scholarly journals Assembly of different isoforms of actin and tropomyosin into the skeletal tropomyosin-enriched microfilaments during differentiation of muscle cells in vitro.

1986 ◽  
Vol 103 (6) ◽  
pp. 2173-2183 ◽  
Author(s):  
J J Lin ◽  
J L Lin
Keyword(s):  
Monoclonal Antibody ◽  
Striated Muscle ◽  
Chicken Embryo Fibroblast ◽  
Muscle Cells ◽  
Electron Microscopic ◽  
Thin Filaments ◽  
Microscopic Studies ◽  
The One ◽  
Muscle Isoforms

We have used a monoclonal antibody (CL2) directed against striated muscle isoforms of tropomyosin to selectively isolate a class of microfilaments (skeletal tropomyosin-enriched microfilaments) from differentiating muscle cells. This class of microfilaments differed from the one (tropomyosin-enriched microfilaments) isolated from the same cells by a monoclonal antibody (LCK16) recognizing all isoforms of muscle and nonmuscle tropomyosin. In myoblasts, the skeletal tropomyosin-enriched microfilaments had a higher content of alpha-actin and phosphorylated isoforms of tropomyosin as compared with the tropomyosin-enriched microfilaments. Moreover, besides muscle isoforms of actin and tropomyosin, significant amounts of nonmuscle isoforms of actin and tropomyosin were found in the skeletal tropomyosin-enriched microfilaments of myoblasts and myotubes. These results suggest that different isoforms of actin and tropomyosin can assemble into the same set of microfilaments, presumably pre-existing microfilaments, to form the skeletal tropomyosin-enriched microfilaments, which will eventually become the thin filaments of myofibrils. Therefore, the skeletal tropomyosin-enriched microfilaments detected here may represent an intermediate class of microfilaments formed during thin filament maturation. Electron microscopic studies of the isolated microfilaments from myoblasts and myotubes showed periodic localization of tropomyosin molecules along the microfilaments. The tropomyosin periodicity in the microfilaments of myoblasts and myotubes was 35 and 37 nm, respectively, whereas the nonmuscle tropomyosin along chicken embryo fibroblast microfilaments had a 34-nm repeat.

Cytoplasmic Granules in Lymphocytes from Rats Dosed with SK&F 14336-D

1971 ◽  
Vol 29 ◽  
pp. 556-557
Author(s):  
T. G. Merrill ◽  
B. J. Payne ◽  
A. J. Tousimis
Keyword(s):  
Lipid Storage ◽  
Pokeweed Mitogen ◽  
Electron Microscopic ◽  
Cytoplasmic Granules ◽  
Storage Diseases ◽  
Tay Sachs Disease ◽  
Large Numbers ◽  
Microscopic Studies ◽  
Neurovisceral Lipidosis

Rats given SK&F 14336-D (9-[3-Dimethylamino propyl]-2-chloroacridane), a tranquilizing drug, developed an increased number of vacuolated lymphocytes as observed by light microscopy. Vacuoles in peripheral blood of rats and humans apparently are rare and are not usually reported in differential counts. Transforming agents such as phytohemagglutinin and pokeweed mitogen induce similar vacuoles in in vitro cultures of lymphocytes. These vacuoles have also been reported in some of the lipid-storage diseases of humans such as amaurotic familial idiocy, familial neurovisceral lipidosis, lipomucopolysaccharidosis and sphingomyelinosis. Electron microscopic studies of Tay-Sachs' disease and of chloroquine treated swine have demonstrated large numbers of “membranous cytoplasmic granules” in the cytoplasm of neurons, in addition to lymphocytes. The present study was undertaken with the purpose of characterizing the membranous inclusions and developing an experimental animal model which may be used for the study of lipid storage diseases.

Abstract WMP31: Dedifferentiation of Smooth Muscle Cells and its Potential Contribution to Pathogenesis of Intracranial Aneurysms

Stroke ◽  
2020 ◽  
Vol 51 (Suppl_1) ◽  
Author(s):  
Mieko Oka ◽  
Nobuhiko Ohno ◽  
Takakazu Kawamata ◽  
Tomohiro Aoki
Keyword(s):  
Endothelial Cells ◽  
Smooth Muscle ◽  
Smooth Muscle Cells ◽  
Inflammatory Responses ◽  
Muscle Cells ◽  
Inflammatory Factors ◽  
Potential Contribution ◽  
Electron Microscopic

