scholarly journals Mitotic post-translational modifications of histones promote chromatin compaction in vitro

Open Biology ◽  
2017 ◽  
Vol 7 (9) ◽  
pp. 170076 ◽  
Author(s):  
Alisa Zhiteneva ◽  
Juan Jose Bonfiglio ◽  
Alexandr Makarov ◽  
Thomas Colby ◽  
Paola Vagnarelli ◽  
...  
Keyword(s):  
Histone Modifications ◽  
Cell Biology ◽  
Transmission Electron Microscopy Analysis ◽  
Histone Proteins ◽  
Post Translational Modifications ◽  
Chromatin Compaction ◽  
Electron Microscopy Analysis ◽  
Core Histones ◽  
Mitotic Chromatin

How eukaryotic chromosomes are compacted during mitosis has been a leading question in cell biology since the nineteenth century. Non-histone proteins such as condensin complexes contribute to chromosome shaping, but appear not to be necessary for mitotic chromatin compaction. Histone modifications are known to affect chromatin structure. As histones undergo major changes in their post-translational modifications during mitotic entry, we speculated that the spectrum of cell-cycle-specific histone modifications might contribute to chromosome compaction during mitosis. To test this hypothesis, we isolated core histones from interphase and mitotic cells and reconstituted chromatin with them. We used mass spectrometry to show that key post-translational modifications remained intact during our isolation procedure. Light, atomic force and transmission electron microscopy analysis showed that chromatin assembled from mitotic histones has a much greater tendency to aggregate than chromatin assembled from interphase histones, even under low magnesium conditions where interphase chromatin remains as separate beads-on-a-string structures. These observations are consistent with the hypothesis that mitotic chromosome formation is a two-stage process with changes in the spectrum of histone post-translational modifications driving mitotic chromatin compaction, while the action of non-histone proteins such as condensin may then shape the condensed chromosomes into their classic mitotic morphology.

A Brief History of the Discovery of Amelogenin Nanoribbons In Vitro and In Vivo

2021 ◽  
pp. 002203452110434
Author(s):  
Y. Bai ◽  
J. Bonde ◽  
K.M.M. Carneiro ◽  
Y. Zhang ◽  
W. Li ◽  
...  
Keyword(s):  
Recombinant Proteins ◽  
Protein Structures ◽  
Historical Review ◽  
Limited Information ◽  
Transmission Electron Microscopy Analysis ◽  
Electron Microscopy Analysis ◽  
History Of ◽  
The 1960S

Without evidence for an organic framework, biological and biochemical processes observed during amelogenesis provided limited information on how extracellular matrix proteins control the development of the complex fibrous architecture of human enamel. Over a decade ago, amelogenin nanoribbons were first observed from recombinant proteins during in vitro mineralization experiments in our laboratory. In enamel from mice lacking the enzyme kallikrein 4 (KLK4), we later uncovered ribbon-like protein structures that matched the morphology, width, and thickness of the nanoribbons assembled by recombinant proteins. Interestingly, similar structures had already been described since the 1960s, when enamel sections from various mammals were demineralized and stained for transmission electron microscopy analysis. However, at that time, researchers were not aware of the ability of amelogenin to form nanoribbons and instead associated the filamentous nanostructures with possible imprints of mineral ribbons in the gel-like matrix of developing enamel. Further evidence for the significance of amelogenin nanoribbons for enamel development was stipulated when recent mineralization experiments succeeded in templating and orienting the growth of apatite ribbons along the protein nanoribbon framework. This article provides a brief historical review of the discovery of amelogenin nanoribbons in our laboratory in the context of reports by others on similar structures in the developing enamel matrix.

scholarly journals Cellular Effects of Reversed Amidines on Trypanosoma cruzi

2007 ◽  
Vol 51 (11) ◽  
pp. 3803-3809 ◽  
Author(s):  
C. F. Silva ◽  
M. B. Meuser ◽  
E. M. De Souza ◽  
M. N. L. Meirelles ◽  
C. E. Stephens ◽  
...  
Keyword(s):  
Trypanosoma Cruzi ◽  
Chagas Disease ◽  
In Vivo Evaluation ◽  
Transmission Electron Microscopy Analysis ◽  
Dna Minor Groove ◽  
Cellular Effects ◽  
Transmission Electron ◽  
Electron Microscopy Analysis

