Steps of nuclear pore complex disassembly and reassembly during mitosis in earlyDrosophilaembryos

2001 ◽  
Vol 114 (20) ◽  
pp. 3607-3618 ◽  
Author(s):  
Elena Kiseleva ◽  
Sandra Rutherford ◽  
Laura M. Cotter ◽  
Terence D. Allen ◽  
Martin W. Goldberg
Keyword(s):  
Electron Microscopy ◽  
Nuclear Pore Complex ◽  
Drosophila Embryo ◽  
Nuclear Pore ◽  
Polar Regions ◽  
Transmission Electron ◽  
Vitro Model ◽  
First Time

The mechanisms of nuclear pore complex (NPC) assembly and disassembly during mitosis in vivo are not well defined. To address this and to identify the steps of the NPC disassembly and assembly, we investigated Drosophila embryo nuclear structure at the syncytial stage of early development using field emission scanning electron microscopy (FESEM), a high resolution surface imaging technique, and transmission electron microscopy. Nuclear division in syncytial embryos is characterized by semi-closed mitosis, during which the nuclear membranes are ruptured only at the polar regions and are arranged into an inner double membrane surrounded by an additional ‘spindle envelope’. FESEM analysis of the steps of this process as viewed on the surface of the dividing nucleus confirm our previous in vitro model for the assembly of the NPCs via a series of structural intermediates, showing for the first time a temporal progression from one intermediate to the next. Nascent NPCs initially appear to form at the site of fusion between the mitotic nuclear envelope and the overlying spindle membrane. A model for NPC disassembly is offered that starts with the release of the central transporter and the removal of the cytoplasmic ring subunits before the star ring.

scholarly journals Schwann Cell Autophagy and Necrosis as Mechanisms of Cell Death by Acanthamoeba

Pathogens ◽  
2020 ◽  
Vol 9 (6) ◽  
pp. 458
Author(s):  
Ismael Castelan-Ramírez ◽  
Lizbeth Salazar-Villatoro ◽  
Bibiana Chávez-Munguía ◽  
Citlaltepetl Salinas-Lara ◽  
Carlos Sánchez-Garibay ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Confocal Microscopy ◽  
Rattus Norvegicus ◽  
Multilamellar Bodies ◽  
Transmission Electron ◽  
Granulomatous Amoebic Encephalitis ◽  
Etiological Agents ◽  
First Time

Amoebae of the genus Acanthamoeba are etiological agents of granulomatous amoebic encephalitis (GAE). Recently, through an in vivo GAE model, Acanthamoeba trophozoites were immunolocalized in contact with the peripheral nervous system (PNS) cells—Schwann cells (SC). In this study, we analyzed in greater detail the in vitro early morphological events (1, 2, 3, and 4 h) during the interaction of A. culbertsoni trophozoites (ATCC 30171) with SC from Rattus norvegicus (ATCC CRL-2941). Samples were processed for scanning and transmission electron microscopy as well as confocal microscopy. After 1 h of interaction, amoebae were observed to be adhered to the SC cultures, emitting sucker-like structures associated with micro-phagocytic channels. In addition, evidence of necrosis was identified since edematous organelles as well as multivesicular and multilamellar bodies characteristics of autophagy were detected. At 2 h, trophozoites migrated beneath the SC culture in which necrosis and autophagy persisted. By 3 and 4 h, extensive lytic zones were observed. SC necrosis was confirmed by confocal microscopy. We reported for the first time the induction of autophagic and necrotic processes in PNS cells, associated in part with the contact-dependent pathogenic mechanisms of A. culbertsoni trophozoites.

scholarly journals Cemp1-p3 Peptide Promotes the Transformation of Octacalcium Phosphate into Hydroxyapatite Crystals

Crystals ◽  
2020 ◽  
Vol 10 (12) ◽  
pp. 1131
Author(s):  
Maricela Santana ◽  
Gonzalo Montoya ◽  
Raúl Herrera ◽  
Lía Hoz ◽  
Enrique Romo ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Octacalcium Phosphate ◽  
Sequence Motifs ◽  
Simple Method ◽  
Transmission Electron ◽  
Precursor Phase ◽  
Mineralized Tissues ◽  
Potent Growth