Introduction: Intracranial aneurysm (IA) affects 1 to 5 % in general public and becomes the primary cause of subarachnoid hemorrhage, the most severe form of stroke. However, currently, no drug therapy is available for IAs to prevent progression and rupture of lesions. Elucidation of mechanisms underlying the disease is thus mandatory. Considering the important role of vascular smooth muscle cells (SMCs) in the maintenance of stiffness of arterial walls and also in the pathogenesis of atherosclerosis via mediating inflammatory responses, we in the present study analyzed morphological or phenotypical changes of SMCs during the disease development in the lesions. Methods: We subjected rats to an IA model in which lesions are induced by increase of hemodynamic force loading on intracranial arterial bifurcations and performed histopathological analyses of induced lesions including the electron microscopic examination. We then immunostained specimens from induced lesions to explore factors responsible for dedifferentiation or migration of SMCs. In vitro study was also done to examine effect of some candidate factors on dedifferentiation or migration of cultured SMCs. Results: We first found the accumulation of SMCs underneath the endothelial cell layer mainly at the neck portion of the lesion. These cells was positive for the embryonic form of myosin heavy chain, a marker for the dedifferentiated SMCs, and the expression of pro-inflammatory factors like TNF-α. In immunostaining to explore the potential factor regulating the dedifferentiation of SMCs, we found that Platelet-derived growth factor-BB (PDGF-BB) was expressed in endothelial cells at the neck portion of IA walls. Consistently, recombinant PDGF-BB could promote the dedifferentiate of SMCs and chemo-attracted them in in vitro. Finally, in the stenosis model of the carotid artery, PDGF-BB expression was induced in endothelial cells in which high wall shear stress was loaded and the dedifferentiation of SMCs occurred there. Conclusions: The findings from the present study imply the role of dedifferentiated SMCs partially recruited by PDGF-BB from endothelial cells in the formation of inflammatory microenvironment at the neck portion of IA walls, leading to the progression of the disease.

Estramustine binds MAP-2 to inhibit microtubule assembly in vitro

1988 ◽  
Vol 89 (3) ◽  
pp. 331-342
Author(s):  
M.E. Stearns ◽  
K.D. Tew
Keyword(s):  
Linear Regression Analysis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Gel Filtration ◽  
Microtubule Assembly ◽  
Scatchard Analysis ◽  
Binding Assays ◽  
Electron Microscopic ◽  
Associated Proteins ◽  
Microscopic Studies

We have investigated the ability of estramustine to bind to rat brain microtubule-associated proteins (MAPs) and purified MAP-2 in vitro. [3H]estramustine's relative affinity for tubulin and MAPs was assessed by gel filtration chromatography, immunoprecipitation and binding assays. Scatchard analysis demonstrated a specific affinity of the drug for MAP-2. Calculations from kinetic parameters and non-linear regression analysis gave a Kd of 15 microM, and a Bmax of 3.4 × 10(−7)M ml-1. Extrapolation of this value suggested that each MAP-2 molecule binds approximately 20 molecules of estramustine. Microtubule assembly studies and SDS-polyacrylamide gel electrophoresis revealed that at 20–60 microM levels, estramustine inhibited the association of MAPs with taxol microtubules. Turbidity (A350) studies further demonstrated that 20–60 microM-estramustine inhibited MAP-2-driven tubulin assembly and produced microtubule disassembly. Electron-microscopic studies confirmed the centrifugation and turbidity results. The data demonstrated that estramustine can bind MAPs and MAP-2 specifically, thereby inhibiting microtubule assembly.

Electron microscopic studies on the structure of motile primordial germ cells of Xenopus laevis in vitro

Development ◽  
1978 ◽  
Vol 46 (1) ◽  
pp. 119-133
Author(s):  
Janet Heasman ◽  
C. C. Wylie
Keyword(s):  
Xenopus Laevis ◽  
Germ Cells ◽  
Primordial Germ Cells ◽  
Structural Features ◽  
Culture Conditions ◽  
Structural Basis ◽  
Electron Microscopic ◽  
Microscopic Studies

Primordial germ cells (PGCs) of Xenopus laevis have been isolated from early embryos and kept alive in vitro, in order to study the structural basis of their motility, using the transmission and scanning electron microscope. The culture conditions used mimicked as closely as possible the in vivo environment of migrating PGCs, in that isolated PGCs were seeded onto monolayers of amphibian mesentery cells. In these conditions we have demonstrated that: (a) No significant differences were found between the morphology of PGCs in vitro and in vivo. (b) Structural features involved in PGC movement in vitro include (i) the presence of a filamentous substructure, (ii) filopodial and blunt cell processes, (iii) cell surface specializations. These features are also characteristic of migratory PGCs studied in vivo. (c) PGCs in vitro have powers of invasion similar to those of migrating PGCs in vivo. They occasionally become completely surrounded by cells of the monolayer and, in this situation, bear striking resemblance to PGCs moving between mesentery cells to the site of the developing gonad in stage-44 tadpoles. We conclude that as far as it is possible to assess, the behaviour of isolated PGCs in these in vitro conditions mimics their activities in vivo. This allows us to study the ultrastructural basis of their migration.