ABSTRACT Aromatic diamidines represent a class of DNA minor groove-binding ligands that exhibit high levels of antiparasitic activity. Since the chemotherapy for Chagas' disease is still an unsolved problem and previous reports on diamidines and related analogues show that they have high levels of activity against Trypanosoma cruzi infection both in vitro and in vivo, our present aim was to evaluate the cellular effects in vitro of three reversed amidines (DB889, DB702, and DB786) and one diguanidine (DB711) against both amastigotes and bloodstream trypomastigotes of T. cruzi, the etiological agent of Chagas ' disease. Our data show that the reversed amidines have higher levels of activity than the diguanidine, with the order of trypanocidal activities being as follows: DB889 > DB702 > DB786 > DB711. Transmission electron microscopy analysis showed that the reversed amidines induced many alterations in the nuclear morphology, swelling of the endoplasmic reticulum and Golgi structures, and consistent damage in the mitochondria and kinetoplasts of the parasites. Interestingly, in trypomastigotes treated with the reversed amidine DB889, multiple axoneme structures (flagellar microtubules) were noted. Flow cytometry analysis confirmed that the treated parasites presented an important loss of the mitochondrial membrane potential, as revealed by a decrease in rhodamine 123 fluorescence. Our results show that the reversed amidines have promising activities against the relevant mammalian forms of T. cruzi and display high trypanocidal effects at very low doses. This is especially the case for DB889, which merits further in vivo evaluation.

scholarly journals FABRICATION, CHARACTERIZATION, AND IN VITRO EVALUATION OF PEGYLATED GLYCERIDE LABRASOL® NANOSTRUCTURED LIPID CARRIER COMPOSITES OF METHOTREXATE: THE PATHWAY TO EFFECTIVE CANCER THERAPY

2019 ◽  
pp. 229-237 ◽  
Author(s):  
RADHARANI PANDA ◽  
KETOUSETUO KUOTSU
Keyword(s):  
Sustained Release ◽  
Drug Loading ◽  
In Vitro Release ◽  
Entrapment Efficiency ◽  
Nanostructured Lipid Carrier ◽  
Transmission Electron Microscopy Analysis ◽  
Transmission Electron ◽  
Electron Microscopy Analysis ◽  
High Entrapment Efficiency

Objective: The objective of the current study is to optimize and evaluate the potential of polyethylene glycolylated (PEG) glyceride Labrasol® nanostructured lipid carrier (NLC) composites of methotrexate (MTX) to achieve enhanced sustained release delivery in cancer treatment. Materials and Methods: MTX-NLC was successfully prepared by hot melt emulsification and probe sonication method for spatial and controlled release of this therapeutic agent. Results: The solubility screening of MTX and lipids resulted in the selection of Monostearin as solid lipid, PEGylated glyceride Labrasol® and olive oil as liquid lipids for the formulation of MTX-loaded NLC composites. Particle size, zeta potential, and polydispersity index of both the composites were confirmed using dynamic light scattering, whereby Labrasol® MTX-NLC showed high entrapment efficiency and drug loading. A spherical particle shape with smooth surface of all the composites was confirmed from the scanning electron microscope and transmission electron microscopy analysis. Labrasol® MTX-NLC showed remarkably increased cytotoxic response, augmented cellular uptake, and low half maximal inhibitory concentration value in MCF-7 cells. In vitro release study confirmed that encapsulation of MTX in PEGylated glyceride Labrasol® MTX-NLC resulted in enhanced sustained release of MTX for a period of 48 h. Conclusion: The present study establishes that PEGylated glyceride Labrasol® MTX-NLC can be considered as a promising anticancer delivery system, thereby improving antitumor efficacy of the drug.

Autogenous regulation of histone mRNA decay by histone proteins in a cell-free system

1987 ◽  
Vol 7 (12) ◽  
pp. 4345-4356
Author(s):  
S W Peltz ◽  
J Ross
Keyword(s):  
Protein Interactions ◽  
Mrna Decay ◽  
Mrna Turnover ◽  
Histone Mrna ◽  
Globin Mrna ◽  
Histone Proteins ◽  
Free System ◽  
Gamma Globin ◽  
Core Histones

We tested the hypothesis that histone mRNA turnover is accelerated in the presence of free histone proteins. In an in vitro mRNA decay system, histone mRNA was degraded four- to sixfold faster in reaction mixtures containing core histones and a cytoplasmic S130 fraction than in reaction mixtures lacking these components. The decay rate did not change significantly when histones or S130 was added separately, suggesting either that the histones were modified and thereby activated by S130 or that additional factors besides histones were required. RecA, SSB (single-stranded binding), and histone proteins all formed complexes with histone mRNA, but only histones induced accelerated histone mRNA turnover. Therefore, the effect was not the result of random RNA-protein interactions. Moreover, histone proteins did not induce increased degradation of gamma globin mRNA, c-myc mRNA, or total poly(A)- or poly(A)+ polysomal mRNAs. This autoregulatory mechanism is consistent with the observed accumulation of cytoplasmic histone proteins in cells after DNA synthesis stops, and it can account, in part, for the rapid disappearance of histone mRNA at the end of S phase.

