scholarly journals Subaxolemmal cytoskeleton in squid giant axon. II. Morphological identification of microtubule- and microfilament-associated domains of axolemma.

1986 ◽  
Vol 102 (5) ◽  
pp. 1710-1725 ◽  
Author(s):  
S Tsukita ◽  
S Tsukita ◽  
T Kobayashi ◽  
G Matsumoto
Keyword(s):  
Electron Microscopy ◽  
Three Dimensional ◽  
Preceding Paper ◽  
Microscopic Analysis ◽  
Giant Axon ◽  
Squid Giant Axon ◽  
Molecular Organization ◽  
Fixation Method ◽  
Morphological Identification

In the preceding paper (Kobayashi, T., S. Tsukita, S. Tsukita, Y. Yamamoto, and G. Matsumoto, 1986, J. Cell Biol., 102:1710-1725), we demonstrated biochemically that the subaxolemmal cytoskeleton of the squid giant axon was highly specialized and mainly composed of tubulin, actin, axolinin, and a 255-kD protein. In this paper, we analyzed morphologically the molecular organization of the subaxolemmal cytoskeleton in situ. For thin section electron microscopy, the subaxolemmal cytoskeleton was chemically fixed by the intraaxonal perfusion of the fixative containing tannic acid. With this fixation method, the ultrastructural integrity was well preserved. For freeze-etch replica electron microscopy, the intraaxonally perfused axon was opened and rapidly frozen by touching its inner surface against a cooled copper block (4 degrees K), thus permitting the direct stereoscopic observation of the cytoplasmic surface of the axolemma. Using these techniques, it became clear that the major constituents of the subaxolemmal cytoskeleton were microfilaments and microtubules. The microfilaments were observed to be associated with the axolemma through a specialized meshwork of thin strands, forming spot-like clusters just beneath the axolemma. These filaments were decorated with heavy meromyosin showing a characteristic arrowhead appearance. The microtubules were seen to run parallel to the axolemma and embedded in the fine three-dimensional meshwork of thin strands. In vitro observations of the aggregates of axolinin and immunoelectron microscopic analysis showed that this fine meshwork around microtubules mainly consisted of axolinin. Some microtubules grazed along the axolemma and associated laterally with it through slender strands. Therefore, we were led to conclude that the axolemma of the squid giant axon was specialized into two domains (microtubule- and microfilament-associated domains) by its underlying cytoskeletons.

Neurofilaments and Paired Helical Filaments

1985 ◽  
Vol 43 ◽  
pp. 740-743
Author(s):  
J. Metuzals
Keyword(s):  
Animal Model ◽  
Posttranslational Modifications ◽  
Posttranslational Modification ◽  
Giant Axon ◽  
Paired Helical Filaments ◽  
Squid Giant Axon ◽  
In Vitro Experiments ◽  
Thin Sections ◽  
Structural Relationships

It has been demonstrated that the neurofibrillary tangles in biopsies of Alzheimer patients, composed of typical paired helical filaments (PHF), consist also of typical neurofilaments (NF) and 15nm wide filaments. Close structural relationships, and even continuity between NF and PHF, have been observed. In this paper, such relationships are investigated from the standpoint that the PHF are formed through posttranslational modifications of NF. To investigate the validity of the posttranslational modification hypothesis of PHF formation, we have identified in thin sections from frontal lobe biopsies of Alzheimer patients all existing conformations of NF and PHF and ordered these conformations in a hypothetical sequence. However, only experiments with animal model preparations will prove or disprove the validity of the interpretations of static structural observations made on patients. For this purpose, the results of in vitro experiments with the squid giant axon preparations are compared with those obtained from human patients. This approach is essential in discovering etiological factors of Alzheimer's disease and its early diagnosis.

In-vitro production and pre-validation of lyophilized canine platelet-rich plasma for therapeutic use

2021 ◽  
Vol 41 ◽  
Author(s):  
Natália P.P. Freitas ◽  
Maria Márcia M.S. Maior ◽  
Beatriz A.P. Silva ◽  
Marcus R.L. Bezerra ◽  
José F. Nunes ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Cell Proliferation ◽  
Growth Factors ◽  
Platelet Rich Plasma ◽  
Three Dimensional ◽  
In Vitro Production ◽  
Mesh Structure ◽  
Prp Gel

