
pp. 233-242 ◽  
Author(s):  
B. G. Wilkes ◽  
Elizabeth T. Palmer
Keyword(s):  
Physiological Activity ◽  
Outer Region ◽  
Living Cells ◽  
Yeast Cells ◽  
Live Cells ◽  
Intracellular Enzyme ◽  
Relationship Of ◽  
Kinetics Of

1. The pH-activity relationship of invertase has been studied in vivo and in vitro under identical external environmental conditions. 2. The effect of changing (H+) upon the sucroclastic activity of living cells of S. cerevisiae and of invertase solutions obtained therefrom has been found, within experimental error, to be identical. 3. The region of living yeast cells in which invertase exerts its physiological activity changes its pH freely and to the same extent as that of the suspending medium. It is suggested that this may indicate that this intracellular enzyme may perform its work somewhere in the outer region of the cell. 4. In using live cells containing maltase, no evidence of increased sucroclastic activity around pH 6.9, due to the action of Weidenhagen's α-glucosidase (maltase), was found.

Synthetic Riboswitches for the Analysis of tRNA Processing by eukaryotic RNase P Enzymes

RNA ◽  
2022 ◽  
pp. rna.078814.121
Author(s):  
Anna Ender ◽  
Nadine Grafl ◽  
Tim Kolberg ◽  
Sven Findeiss ◽  
Peter F. Stadler ◽  
...  
Keyword(s):  
Homo Sapiens ◽  
Rnase P ◽  
Single Stranded Region ◽  
Domains Of Life ◽  
The Individual ◽  
The Impact ◽  
Individual Enzyme ◽  
Trna Precursor

Removal of the 5' leader region is an essential step in the maturation of tRNA molecules in all domains of life. This reaction is catalyzed by various RNase P activities, ranging from ribonucleoproteins with ribozyme activity to protein-only forms. In Escherichia coli, the efficiency of RNase P mediated cleavage can be controlled by computationally designed riboswitch elements in a ligand-dependent way, where the 5' leader sequence of a tRNA precursor is either sequestered in a hairpin structure or presented as a single-stranded region accessible for maturation. In the presented work, the regulatory potential of such artificial constructs is tested on different forms of eukaryotic RNase P enzymes – two protein-only RNase P enzymes (PRORP1 and PRORP2) from Arabidopsis thaliana and the ribonucleoprotein of Homo sapiens. The PRORP enzymes were analyzed in vitro as well as in vivo in a bacterial RNase P complementation system. We also tested in HEK293T cells whether the riboswitches remain functional with human nuclear RNase P. While the regulatory principle of the synthetic riboswitches applies for all tested RNase P enzymes, the results also show differences in the substrate requirements of the individual enzyme versions. Hence, such designed RNase P riboswitches represent a novel tool to investigate the impact of the structural composition of the 5'-leader on substrate recognition by different types of RNase P enzymes.

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