Role of histone modifications in defining chromatin structure and function

2008 ◽  
Vol 389 (4) ◽  
pp. 353-363 ◽  
Author(s):  
Kathy A. Gelato ◽  
Wolfgang Fischle
Keyword(s):  
Histone Modifications ◽  
Chromatin Structure ◽  
Eukaryotic Cell ◽  
Structure And Function ◽  
Cell Nuclei ◽  
Histone Proteins ◽  
Post Translational Modifications ◽  
And Function ◽  
Molecular Components

AbstractChromosomes in eukaryotic cell nuclei are not uniformly organized, but rather contain distinct chromatin elements, with each state having a defined biochemical structure and biological function. These are recognizable by their distinct architectures and molecular components, which can change in response to cellular stimuli or metabolic requirements. Chromatin elements are characterized by the fundamental histone and DNA components, as well as other associated non-histone proteins and factors. Post-translational modifications of histone proteins in particular often correlate with a specific chromatin structure and function. Patterns of histone modifications are implicated as having a role in directing the level of chromatin compaction, as well as playing roles in multiple functional pathways directing the readout of distinct regions of the genome. We review the properties of various chromatin elements and the apparent links of histone modifications with chromatin organization and functional output.

scholarly journals Autogenous regulation of histone mRNA decay by histone proteins in a cell-free system.

1987 ◽  
Vol 7 (12) ◽  
pp. 4345-4356 ◽  
Author(s):  
S W Peltz ◽  
J Ross
Keyword(s):  
Protein Interactions ◽  
Mrna Decay ◽  
Mrna Turnover ◽  
Histone Mrna ◽  
Globin Mrna ◽  
Histone Proteins ◽  
Free System ◽  
Gamma Globin ◽  
Core Histones

We tested the hypothesis that histone mRNA turnover is accelerated in the presence of free histone proteins. In an in vitro mRNA decay system, histone mRNA was degraded four- to sixfold faster in reaction mixtures containing core histones and a cytoplasmic S130 fraction than in reaction mixtures lacking these components. The decay rate did not change significantly when histones or S130 was added separately, suggesting either that the histones were modified and thereby activated by S130 or that additional factors besides histones were required. RecA, SSB (single-stranded binding), and histone proteins all formed complexes with histone mRNA, but only histones induced accelerated histone mRNA turnover. Therefore, the effect was not the result of random RNA-protein interactions. Moreover, histone proteins did not induce increased degradation of gamma globin mRNA, c-myc mRNA, or total poly(A)- or poly(A)+ polysomal mRNAs. This autoregulatory mechanism is consistent with the observed accumulation of cytoplasmic histone proteins in cells after DNA synthesis stops, and it can account, in part, for the rapid disappearance of histone mRNA at the end of S phase.

scholarly journals Cross-seeding Controls Aβ Fibril Populations and Resulting Function

2021 ◽  
Author(s):  
Henry S Pan ◽  
Michael J Lucas ◽  
Eric J Verbeke ◽  
Gina M Partipilo ◽  
Ethan C Helfman ◽  
...  
Keyword(s):  
Neuroblastoma Cell ◽  
Population Level ◽  
Secondary Nucleation ◽  
Transmission Electron Microscopy Analysis ◽  
Structural Distribution ◽  
Fibril Structure ◽  
Transmission Electron ◽  
Electron Microscopy Analysis