Dental cementum contains unique molecules that regulate the mineralization process in vitro and in vivo, such as cementum protein 1 (CEMP1). This protein possesses amino acid sequence motifs like the human recombinant CEMP1 with biological activity. This novel cementum protein 1-derived peptide (CEMP1-p3, from the CEMP1’s N-terminal domain: (QPLPKGCAAVKAEVGIPAPH), consists of 20 amino acids. Hydroxyapatite (HA) crystals could be obtained through the combination of the amorphous precursor phase and macromolecules such as proteins and peptides. We used a simple method to synthesize peptide/hydroxyapatite nanocomposites using OCP and CEMP1-p3. The characterization of the crystals through scanning electron microscopy (SEM), powder X-ray diffraction (XRD), high--resolution transmission electron microscopy (HRTEM), and Raman spectroscopy revealed that CEMP1-p3 transformed OCP into hydroxyapatite (HA) under constant ionic strength and in a buffered solution. CEMP1-p3 binds and highly adsorbs to OCP and is a potent growth stimulator of OCP crystals. CEMP1-p3 fosters the transformation of OCP into HA crystals with crystalline planes (300) and (004) that correspond to the cell of hexagonal HA. Octacalcium phosphate crystals treated with CEMP1-p3 grown in simulated physiological buffer acquired hexagonal arrangement corresponding to HA. These findings provide new insights into the potential application of CEMP1-p3 on possible biomimetic approaches to generate materials for the repair and regeneration of mineralized tissues, or restorative materials in the orthopedic field.

scholarly journals Human Islet Amyloid Polypeptide Fibril Binding to Catalase: A Transmission Electron Microscopy and Microplate Study

2010 ◽  
Vol 10 ◽  
pp. 879-893 ◽  
Author(s):  
Nathaniel G. N. Milton ◽  
J. Robin Harris
Keyword(s):  
Electron Microscopy ◽  
Transmission Electron Microscopy ◽  
Islet Amyloid Polypeptide ◽  
Amyloid Β ◽  
Human Islet ◽  
Islet Amyloid ◽  
Human Islet Amyloid Polypeptide ◽  
Transmission Electron

The diabetes-associated human islet amyloid polypeptide (IAPP) is a 37-amino-acid peptide that forms fibrilsin vitroandin vivo. Human IAPP fibrils are toxic in a similar manner to Alzheimer's amyloid-β (Aβ) and prion protein (PrP) fibrils. Previous studies have shown that catalase binds to Aβ fibrils and appears to recognize a region containing the Gly-Ala-Ile-Ile sequence that is similar to the Gly-Ala-Ile-Leu sequence found in human IAPP residues 24-27. This study presents a transmission electron microscopy (TEM)—based analysis of fibril formation and the binding of human erythrocyte catalase to IAPP fibrils. The results show that human IAPP 1-37, 8-37, and 20-29 peptides form fibrils with diverse and polymorphic structures. All three forms of IAPP bound catalase, and complexes of IAPP 1-37 or 8-37 with catalase were identified by immunoassay. The binding of biotinylated IAPP to catalase was high affinity with a KDof 0.77nM, and could be inhibited by either human or rat IAPP 1-37 and 8-37 forms. Fibrils formed by the PrP 118-135 peptide with a Gly-Ala-Val-Val sequence also bound catalase. These results suggest that catalase recognizes a Gly-Ala-Ile-Leu—like sequence in amyloid fibril-forming peptides. For IAPP 1-37 and 8-37, the catalase binding was primarily directed towards fibrillar rather than ribbon-like structures, suggesting differences in the accessibility of the human IAPP 24-27 Gly-Ala-Ile-Leu region. This suggests that catalase may be able to discriminate between different structural forms of IAPP fibrils. The ability of catalase to bind IAPP, Aβ, and PrP fibrils demonstrates the presence of similar accessible structural motifs that may be targets for antiamyloid therapeutic development.

scholarly journals Optimization of Innovative Three-Dimensionally-Structured Hybrid Vesicles to Improve the Cutaneous Delivery of Clotrimazole for the Treatment of Topical Candidiasis

Pharmaceutics ◽  
2019 ◽  
Vol 11 (6) ◽  
pp. 263 ◽  
Author(s):  
Maria Letizia Manca ◽  
Iris Usach ◽  
José Esteban Peris ◽  
Antonella Ibba ◽  
Germano Orrù ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Transmission Electron Microscopy ◽  
Chemical Properties ◽  
Chemical Characterization ◽  
Yeast Cells ◽  
Unilamellar Vesicles ◽  
Transmission Electron ◽  
Physico Chemical