ABSTRACT: Platelet-rich plasma (PRP) has been considered a promising therapeutic alternative, since platelets are rich in growth factors that are used in the Regenerative Medicine field. However, fresh PRP cannot be stored for long periods. This study aimed to develop a protocol for obtaining lyophilized canine PRP capable of maintaining viability after its reconstitution. For that purpose, canine PRP extraction and lyophilization protocols were initially tested. Subsequently, assays were carried out to quantify the growth factors VEGF and TGF-β, before and after the lyophilization process, gelation test and the three-dimensional gel structure analysis of the reconstituted lyophilized PRP by electron microscopy, as well as in vitro cell proliferation test in lyophilized PRP gel. Additionally, the immunogenicity test was performed, using allogeneic samples of lyophilized PRP. The results showed that the lyophilized PRP had adequate therapeutic concentrations of growth factors VEGF and TGF-β (9.1pg/mL and 6161.6pg/mL, respectively). The reconstituted PRP gel after lyophilization showed an in vitro durability of 10 days. Its electron microscopy structure was similar to that of fresh PRP. In the cell proliferation test, an intense division process was verified in mesenchymal stem cells (MSCs) through the three-dimensional mesh structure of the lyophilized PRP gel. The immunogenicity test showed no evidence of an immune reaction. The findings were promising, suggesting the possibility of having a lyophilized canine PRP that can be marketed. New in vivo and in vitro studies must be carried out for therapeutic confirmation.

Scanning electron microscopy of mouse ciliated oviduct and tracheal epithelium infected in vitro with Bordetella pertussis

1983 ◽  
Vol 29 (4) ◽  
pp. 415-420 ◽  
Author(s):  
Lauren B. Opremcak ◽  
Melvin S. Rheins
Keyword(s):  
Electron Microscopy ◽  
Scanning Electron Microscopy ◽  
Organ Culture ◽  
Bordetella Pertussis ◽  
Tracheal Ring ◽  
Fixation Method ◽  
Bacterial Cells ◽  
Ciliated Cells ◽  
Scanning Electron

Infection of mouse tracheal organ culture with Bordetella pertussis resulted in ciliostasis within 36 h. Scanning electron microscopy revealed that B. pertussis attached exclusively to ciliated cells but did not induce expulsion of this cell type at a test interval of 48 h. Mouse oviduct organ culture infected with B. pertussis demonstrated the same strict tropism for ciliated cells as in the tracheal ring system. Only ciliated cells were parasitized, becoming heavily colonized 48 h postinfection. Infected ciliated oviduct cells were not extruded. A fixation method which enhances fine structure was used in the scanning electron microscope studies. Bacterial fimbriae were not observed as the method of attachment of B. pertussis to cilia but fine fibers were seen extending between cilia and bacterial cells.

Microscopic Analysis of Porous Biodegradable Scaffolds for Tissue Engineering

2000 ◽  
Vol 6 (S2) ◽  
pp. 988-989
Author(s):  
F. Buevich ◽  
S. Pulapura ◽  
J. Kohn
Keyword(s):  
Tissue Regeneration ◽  
Combinatorial Library ◽  
Three Dimensional ◽  
Microscopic Analysis ◽  
Porous Scaffolds ◽  
Biodegradable Scaffolds ◽  
Condensation Polymers ◽  
Scaffold Architecture ◽  
Useful Lifetime

Introduction: There is considerable interest in the use of three-dimensional porous scaffolds for tissue regeneration. The presence of an interconnected framework of pores with large surface area facilitates the formation of extracellular matrix and permits cellular ingrowth into implanted structures. For scaffolds to be useful for tissue regeneration, they must maintain good dimensional stability during the lifetime of the implant. While the initial scaffold architecture is often well characterized, a systematic study of the influence of incubation on the scaffold architecture is critical to ensure that the scaffolds retain their interconnected network of pores during their useful lifetime. Herein, we report on the evaluation of the architecture of polyarylate scaffolds and their stability under in vitro conditions using scanning electron microscopy (SEM).The polymers used in this study were selected from a library of degradable polyarylates. This library is the first reported combinatorial library of biodegradable condensation polymers.

Fine structure and molecular organization of the periostracum in a gastropod mollusc Buccinum undatum L. and its relation to similar structural protein systems in other invertebrates

1978 ◽  
Vol 283 (998) ◽  
pp. 417-459 ◽  
Keyword(s):  
Electron Microscopy ◽  
Cholesteric Liquid Crystal ◽  
Three Dimensional ◽  
Coiled Coil ◽  
Longitudinal Axis ◽  
Molecular Organization ◽  
Structural Protein ◽  
Buccinum Undatum ◽  
Basic Features ◽  
Smooth Transitions