Amyloid peptides nucleate from monomers to aggregate into fibrils through primary nucleation; pre-existing fibrils can then act as seeds for additional monomers to fibrillize through secondary nucleation. Both nucleation processes can occur simultaneously, yielding a distribution of fibril polymorphs that can generate a spectrum of neurodegenerative effects. Understanding the mechanisms driving polymorph structural distribution during both nucleation processes is important for uncovering fibril structure-function relationships, as well creating polymorph distributions in vitro that better match distributions found in vivo. Here, we explore how cross-seeding WT Aβ1-40 with Aβ1-40 mutants E22G (Arctic) and E22Δ (Osaka), as well as with WT Aβ1-42 affects the distribution of fibril structural polymorphs, and how changes in structural distribution impact toxicity. Transmission electron microscopy analysis reveals that fibril seeds derived from mutants of Aβ1-40 impart their structure to WT Aβ1-40 monomer during secondary nucleation, but WT Aβ1-40 fibril seeds do not affect the structure of fibrils assembled from mutant Aβ1-40 monomers, despite kinetics data indicating accelerated aggregation when cross-seeding of any combination of mutants. Additionally, WT Aβ1-40 fibrils seeded with mutant fibrils to produce similar structural distributions to the mutant seeds also produced similar cytotoxicity on neuroblastoma cell lines. This indicates that mutant fibril seeds not only impart their structure to growing WT Aβ1-40 aggregates, but they also impart cytotoxic properties. Our findings provide clear evidence that there is a relationship between fibril structure and phenotype on a polymorph population level, and that these properties can be passed on through secondary nucleation of succeeding generations of fibrils.

scholarly journals Reduced USP22 Expression Impairs Mitotic Removal of H2B Monoubiquitination, Alters Chromatin Compaction and Induces Chromosome Instability That May Promote Oncogenesis

Cancers ◽  
2021 ◽  
Vol 13 (5) ◽  
pp. 1043
Author(s):  
Lucile M. Jeusset ◽  
Brent J. Guppy ◽  
Zelda Lichtensztejn ◽  
Darin McDonald ◽  
Kirk J. McManus
Keyword(s):  
Chromosome Segregation ◽  
Molecular Mechanisms ◽  
Chromosome Instability ◽  
Critical Determinant ◽  
Chromatin Compaction ◽  
Cancer Pathogenesis ◽  
Mitotic Chromatin ◽  
Early Mitosis

Chromosome instability (CIN) is an enabling feature of oncogenesis associated with poor patient outcomes, whose genetic determinants remain largely unknown. As mitotic chromatin compaction defects can compromise the accuracy of chromosome segregation into daughter cells and drive CIN, characterizing the molecular mechanisms ensuring accurate chromatin compaction may identify novel CIN genes. In vitro, histone H2B monoubiquitination at lysine 120 (H2Bub1) impairs chromatin compaction, while in vivo H2Bub1 is rapidly depleted from chromatin upon entry into mitosis, suggesting that H2Bub1 removal may be a pre-requisite for mitotic fidelity. The deubiquitinating enzyme USP22 catalyzes H2Bub1 removal in interphase and may also be required for H2Bub1 removal in early mitosis to maintain chromosome stability. In this study, we demonstrate that siRNA-mediated USP22 depletion increases H2Bub1 levels in early mitosis and induces CIN phenotypes associated with mitotic chromatin compaction defects revealed by super-resolution microscopy. Moreover, USP22-knockout models exhibit continuously changing chromosome complements over time. These data identify mitotic removal of H2Bub1 as a critical determinant of chromatin compaction and faithful chromosome segregation. We further demonstrate that USP22 is a CIN gene, indicating that USP22 deletions, which are frequent in many tumor types, may drive genetic heterogeneity and contribute to cancer pathogenesis.

Correlative microscopy in distrubuted (client/server) computing environments: tools for catalyzing the rapid dissemination of information about the cell biology of the human prostate

1995 ◽  
Vol 53 ◽  
pp. 682-683
Author(s):  
A. Hakam ◽  
J.T. Gau ◽  
M.L. Grove ◽  
B.A. Evans ◽  
M. Shuman ◽  
...  
Keyword(s):  
United States ◽  
Cell Biology ◽  
Tissue Bank ◽  
The United States ◽  
Prostate Adenocarcinoma ◽  
Correlative Microscopy ◽  
Client Server ◽  
Critical Elements ◽  
Transplant Organ

Prostate adenocarcinoma is the most common malignant tumor of men in the United States and is the third leading cause of death in men. Despite attempts at early detection, there will be 244,000 new cases and 44,000 deaths from the disease in the United States in 1995. Therapeutic progress against this disease is hindered by an incomplete understanding of prostate epithelial cell biology, the availability of human tissues for in vitro experimentation, slow dissemination of information between prostate cancer research teams and the increasing pressure to “ stretch” research dollars at the same time staff reductions are occurring.To meet these challenges, we have used the correlative microscopy (CM) and client/server (C/S) computing to increase productivity while decreasing costs. Critical elements of our program are as follows:1) Establishing the Western Pennsylvania Genitourinary (GU) Tissue Bank which includes >100 prostates from patients with prostate adenocarcinoma as well as >20 normal prostates from transplant organ donors.

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