scholarly journals Myosin VI: cellular functions and motor properties

2004 ◽  
Vol 359 (1452) ◽  
pp. 1931-1944 ◽  
Author(s):  
K. C. Holmes ◽  
D. R. Trentham ◽  
R. Simmons ◽  
Rhys Roberts ◽  
Ida Lister ◽  
...  
Keyword(s):  
Optical Tweezers ◽  
Leading Edge ◽  
Force Transducer ◽  
Myosin Vi ◽  
Cellular Functions ◽  
Electron Microscopic ◽  
Coated Pits ◽  
Microscopic Studies ◽  
Golgi Network

Myosin VI has been localized in membrane ruffles at the leading edge of cells, at the trans–Golgi network compartment of the Golgi complex and in clathrin–coated pits or vesicles, indicating that it functions in a wide variety of intracellular processes. Myosin VI moves along actin filaments towards their minus end, which is the opposite direction to all of the other myosins so far studied (to our knowledge), and is therefore thought to have unique properties and functions. To investigate the cellular roles of myosin VI, we identified various myosin VI binding partners and are currently characterizing their interactions within the cell. As an alternative approach, we have expressed and purified full–length myosin VI and studied its in vitro properties. Previous studies assumed that myosin VI was a dimer, but our biochemical, biophysical and electron microscopic studies reveal that myosin VI can exist as a stable monomer. We observed, using an optical tweezers force transducer, that monomeric myosin VI is a non–processive motor which, despite a relatively short lever arm, generates a large working stroke of 18 nm. Whether monomer and/or dimer forms of myosin VI exist in cells and their possible functions will be discussed.

scholarly journals Biotinylphallotoxins: preparation and use as actin probes.

1989 ◽  
Vol 37 (7) ◽  
pp. 1035-1045 ◽  
Author(s):  
H Faulstich ◽  
S Zobeley ◽  
U Bentrup ◽  
B M Jockusch
Keyword(s):  
Actin Filaments ◽  
Ultrastructural Level ◽  
Microscopic Level ◽  
Electron Microscopic Level ◽  
Electron Microscopic ◽  
Permeabilized Cells ◽  
High Affinity ◽  
Double Label ◽  
The One

We describe the synthesis of four phalloidin derivatives conjugated with biotin. An aminomethyldithiolane derivative of ketophalloidin was used as a reactive starter compound, and biotin residues were coupled to this molecule either directly, separated by spacer chains comprised of one or two glycyl residues, or of a 12-atom long chain constructed from succinic acid and hexamethylendiamine. Although all products still displayed a high affinity for F-actin, as seen in competition experiments with [3H]-demethylphalloidin, only the one with the longest spacer (BHPP) showed specific and high-affinity decoration of actin filaments in permeabilized cells, in conjunction with FITC-coupled avidin and fluorescence microscopy. Combined with gold-streptavidin, BHPP decorated the actin filament system at the light and electron microscopic level faithfully and with satisfactory density. Actin filaments polymerized in vitro from purified protein were not as densely labeled as had been expected. However, in all these experiments the new phalloidin probe, when combined with avidin or streptavidin, yielded clear and highly specific labeling of F-actin. Therefore, this system is useful to identify and localize actin unambiguously in microfilaments, independent of actin antibodies, and should facilitate double-label experiments on cytoskeletal components at the ultrastructural level.

scholarly journals Accelerated myeloblast destruction and abnormal lysosome disruption in cultured bone marrow: association with indolent acute myelogenous leukemia

Blood ◽  
1976 ◽  
Vol 48 (1) ◽  
pp. 23-32 ◽  
Author(s):  
RF Branda ◽  
HS Jacob ◽  
SD Douglas ◽  
CF Moldow ◽  
RR Puumala
Keyword(s):  
Bone Marrow ◽  
Acute Myelogenous Leukemia ◽  
Myelogenous Leukemia ◽  
Leukemic Cells ◽  
Electron Microscopic ◽  
Granule Formation ◽  
Microscopic Studies ◽  
Acute Myelogenous ◽  
Primary Granules

Abstract Despite no chemotherapy and a marrow morphologically typical of frank relapse, an acute myelogenous leukemia (AML) patient survived for nearly 1 yr. During this time she remained asymptomatic and maintained nearly normal levels of platelets and hemoglobin. Cytochemical and electron microscopic studies of her bone marrow in liquid culture revealed on several occasions a unique maturational sequence in that leukemic cells differentiated to form morphologically abnormal primary granules which appeared to rupture and cause cytolysis of these cells. In these cultures, blasts rapidly disappeared and were replaced by more mature granulocytes, in contrast to observations in cultures derived from five other patients with AML in relapse which showed persistently elevated blast counts with no evidence of maturation in vitro. These findings support the concept that in AML cell maturation is regularly impaired and in some cases also aberrant. In addition, the abnormal granule formation with autolysis of the leukemic cells observed in one patient may explain both the early cell death in vitro and this patient's relatively indolent clinical course. Similar in vitro studies may help predict atypical clinical courses in patients with AML and facilitate design of appropriate chemotherapy.