New three-dimensionally-structured hybrid phospholipid vesicles, able to load clotrimazole in a high amount (10 mg/mL), were obtained for the first time in this work by significantly reducing the amount of water (≤10%), which was replaced with a mixture of glycerol and ethanol (≈90%). A pre-formulation study was carried out to evaluate the effect of both the composition of the hydrating medium and the concentration of the phospholipid on the physico-chemical properties of hybrid vesicles. Four different three-dimensionally-structured hybrid vesicles were selected as ideal systems for the topical application of clotrimazole. An extensive physico-chemical characterization performed using transmission electron microscopy (TEM), cryogenic transmission electron microscopy (cryo-TEM), 31P-NMR, and small-angle X-ray scattering (SAXS) displayed the formation of small, multi-, and unilamellar vesicles very close to each other, and was capable of forming a three-dimensional network, which stabilized the dispersion. Additionally, the dilution of the dispersion with water reduced the interactions between vesicles, leading to the formation of single unilamellar vesicles. The evaluation of the in vitro percutaneous delivery of clotrimazole showed an improved drug deposition in the skin strata provided by the three-dimensionally-structured vesicles with respect to the commercial cream (Canesten®) used as a reference. Hybrid vesicles were highly biocompatible and showed a significant antifungal activity in vitro, greater than the commercial cream Canesten®. The antimycotic efficacy of formulations was confirmed by the reduced proliferation of the yeast cells at the site of infection in vivo. In light of these results, clotrimazole-loaded, three-dimensionally-structured hybrid vesicles appear to be one of the most innovative and promising formulations for the treatment of candidiasis infections.

Yolk platelet degradation in preemergence Artemia embryos: response to protons in vivo and in vitro

1987 ◽  
Vol 252 (4) ◽  
pp. R774-R781 ◽  
Author(s):  
P. J. Utterback ◽  
S. C. Hand
Keyword(s):  
Electron Microscopy ◽  
Transmission Electron Microscopy ◽  
Dry Weight ◽  
Platelet Protein ◽  
Transmission Electron ◽  
Structural Differences ◽  
Alkaline Conditions ◽  
Yolk Platelet

Alteration of intracellular pH (pHi) influences yolk platelet degradation during preemergence development in Artemia embryos. Cysts incubated for 10 h under conditions of aerobic development (aqueous medium equilibrated with 60% N2-40% O2, pHi greater than or equal to 7.9) exhibit a significant decrease in numbers of yolk platelets and platelet protein. In contrast, cysts incubated for 10 h under aerobic acidosis (60% CO2-40% O2, pHi = 6.8) show no significant decrease in numbers of yolk platelets or platelet protein. When subjected to alkaline conditions in vitro, yolk platelets release protein exponentially as a function of time. The process is essentially complete in 40 min. The extent of protein and lipid release from platelets increases markedly as pH of the medium is raised in increments from 6.3 to 8.0. Concomitant with these changes are reduction (50%) in platelet dry weight and reduction (21%) in platelet diameter. Transmission electron microscopy does not reveal major structural differences between isolated yolk platelets and those contained in hydrated embryos. The proton effects on platelet composition and size detected in vitro may explain in part the mechanism of platelet degradation observed during aerobic development and its suppression under conditions of acidic pHi.

scholarly journals The production of mature oocytes from adult ovaries following primary follicle culture in a marsupial

Reproduction ◽  
2009 ◽  
Vol 138 (2) ◽  
pp. 247-255 ◽  
Author(s):  
A Nation ◽  
L Selwood
Keyword(s):  
Normal Growth ◽  
Primary Follicle ◽  
Follicle Culture ◽  
Nuclear Maturation ◽  
Transmission Electron ◽  
Progressive Growth ◽  
Sminthopsis Macroura ◽  
First Time

A model marsupial culture system has been developed whereby individual primary follicles, obtained from adult ovaries, can be grown in vitro to the antral stage and oocytes retrieved from these follicles can achieve nuclear maturation (metaphase II) in the presence of LH. Primary follicles isolated from adult Sminthopsis macroura ovaries were cultured individually in one of four systems: microdrops under oil, upright, inverted, or roller culture. After 6 days of culture, cumulus–oocyte complexes (COCs) were excised from early antral follicles and incubated for an additional 24 h to assess meiotic competence and the effects of LH and lithium on oocyte maturation. Histology and transmission electron microscopy established normal in vivo standards and verified oocyte and follicular integrity following culture. On day 6 of culture, follicle viability was significantly greater in the inverted system (73%) than in the other three systems (10–46%). The inverted system was the most effective in supporting development with follicles demonstrating progressive growth during culture and showing antral signs by day 4. Meiotic resumption during COC culture was facilitated by LH, but hindered by lithium. The ability to resume meiosis and progress to metaphase II was equivalent in oocytes retrieved following follicle culture and those matured in vivo. This study highlights the importance of oxygen and nutrient availability during marsupial follicle culture, and demonstrates for the first time that primary follicles isolated from adult mammalian ovaries can undergo normal growth and development in vitro, to produce mature, meiotically competent oocytes.

