scholarly journals CYTOCHEMICAL LOCALIZATION OF CHOLINESTERASE IN EMBRYONIC RABBIT CARDIAC MUSCLE

1970 ◽  
Vol 18 (1) ◽  
pp. 38-43 ◽  
Author(s):  
MARTIN HAGOPIAN ◽  
VIRGINIA M. TENNYSON ◽  
DAVID SPIRO
Keyword(s):  
Endoplasmic Reticulum ◽  
Sarcoplasmic Reticulum ◽  
Nuclear Envelope ◽  
Cardiac Muscle ◽  
Golgi Complex ◽  
Cardiac Myocyte ◽  
Cholinesterase Activity ◽  
Cytochemical Localization ◽  
Contractile Tissue ◽  
Rabbit Embryos

Cardiac muscle of rabbit embryos from day 9 through day 18 of gestation was studied by a modification of the Koelle-Friedenwald copper thiocholine technique for the localization of cholinesterase activity. In the earlier stages of development a cholinesterase, presumed to be acetylcholinesterase from its substrate and inhibitor specificity, is found in the abundant, randomly dispersed rough surfaced endoplasmic reticulum of the myoblast. Cytochemical end product is also occasionally found in the nuclear envelope, Golgi complex and subsarcolemmal cisternae. The localization of the enzyme first in the rough surfaced endoplasmic reticulum and Golgi complex and at later stages in the sarcoplasmic reticulum is significant with regard to the differentiation of the cardiac myocyte. Furthermore, the presence of this enzyme in contractile tissue before the appearance of nerves or nerve endings demonstrates an acetylcholine-cholinesterase system of myogenic origin.

scholarly journals THE LOCALIZATION OF CHOLINESTERASE ACTIVITY IN RAT CARDIAC MUSCLE BY ELECTRON MICROSCOPY

1964 ◽  
Vol 23 (2) ◽  
pp. 217-232 ◽  
Author(s):  
Morris J. Karnovsky
Keyword(s):  
Electron Microscopy ◽  
Endoplasmic Reticulum ◽  
Sarcoplasmic Reticulum ◽  
Enzymatic Activity ◽  
Cardiac Muscle ◽  
Heart Muscle ◽  
Cholinesterase Activity ◽  
Physiological Role ◽  
Ultrastructural Level ◽  
Formalin Fixed

A method has been developed for localizing sites of cholinesterase activity in rat cardiac muscle by electron microscopy. The method utilizes thiocholine esters as substrates, and is believed to be dependent on the reduction of ferricyanide to ferrocyanide by thiocholine released by enzymatic activity. The ferrocyanide thus formed is captured by copper to form fine, electron-opaque deposits of copper ferrocyanide, which sharply delineate sites of enzymatic activity at the ultrastructural level. Cholinesterase activity in formalin-fixed heart muscle was localized: (a) in longitudinal elements of the sarcoplasmic reticulum, but not in the T, or transverse, elements; and (b) in the A band, with virtually no activity noted in the M band, or in the H zone. The I band was also negative. No activity was detected in the sarcolemma, or in invaginations of the sarcolemma at the level of the Z band. The perinuclear element of the sarcoplasmic (endoplasmic) reticulum was frequently strongly positive. Activity at all sites was completely abolished by omitting the substrates, or by inhibition with eserine 10-4 M and diisopropylfluorophosphate 10-5 M. Eserine 10-5 M completely inhibited reaction in the sarcoplasmic reticulum, and virtually abolished that in the A band. These observations, together with the use of the relatively specific substrates and suitable controls to eliminate non-enzymatic staining, indicate that cholinesterase activity was being demonstrated. The activity in rat heart against different substrates was that of non-specific cholinesterases, in accordance with biochemical data. The activity in the A band was considered to be probably due to myosincholinesterase. It is proposed that the localization of cholinesterases in myocardium at the ultrastructural level should be taken into account in considering the possible functions of these myocardial enzymes, and it is hoped that knowledge of their localization will open up new avenues of approach in considering their physiological role in myocardium, which at present is not definitely known.

scholarly journals Endoplasmic reticulum-to-Golgi transitions upon herpes virus infection