scholarly journals Platelet Satellitism

Blood ◽  
1974 ◽  
Vol 43 (6) ◽  
pp. 831-836 ◽  
Author(s):  
Carl R. Kjeldsberg ◽  
John Swanson
Keyword(s):  
Polymorphonuclear Leukocytes ◽  
Plasma Membranes ◽  
Clinical Importance ◽  
Electron Microscopic ◽  
Normal Platelet ◽  
Microscopic Studies ◽  
Platelet Satellitism ◽  
Platelet Functions

Abstract Platelet adherence to polymorphonuclear leukocytes, or so-called platelet satellitism, has, to our knowledge, been reported in only four patients. We had the opportunity to study this phenomenon in two patients. Platelet satellitism was only seen in EDTA anticoagulated blood, and the platelets were seen to surround polymorphonuclear leukocytes only. Electron microscopic studies demonstrated focally opposed regions of platelet and neutrophil plasma membranes. Phagocytosis of platelets was also observed. In vivo and in vitro platelet functions were normal. Platelet satellitism is an in vitro phenomenon, the cause of which is unknown. We are unable to relate it to functional abnormalitles of the blood, the clinical condition of the patient, or to drugs. This phenomenon has some clinical importance in that it causes spurious thrombocytopenia.

Structural and functional study of control of canine tracheal smooth muscle

1980 ◽  
Vol 238 (1) ◽  
pp. C27-C33 ◽  
Author(s):  
M. S. Kannan ◽  
E. E. Daniel
Keyword(s):  
Smooth Muscle ◽  
Electrical Field Stimulation ◽  
Sympathetic Nerves ◽  
Tracheal Smooth Muscle ◽  
Functional Study ◽  
Field Stimulation ◽  
Electron Microscopic ◽  
Microscopic Studies ◽  
Stimulation Of

The structural bases for myogenic and neurogenic control of canine tracheal smooth muscle were studied. At optimum lengths, strips of muscle showed insignificant neurogenic or myogenic tone. Atropine and/or tetrodotoxin blocked the contractile responses elicited on electrical field stimulation of intrinsic nerves. After raising the tone with tetraethylammonium ion and in the presence of atropine, field stimulation of nerves caused a relaxation, a major component of which was blocked by propranolol and/or tetrodotoxin, suggesting an effect mediated through interaction of mediator released from sympathetic nerves with beta-adrenergic receptors. Electron microscopic studies revealed gap junctions between extensions of smooth-muscle cells and a sparse innervation. The axonal varicosities, corresponding to cholinergic (predominantly) and adrenergic (occasionally) nerves, were seen predominantly in the clefts between cell bundles. The physiological responses were compared with the morphological features. Although this muscle exhibits multiunit behavior in vitro, implying that nerves initiate the coordinate activity, its ultrastructural features suggest a potential for single-unit behavior.

scholarly journals THE RELATIONSHIP BETWEEN MYOFILAMENT PACKING DENSITY AND SARCOMERE LENGTH IN FROG STRIATED MUSCLE

1967 ◽  
Vol 33 (2) ◽  
pp. 255-263 ◽  
Author(s):  
Philip W. Brandt ◽  
Enrique Lopez ◽  
John P. Reuben ◽  
Harry Grundfest
Keyword(s):  
Cross Sections ◽  
Packing Density ◽  
Sarcomere Length ◽  
Striated Muscle ◽  
X Ray Diffraction ◽  
Electron Microscopic ◽  
Semitendinosus Muscle ◽  
Direct Function ◽  
Microscopic Studies

In cross-sections of single fibers from the frog semitendinosus muscle the number of thick myofilaments per unit area (packing density) is a direct function of the sarcomere length. Our data, derived from electron microscopic studies, fit well with other data derived from in vivo, low-angle X-ray diffraction studies of whole semitendinosus muscles. The data are consistent with the assumption that the sarcomere of a fibril maintains a constant volume during changes in sarcomere length. The myofilament lattice, therefore, expands as the sarcomere shortens. Since the distance between adjacent myofilaments is an inverse square root function of sarcomere length, the interaction of the thick and the thin myofilaments during sarcomere shortening may occur over distances which increase 70 A or more. The "expanding-sarcomere, sliding-filament" model of sarcomere shortening is discussed in terms of the current concepts of muscle architecture and contraction.

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