scholarly journals CARMUSTINE LOADED NANOSIZE LIPID VESICLES SHOWED PREFERENTIAL CYTOTOXICITY AND INTERNALIZATION IN U87MG CELL LINE ALONG WITH IMPROVED PHARMACOKINETIC PROFILE IN MICE: A STRATEGY FOR TREATMENT OF GLIOMA

2020 ◽  
pp. 240-248
Author(s):  
BHABANI SANKAR SATAPATHY ◽  
JNANRANJAN PANDA
Keyword(s):  
Electron Microscopy ◽  
Cell Line ◽  
Drug Loading ◽  
Lipid Vesicles ◽  
Pharmacokinetic Profile ◽  
In Vitro Cytotoxicity ◽  
Pharmacokinetic Data ◽  
Transmission Electron

Objective: Successful treatment of glioma still remains a tough challenge. The present study aims at the development and evaluation of carmustine loaded nanosize phospholipid vesicles (CNLVs) for the treatment of glioma. Methods: The experimental NLVs were developed by conventional lipid layer hydration technique and were characterized by different in vitro tools such as diffraction light scattering (DLS), zeta potential, field emission scanning electron microscopy (FESEM), cryo-transmission electron microscopy (cryo-TEM), in vitro drug loading capacity, drug release study etc. In vitro cytotoxicity and cellular uptake of the optimized drug-loaded NLVs were carried out in U87MG human glioblastoma cell line. In vivo pharmacokinetic study was conducted in Swiss albino mice. Results: DLS data showed an average vesicle diameter of 92 nm with narrow size distribution. Optimized CNLVs were spherical in shape with a smooth surface as depicted from FESEM data. Cryo-TEM study confirmed formation of unilamellar vesicles with intact outer bilayer. A reasonable drug loading of 7.8 % was reported for the optimized CNLVs along with a sustained release of CS over a 48 h study period. In vitro cytotoxicity assay revealed a considerable higher toxicity of CNLVs than free drugs in the U87MG cells. Confocal microscopy showed a satisfactory internalization of the optimized drug-loaded NLVs in the tested cell line. Pharmacokinetic data demonstrated an enhanced mean residence time of optimized CNLVs in blood than free drug. Conclusion: Results depicted the potential of experimental CNLVs for the treatment of glioma after further in vivo tests.

Morphometrical Ultrastructural Studies of the Rat's Hepatocytes Under Cooling

1997 ◽  
Vol 3 (S2) ◽  
pp. 245-246
Author(s):  
A.S. Kaprelyants ◽  
A.A. Kaprelyants ◽  
A.N. Reylan ◽  
R.K. Migunova
Keyword(s):  
Electron Microscopy ◽  
Transmission Electron Microscopy ◽  
Electron Microscopic ◽  
Rat Hepatocyte ◽  
Ultrastructural Studies ◽  
Transmission Electron ◽  
Transmission Electron Microscopic ◽  
Morphometric Methods

The aim of given investigation is to study the effect of cooling upon rat hepatocyte structure using transmission electron microscopic and computer morphometric methods. Ultrastructural and morphometrical characteristics of hepatocytes under liver cooling for various levels under in vivo and in vitro conditions were investigated. Vistar rats of 180-250 g were used in the experiment. Liver cooling (in vivo) was performed by means of original cryoapplicator with different probe temperature (1,2). Liver tissue for transmission electron microscopy was fixed in glutaraldehyde fixator on cocadylate buffer and OsO4. Dehydration was completed on acetone (3). Tissue embedding was done into the mixture of Epon/Araldite epoxy rasin. Ultrathin slices were contrasted by the method of Reinolds. Cell viewing and imaging were accomplished by electron microscope at accelerating power of 75kV.Morphometrical and stereometrical analysis was performed using the “Morpho-Tools” original computer system (c) 1994-1996 A.S. Kaprelyants, A.A. Kaprelyants, A.N. Reylan .

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