F1000Research ◽  
2018 ◽  
Vol 6 ◽  
pp. 1804 ◽  
Author(s):  
Peter Wild ◽  
Andres Kaech ◽  
Elisabeth M. Schraner ◽  
Ladina Walser ◽  
Mathias Ackermann
Keyword(s):  
Endoplasmic Reticulum ◽  
Nuclear Envelope ◽  
Golgi Complex ◽  
Nuclear Membrane ◽  
Progeny Virus ◽  
Cytoplasmic Matrix ◽  
Outer Nuclear Membrane ◽  
Nuclear Membranes ◽  
Perinuclear Space ◽  
Hsv 1

Background: Herpesvirus capsids are assembled in the nucleus, translocated to the perinuclear space by budding, acquiring tegument and envelope, or released to the cytoplasm via impaired nuclear envelope. One model proposes that envelopment, “de-envelopment” and “re-envelopment” is essential for production of infectious virus. Glycoproteins gB/gH were reported to be essential for de-envelopment, by fusion of the “primary” envelope with the outer nuclear membrane. Yet, a high proportion of enveloped virions generated from genomes with deleted gB/gH were found in the cytoplasm and extracellular space, suggesting the existence of alternative exit routes.Methods: We investigated the relatedness between the nuclear envelope and membranes of the endoplasmic reticulum and Golgi complex, in cells infected with either herpes simplex virus 1 (HSV-1) or a Us3 deletion mutant thereof, or with bovine herpesvirus 1 (BoHV-1) by transmission and scanning electron microscopy, employing freezing technique protocols.Results:  The Golgi complex is a compact entity in a juxtanuclear position covered by a membrane on thecisface. Golgi membranes merge with membranes of the endoplasmic reticulum forming an entity with the perinuclear space. All compartments contained enveloped virions. After treatment with brefeldin A, HSV-1 virions aggregated in the perinuclear space and endoplasmic reticulum, while infectious progeny virus was still produced.Conclusions: The data suggest that virions derived by budding at nuclear membranes are intraluminally transported from the perinuclear space via Golgi -endoplasmic reticulum transitions into Golgi cisternae for packaging. Virions derived by budding at nuclear membranes are infective like Us3 deletion mutants, which  accumulate in the perinuclear space. Therefore, i) de-envelopment followed by re-envelopment is not essential for production of infective progeny virus, ii) the process taking place at the outer nuclear membrane is budding not fusion, and iii) naked capsids gain access to the cytoplasmic matrix via impaired nuclear envelope as reported earlier.

scholarly journals Vesicle budding from endoplasmic reticulum is involved in calsequestrin routing to sarcoplasmic reticulum of skeletal muscles

2004 ◽  
Vol 379 (2) ◽  
pp. 505-512 ◽  
Author(s):  
Alessandra NORI ◽  
Elena BORTOLOSO ◽  
Federica FRASSON ◽  
Giorgia VALLE ◽  
Pompeo VOLPE
Keyword(s):  
Skeletal Muscle ◽  
Endoplasmic Reticulum ◽  
Sarcoplasmic Reticulum ◽  
Golgi Complex ◽  
Skeletal Muscles ◽  
Muscle Fibres ◽  
Vesicle Budding ◽  
Er Exit Sites ◽  
Skeletal Muscle Fibres ◽  
Mutant Forms

CS (calsequestrin) is an acidic glycoprotein of the SR (sarcoplasmic reticulum) lumen and plays a crucial role in the storage of Ca2+ and in excitation–contraction coupling of skeletal muscles. CS is synthesized in the ER (endoplasmic reticulum) and is targeted to the TC (terminal cisternae) of SR via mechanisms still largely unknown, but probably involving vesicle transport through the Golgi complex. In the present study, two mutant forms of Sar1 and ARF1 (ADP-ribosylation factor 1) were used to disrupt cargo exit from ER-exit sites and intra-Golgi trafficking in skeletal-muscle fibres respectively. Co-expression of Sar1-H79G (His79→Gly) and recombinant, epitope-tagged CS, CSHA1 (where HA1 stands for nine-amino-acid epitope of the viral haemagglutinin 1), barred segregation of CSHA1 to TC. On the other hand, expression of ARF1-N126I altered the subcellular localization of GM130, a cis-medial Golgi protein in skeletal-muscle fibres and myotubes, without interfering with CSHA1 targeting to either TC or developing SR. Thus active budding from ER-exit sites appears to be involved in CS targeting and routing, but these processes are insensitive to modification of intracellular vesicle trafficking and Golgi complex disruption caused by the mutant ARF1-N126I. It also appears that CS routing from ER to SR does not involve classical secretory pathways through ER–Golgi intermediate compartments, cis-medial Golgi and trans-Golgi network.