The horny layer of periostracum which covers the shell of Buccinum undatum L. has been studied by a combination of the fine structural techniques including high resolution transmission electron microscopy and scanning electron microscopy as well as by chemical analysis and X-ray diffraction. It has been found that the main structural component is a tectin type protein with globular and probably coiled-coil α-helical regions accompanied by a small amount of polysaccharide. Much of the periostracum is built up of protein sheets superposed in a regular manner and stabilized by some type of covalent cross-linking involving aromatic molecules. The protein is one of the class of structural macromolecules called scleroproteins. Each sheet of protein is made up of molecular sub-units which have a characteristic dumb-bell shape and which are about 32 nm long and 6.5 nm wide at their globular ends. End-to-end long-axis aggregation of these units produces filaments which aggregate further by side-to-side association into ribbons and ultimately sheets. The side-to-side association is always in register and hence the sheets have a major transverse striation repeating at 32 nm intervals. The protein sheets can be ascribed a longitudinal axis in terms of the direction of their component filaments. On this basis it can be shown that successive superposed sheets are rotated in a horizontal plane through an angle of 20-25° relative to one another, in a constant direction either clockwise or anticlockwise. Such helicoidal organization is of the cholesteric liquid crystal type which is often found in a biological context, e.g. chitin fibril disposition in arthropod cuticle. This helicoidal layering of the protein sheets is manifested in oblique sections of periostracum as repeated parabolic lamellae. Irregularities in the form of the parabolic lamellae can be accounted for on the basis of the curvature and extensive folding of the periostracum. The outer and innermost layers of the periostracum tend not to show helicoidal organization but exhibit a different aggregation mode of the dumb-bell-shaped units into a three-dimensional hexagonally packed network matrix. This matrix is much interrupted by vacuoles and localized smooth transitions into the ribbon mode of aggregation. This ability to exist in both fibrous and network aggregation states is comparable to that known among the collagens and muscle proteins. The amino acid compositions and conformations of proteins which can form cholesteric helicoidal systems are reviewed and compared with the protein of Buccinum periostracum. This property is apparently confined to alpha helical rod-shaped proteins and globular tektins. The beta conformation does not favour cholesteric organization. The structures and compositions of other molluscan periostraca and periostracum- like structures from other invertebrate phyla are compared with the periostracum of Buccinum . While all periostraca and functionally related structures have certain basic features in common there is a considerable degree of variation at the molecular and organizational levels.

scholarly journals Subaxolemmal cytoskeleton in squid giant axon. I. Biochemical analysis of microtubules, microfilaments, and their associated high-molecular-weight proteins.

1986 ◽  
Vol 102 (5) ◽  
pp. 1699-1709 ◽  
Author(s):  
T Kobayashi ◽  
S Tsukita ◽  
S Tsukita ◽  
Y Yamamoto ◽  
G Matsumoto
Keyword(s):  
Molecular Weight ◽  
High Molecular Weight ◽  
Actin Filaments ◽  
Giant Axon ◽  
Protein Molecule ◽  
Biochemical Properties ◽  
Cross Reactivity ◽  
Squid Giant Axon ◽  
Molecular Organization ◽  
Nacl Solution

Using the squid giant axon, we analyzed biochemically the molecular organization of the axonal cytoskeleton underlying the axolemma (subaxolemmal cytoskeleton). The preparation enriched in the subaxolemmal cytoskeleton was obtained by squeezing out the central part of the axoplasm using a roller. The electrophoretic banding pattern of the subaxolemmal cytoskeleton was characterized by large amounts of two high-molecular-weight (HMW) proteins (260 and 255 kD). The alpha, beta-tubulin, actin, and some other proteins were also its major constituents. The 260-kD protein is known to play an important role in maintaining the excitability of the axolemma (Matsumoto, G., M. Ichikawa, A. Tasaki, H. Murofushi, and H. Sakai, 1983, J. Membr. Biol., 77:77-91) and was recently designated "axolinin" (Sakai, H., G. Matsumoto, and H. Murofushi, 1985, Adv. Biophys., 19:43-89). We purified axolinin and the 255-kD protein in their native forms and further characterized their biochemical properties. The purified axolinin was soluble in 0.6 M NaCl solution but insoluble in 0.1 M NaCl solution. It co-sedimented with microtubules but not with actin filaments. In low-angle rotary-shadowing electron microscopy, the axolinin molecule in 0.6 M NaCl solution looked like a straight rod approximately 105 nm in length with a globular head at one end. On the other hand, the purified 255-kD protein was soluble in both 0.1 and 0.6 M NaCl solution and co-sedimented with actin filaments but not with microtubules. The 255-kD protein molecule appeared as a characteristic horseshoe-shaped structure approximately 35 nm in diameter. Furthermore, the 255-kD protein showed no cross-reactivity to the anti-axolinin antibody. Taken together, these characteristics lead us to conclude that the subaxolemmal cytoskeleton in the squid giant axon is highly specialized, and is mainly composed of microtubules and a microtubule-associated HMW protein (axolinin), and actin filaments and an actin filament-associated HMW protein (255-kD protein).

