In Vivo Cryotechniques for Preparation of Animal Tissues for Immunoelectron Microscopy

2010 ◽  
pp. 167-179 ◽  
Author(s):  
Shinichi Ohno ◽  
Nobuhiko Ohno ◽  
Nobuo Terada ◽  
Sei Saitoh ◽  
Yurika Saitoh ◽  
...  
Keyword(s):  
Immunoelectron Microscopy ◽  
Animal Tissues

scholarly journals Membrane Translocation of 15-Lipoxygenase in Hematopoietic Cells Is Calcium-Dependent and Activates the Oxygenase Activity of the Enzyme

Blood ◽  
1998 ◽  
Vol 91 (1) ◽  
pp. 64-74 ◽  
Author(s):  
Roland Brinckmann ◽  
Kerstin Schnurr ◽  
Dagmar Heydeck ◽  
Thomas Rosenbach ◽  
Gerhard Kolde ◽  
...  
Keyword(s):  
Fatty Acid ◽  
Immunoelectron Microscopy ◽  
Membrane Lipids ◽  
Membrane Binding ◽  
Hematopoietic Cells ◽  
Cell Fractionation ◽  
Calcium Dependent ◽  
Oxygenase Activity ◽  
Lipoxygenase Products

Abstract Mammalian 15-lipoxygenases, which have been implicated in the differentiation of hematopoietic cells are commonly regarded as cytosolic enzymes. Studying the interaction of the purified rabbit reticulocyte 15-lipoxygenase with various types of biomembranes, we found that the enzyme binds to biomembranes when calcium is present in the incubation mixture. Under these conditions, an oxidation of the membrane lipids was observed. The membrane binding was reversible and led to an increase in the fatty acid oxygenase activity of the enzyme. To find out whether such a membrane binding also occurs in vivo, we investigated the intracellular localization of the enzyme in stimulated and resting hematopoietic cells by immunoelectron microscopy, cell fractionation studies and activity assays. In rabbit reticulocytes, the 15-lipoxygenase was localized in the cytosol, but also bound to intracellular membranes. This membrane binding was also reversible and the detection of specific lipoxygenase products in the membrane lipids indicated the in vivo activity of the enzyme on endogenous substrates. Immunoelectron microscopy showed that in interleukin-4 –treated monocytes, the 15-lipoxygenase was localized in the cytosol, but also at the inner side of the plasma membrane and at the cytosolic side of intracellular vesicles. Here again, cell fractionation studies confirmed the in vivo membrane binding of the enzyme. In human eosinophils, which constitutively express the 15-lipoxygenase, the membrane bound share of the enzyme was augmented when the cells were stimulated with calcium ionophore. Only under these conditions, specific lipoxygenase products were detected in the membrane lipids. These data suggest that in hematopoietic cells the cytosolic 15-lipoxygenase translocates reversibly to the cellular membranes. This translocation, which increases the fatty acid oxygenase activity of the enzyme, is calcium-dependent, but may not require a special docking protein.

”In vivo cryotechnique" in combination with replica immunoelectron microscopy for caveolin in smooth muscle cells

1999 ◽  
Vol 112 (6) ◽  
pp. 443-445 ◽  
Author(s):  
I. Takayama ◽  
N. Terada ◽  
T. Baba ◽  
H. Ueda ◽  
Y. Kato ◽  
...  
Keyword(s):  
Smooth Muscle ◽  
Smooth Muscle Cells ◽  
Immunoelectron Microscopy ◽  
Muscle Cells ◽  
In Vivo Cryotechnique

scholarly journals THE PRODUCTION OF BACTERICIDAL SUBSTANCES BY AEROBIC SPORULATING BACILLI

1941 ◽  
Vol 73 (5) ◽  
pp. 629-640 ◽  
Author(s):  
René J. Dubos ◽  
Rollin D. Hotchkiss
Keyword(s):  
Insoluble Fraction ◽  
Physiological Effect ◽  
Antagonistic Activity ◽  
Bacterial Cells ◽  
Animal Tissues ◽  
Gram Positive ◽  
Culture Collections ◽  
Gram Negative