scholarly journals Endoplasmic reticulum-to-Golgi transitions upon herpes virus infection

F1000Research ◽  
2017 ◽  
Vol 6 ◽  
pp. 1804 ◽  
Author(s):  
Peter Wild ◽  
Andres Kaech ◽  
Elisabeth M. Schraner ◽  
Ladina Walser ◽  
Mathias Ackermann
Keyword(s):  
Electron Microscopy ◽  
Scanning Electron Microscopy ◽  
Endoplasmic Reticulum ◽  
Nuclear Envelope ◽  
Golgi Complex ◽  
Progeny Virus ◽  
Perinuclear Space ◽  
Exit Route ◽  
Scanning Electron ◽  
Hsv 1

Background: Herpesvirus capsids are assembled in the nucleus before they are translocated to the perinuclear space by budding, acquiring tegument and envelope, or releasing to the cytoplasm in a “naked” state via impaired nuclear envelope. One model proposes that envelopment, “de-envelopment” and “re-envelopment” are essential steps for production of infectious virus. Glycoproteins gB/gH were reported to be essential for de-envelopment, by fusion of the “primary” envelope with the outer nuclear membrane. Yet, a high proportion of enveloped virions generated from genomes with deleted gB/gH were found in the cytoplasm and extracellular space, suggesting the existence of an alternative exit route.Methods: We investigated the relatedness between the nuclear envelope and membranes of the endoplasmic reticulum and Golgi complex, in cells infected with either herpes simplex virus 1 (HSV-1) or a Us3 deletion mutant thereof, or with bovine herpesvirus 1 (BoHV-1) by transmission and scanning electron microscopy, employing freezing technique protocols that lead to improved spatial and temporal resolution.Results: Scanning electron microscopy showed the Golgi complex as a compact entity in a juxtanuclear position covered by a membrane on thecisface. Transmission electron microscopy revealed that Golgi membranes merge with membranes of the endoplasmic reticulum forming an entity with the perinuclear space. All compartments contained enveloped virions. After treatment with brefeldin A, HSV-1 virions aggregated in the perinuclear space and endoplasmic reticulum, while infectious progeny virus was still produced.Conclusions: The data strongly suggest that virions are intraluminally transported from the perinuclear space via Golgi complex-endoplasmic reticulum transitions into Golgi cisternae for packaging into transport vacuoles. Furthermore, virions derived by budding at nuclear membranes are infective as has been shown for HSV-1 Us3 deletion mutants, which almost entirely accumulate in the perinuclear space. Therefore, de-envelopment followed by re-envelopment is not essential for production of infective progeny virus.

scholarly journals CYTOCHEMICAL LOCALIZATION OF CHOLINESTERASE ACTIVITY IN ADULT RABBIT HEART

1971 ◽  
Vol 19 (6) ◽  
pp. 376-381 ◽  
Author(s):  
MARTIN HAGOPIAN ◽  
VIRGINIA M. TENNYSON
Keyword(s):  
Sarcoplasmic Reticulum ◽  
Papillary Muscle ◽  
Cholinesterase Activity ◽  
Acetylcholinesterase Activity ◽  
Rabbit Heart ◽  
Adult Rabbit ◽  
Cytochemical Localization ◽  
Excitation Contraction Coupling ◽  
Contraction Coupling ◽  
T System

The papillary muscle of the adult rabbit heart was studied by a modification of the Koelle-Friedenwald copper thiocholine technique for the localization of cholinesterase activity. Butyrylcholinesterase (BuChE), identified by its substrate and inhibitor specificity, is found mainly in the terminal sacs of the sarcoplasmic reticulum adjacent to the T system. The localization of the reaction product in this particular site suggests that BuChE may play a role in excitation-contraction coupling in the adult rabbit heart. The present findings are also discussed in comparison with our previous work on the localization of acetylcholinesterase activity in the embryonic rabbit heart.