scholarly journals A Rapid Self-Assembly Peptide Hydrogel for Recruitment and Activation of Immune Cells

Molecules ◽  
2022 ◽  
Vol 27 (2) ◽  
pp. 419
Author(s):  
Ruyue Luo ◽  
Yuan Wan ◽  
Xinyi Luo ◽  
Guicen Liu ◽  
Zhaoxu Li ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Dendritic Cells ◽  
Self Assembly ◽  
Immune Cell ◽  
Three Dimensional ◽  
Hydrophobic Surface ◽  
Inhibitory Effect ◽  
Blue Staining ◽  
Peptide Hydrogel

Self-assembly peptide nanotechnology has attracted much attention due to its regular and orderly structure and diverse functions. Most of the existing self-assembly peptides can form aggregates with specific structures only under specific conditions and their assembly time is relatively long. They have good biocompatibility but no immunogenicity. To optimize it, a self-assembly peptide named DRF3 was designed. It contains a hydrophilic and hydrophobic surface, using two N-terminal arginines, leucine, and two c-terminal aspartate and glutamic acid. Meanwhile, the c-terminal of the peptide was amidated, so that peptide segments were interconnected to increase diversity. Its characterization, biocompatibility, controlled release effect on antigen, immune cell recruitment ability, and antitumor properties were examined here. Congo red/aniline blue staining revealed that peptide hydrogel DRF3 could be immediately gelled in PBS. The stable β-sheet secondary structure of DRF3 was confirmed by circular dichroism spectrum and IR spectra. The observation results of cryo-scanning electron microscopy, transmission electron microscopy, and atomic force microscopy demonstrated that DRF3 formed nanotubule-like and vesicular structures in PBS, and these structures interlaced with each other to form ordered three-dimensional nanofiber structures. Meanwhile, DRF3 showed excellent biocompatibility, could sustainably and slowly release antigens, recruit dendritic cells and promote the maturation of dendritic cells (DCs) in vitro. In addition, DRF3 has a strong inhibitory effect on clear renal cell carcinoma (786-0). These results provide a reliable basis for the application of peptide hydrogels in biomedical and preclinical trials.

scholarly journals Segregation of germline granules in early embryos of Caenorhabditis elegans: an electron microscopic analysis

Development ◽  
1983 ◽  
Vol 73 (1) ◽  
pp. 297-306
Author(s):  
Nurit Wolf ◽  
James Priess ◽  
David Hirsh
Keyword(s):  
Electron Microscopy ◽  
Caenorhabditis Elegans ◽  
Microscopic Analysis ◽  
Precursor Cell ◽  
Electron Microscopic Analysis ◽  
Fixation Method ◽  
Electron Microscopic ◽  
Early Embryos ◽  
Germline Cells

Using an improved fixation method for electron microscopy, we have found germline granules in Caenorhabditis elegans embryos shortly after fertilization and prior to the first cleavage. They are localized in the egg cytoplasm which becomes segregated into the posterior blastomere at the first cleavage. In the following divisions, the granules continue this pattern of asymmetric segregation and are ultimately segregated into the germline precursor cell. The granules are then symmetrically segregated into the germline cells.

Structural analysis of crystalline Ca++ transport ATPase vesicles

1983 ◽  
Vol 41 ◽  
pp. 636-637
Author(s):  
E. Loren Buhle ◽  
Barry E. Knox ◽  
Ueli Aebi
Keyword(s):  
Electron Microscopy ◽  
Sarcoplasmic Reticulum ◽  
Three Dimensional ◽  
Integral Membrane Proteins ◽  
Molecular Organization ◽  
Dimensional Structure ◽  
Crystal Formation ◽  
Transport Atpase ◽  
Ordered Arrays ◽  
Tubular Arrays

In our efforts to study the three-dimensional structure and molecular organization of ion-motive ATPases by electron microscopy and image processing we have systematically investigated conditions which induce the formation of ordered arrays of these integral membrane proteins. Among them we have explored the effect(s) of vanadate and other multi-valent ions on ATPase crystal formation by starting from published procedures [e.g. 1,2]. Here we present some preliminary results concerned with the packing and molecular organization of vanadate-induced crystalline arrays of a Ca++ transport ATPase in tubular rabbit sarcoplasmic reticulum vesicles.The Ca++ activated ATPase from rabbit sarcoplasmic reticulum was purified as published. Induction of ordered tubular arrays of the protein with vanadate was basically achieved as described, except that the protein was slowly dialysed against a vanadate-containing buffer rather than directly adding the vanadate to the protein. Optimal yields of crystalline arrays were obtained after two days of dialysis at 4°C.

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