Butyrylcholinesterase in the Adult Rabbit Heart

1971 ◽  
Vol 29 ◽  
pp. 284-285
Author(s):  
M. Hagopian ◽  
V.M. Tennyson
Keyword(s):  
Endoplasmic Reticulum ◽  
Sarcoplasmic Reticulum ◽  
Papillary Muscle ◽  
Cholinesterase Activity ◽  
Ultrastructural Level ◽  
Rabbit Heart ◽  
Adult Rabbit ◽  
Intercalated Disc ◽  
Complex Forms ◽  
T System

The papillary muscle of the adult rabbit heart was studied by a modification of the copper thiocholine technique for the localization of cholinesterase activity. At the ultrastructural level the muscle shows evidence of butyrylcholinesterase (BuChE) in view of the fact that an electron-opaque reaction product, a copper thiocholine complex, forms when butyrylthiocholine (BuThCh) is used as substrate. The deposition of the reaction product was abolished by preincubation with iso-OMPA (tetraisopropyl pyrophosphortetramide) which specifically inhibits BuChE, but not by BW284C51 (1,5-bis [4-allyldimethylammoniumphenyl] penta-3-one dibromide) which inhibits acetylcholinesterase (AChE). When acetylthiocholine (AThCh) was used as substrate, no end product was found.BuChE activity is seen primarily in the terminal sacs of the sarcoplasmic reticulum (SR) in the region adjacent to the T system (Fig. 1), within the subsarcolemmal vesicles or tubules, and in the endoplasmic reticulum (Fig. 2) of the perinuclear zone. The reaction sites are found occasionally in some areas of the longitudinal elements of the SR, on the nuclear envelope (Fig.2), and in the cisterns which are associated with the intercalated disc. The discs are always negative.

scholarly journals Peroxidase-positive endothelial cells in sinusoids of the mouse liver.

1978 ◽  
Vol 26 (5) ◽  
pp. 409-411 ◽  
Author(s):  
G Stöhr ◽  
W Deimann ◽  
H D Fahimi
Keyword(s):  
Endothelial Cells ◽  
Endoplasmic Reticulum ◽  
Golgi Apparatus ◽  
Nuclear Envelope ◽  
Kupffer Cells ◽  
Peroxidase Activity ◽  
Mouse Liver ◽  
Negative Reaction ◽  
Cytochemical Localization ◽  
Endogenous Peroxidase

The cytochemical localization of endogenous peroxidase activity in sinus lining cells of mouse liver has been investigated. Kupffer cells, as identified by their exclusive ability to phagocytize large (0.8 micron) latex particles, exhibited strong peroxidase activity in nuclear envelope and endoplasmic reticulum. In addition, weak to moderate peroxidase activity was found in 57% of all endothelial cells. The enzyme in endothelial cells was also localized in nuclear envelope and endoplasmic reticulum, with a negative reaction in the Golgi apparatus. These observations indicate that peroxidase staining, as a marker for identification of Kupffer cells in mouse liver, is only of limited value and should be used in conjunction with other methods (e.g., latex phagocytosis).

scholarly journals Varicella-zoster viral glycoprotein envelopment: ultrastructural cytochemical localization.

1986 ◽  
Vol 34 (2) ◽  
pp. 281-284 ◽  
Author(s):  
E A Montalvo ◽  
R T Parmley ◽  
C Grose
Keyword(s):  
Endoplasmic Reticulum ◽  
Nuclear Envelope ◽  
Varicella Zoster Virus ◽  
Nuclear Membrane ◽  
Extracellular Space ◽  
Human Melanoma ◽  
Viral Glycoprotein ◽  
Cytochemical Localization ◽  
Varicella Zoster ◽  
Cytoplasmic Vacuoles

The periodate-thiocarbohydrazide silver proteinate (PA-TCH-SP) method was used to study the envelopment process in varicella-zoster virus-infected human melanoma cells. Viral envelopment could be seen at two sites, the nuclear membrane and at virus-induced intracytoplasmic vacuoles. Virus-associated glycoconjugates were detected by the PA-TCH-SP method at the plasmalemma and on the inner membrane of the intracytoplasmic vacuoles. Virion envelopes acquired at the nuclear membrane were PA-TCH-SP negative, whereas those acquired at intracytoplasmic vacuoles were PA-TCH-SP positive. All virions found inside these vacuoles contained periodate-reactive envelopes. Release of virions into the extracellular space, where virtually all virions were PA-TCH-SP positive, appeared to be via exocytosis. Thus, the PA-TCH-SP method identifies glycoprotein incorporation at specific cytoplasmic vacuoles distinct from nuclear envelope, endoplasmic reticulum, and Golgi lamellae. These results suggest that envelopment within the cytoplasm is a stage in the assembly of the varicella-zoster virion.

scholarly journals Smooth muscle expresses a cardiac/slow muscle isoform of the Ca2+-transport ATPase in its endoplasmic reticulum

1989 ◽  
Vol 257 (1) ◽  
pp. 117-123 ◽  
Author(s):  
F Wuytack ◽  
Y Kanmura ◽  
J A Eggermont ◽  
L Raeymaekers ◽  
J Verbist ◽  
...  
Keyword(s):  
Skeletal Muscle ◽  
Endoplasmic Reticulum ◽  
Smooth Muscle ◽  
Sarcoplasmic Reticulum ◽  
Cardiac Muscle ◽  
Muscle Enzymes ◽  
Transport Atpase ◽  
Slow Twitch ◽  
Transport Atpases ◽  
Fast Twitch

Smooth muscle expresses in its endoplasmic reticulum an isoform of the Ca2+-transport ATPase that is very similar to or identical with that of the cardiac-muscle/slow-twitch skeletal-muscle form. However, this enzyme differs from that found in fast-twitch skeletal muscle. This conclusion is based on two independent sets of observations, namely immunological observations and phosphorylation experiments. Immunoblot experiments show that two different antibody preparations against the Ca2+-transport ATPase of cardiac-muscle sarcoplasmic reticulum also recognize the endoplasmic-reticulum/sarcoplasmic-reticulum enzyme of the smooth muscle and the slow-twitch skeletal muscle whereas they bind very weakly or not at all to the sarcoplasmic-reticulum Ca2+-transport ATPase of the fast-twitch skeletal muscle. Conversely antibodies directed against the fast-twitch skeletal-muscle isoform of the sarcoplasmic-reticulum Ca2+-transport ATPase do not bind to the cardiac-muscle, smooth-muscle or slow-twitch skeletal-muscle enzymes. The phosphorylated tryptic fragments A and A1 of the sarcoplasmic-reticulum Ca2+-transport ATPases have the same apparent Mr values in cardiac muscle, slow-twitch skeletal muscle and smooth muscle, whereas the corresponding fragments in fast-twitch skeletal muscle have lower apparent Mr values. This analytical procedure is a new and easy technique for discrimination between the isoforms of endoplasmic-reticulum/sarcoplasmic-reticulum Ca2+-transport ATPases.

A possible structural basis for the extracellular release of acetylcholinesterase

1975 ◽  
Vol 191 (1103) ◽  
pp. 271-283 ◽  
Keyword(s):  
Endoplasmic Reticulum ◽  
Nuclear Envelope ◽  
Chromaffin Cells ◽  
Plasma Membranes ◽  
Cholinesterase Activity ◽  
Smooth Endoplasmic Reticulum ◽  
Splanchnic Nerve ◽  
Ultrastructural Localization ◽  
Acetylcholinesterase Activity ◽  
Structural Basis

The ultrastructural localization of acetylcholinesterase and non-specific cholinesterase activity has been studied in sections of ox adrenal medulla by cytochemical methods. Non-specific cholinesterase activity, identified by using butyrylthiocholine as substrate and ethopropazine as inhibitor, occurs intracellularly in some adrenaline-containing chromaffin cells: the reaction end-product is deposited within the cisternae of the endoplasmic reticulum and in the nuclear envelope. Reaction end-product of non-specific cholinesterase also occurs in the endoplasmic reticulum of pericytes, around sinusoids and capillaries and within smooth muscle cells. Acetylcholinesterase activity, identified by using acetylthiocholine as substrate and BW 284C51 as inhibitor, occurs in both the splanchnic nerve and in chromaffin cells. Reaction end-product is found at the following sites (i) around myelinated and unmyelinated non-terminal axons of splanchnic nerve, between the axolemma and the Schwann cell membrane; (ii) within the cisternae of axonal smooth endoplasmic reticulum; sometimes these cisternae appear to be connected to the axolemma; (iii) between the axolemmas of preterminal axons and the plasma membranes of chromaffin cells; (iv) between the axolemmas of nerve terminals and the plasma membranes of chromaffin cells, including the synaptic cleft; (v) within cisternae of rough and smooth endoplasmic reticulum, and also within the nuclear envelope, of both adrenaline- and noradrenaline-containing chromaffin cells; (vi) between the plasma membranes of adjacent chromaffin cells, but only when one or both of these cells contain reaction product within the cisternae of its endoplasmic reticulum; these cisternae sometimes appear to be connected to the plasma membrane. These observations raise the question whether the acetylcholinesterase activity released from the perfused adrenal gland might originate from the cisternae of the endoplasmic reticula of splanchnic nerve and/or chromaffin cell.

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