Several species of aerobic sporulating bacilli recently isolated from soil, sewage, manure, and cheese, as well as authentic strains obtained from type culture collections, have been found to exhibit antagonistic activity against unrelated microorganisms. Cultures of these aerobic sporulating bacilli yield an alcohol-soluble, water-insoluble fraction,—tyrothricin,—which is bactericidal for most Gram-positive and Gram-negative microbial species. Two different crystalline products have been separated from tyrothricin. One, which may be called tyrocidine, is bactericidal in vitro for both Gram-positive and Gram-negative species; the other substance, gramicidin, is effective only against Gram-positive microorganisms. In general, tyrocidine behaves like a protoplasmic poison and like other antiseptics, loses much of its activity in the presence of animal tissues. Gramicidin on the contrary exerts a much more subtle physiological effect on the susceptible bacterial cells and, when applied locally at the site of the infection, retains in vivo a striking activity against Gram-positive microorganisms.

scholarly journals Colocalization of microtubule-associated protein 1A and microtubule-associated protein 2 on neuronal microtubules in situ revealed with double-label immunoelectron microscopy.

1987 ◽  
Vol 104 (6) ◽  
pp. 1575-1578 ◽  
Author(s):  
Y Shiomura ◽  
N Hirokawa
Keyword(s):  
Rat Brain ◽  
Immunoelectron Microscopy ◽  
Electron Microscopic ◽  
Gold Particles ◽  
Rabbit Polyclonal Antibody ◽  
Double Label ◽  
Cross Bridges ◽  
Secondary Antibodies

Microtubule-associated protein 1A (MAP1A) and microtubule-associated protein 2 (MAP2) were shown to be colocalized on the same microtubules (MTs) within neuronal cytoskeletons by double-label immunoelectron microscopy. To investigate the electron microscopic disposition of MAP1A and MAP2 and their relationship to MTs in vivo, and to determine whether there are different subsets of MTs which specifically bind either MAP1 or MAP2, we employed a double-label immunogold procedure on rat cerebella using mouse monoclonal antibody against rat brain MAP1A and affinity-purified rabbit polyclonal antibody against rat brain MAP2. MAP1A and MAP2 were identified with secondary antibodies coupled to 10- and 5-nm gold particles, respectively. In Purkinje cell dendrites, both 10- and 5-nm gold particles were observed to be studded on the fuzzy structures attached to the same MTs. Many such structures connected MTs to each other. There was no particular MT which bound either MAP1A or MAP2 alone. Furthermore, there seemed to be no specific regions on MTs where either MAP1A or MAP2 was specifically attached. Hence, we conclude that MAP1A and MAP2 are colocalized on MTs in dendrites and assume that MAP1A and MAP2 have some interrelationship in vivo and that their interactions are responsible for forming the network of cross-bridges between MTs and MTs in neuronal cytoskeletons.

A 32 kDa surface antigen of Theileria parva: characterization and immunization studies

Parasitology ◽  
2000 ◽  
Vol 120 (6) ◽  
pp. 553-564 ◽  
Author(s):  
R. A. SKILTON ◽  
A. J. MUSOKE ◽  
C. W. WELLS ◽  
Y. YAGI ◽  
V. NENE ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Cell Surface ◽  
Immunoelectron Microscopy ◽  
Surface Antigen ◽  
Immunoblot Analysis ◽  
Theileria Parva ◽  
Specific Antibodies ◽  
Major Antigen

Previous studies using monoclonal antibody (mAb) 4C9 specific for a 32 kDa antigen (p32) of Theileria parva demonstrated expression of the antigen on the surface of the sporozoite, making it a potential antigen for sporozoite neutralization. A full-length cDNA encoding the major merozoite/piroplasm surface antigen (mMPSA) of T. parva was cloned and expressed in bacteria. The expressed product reacted strongly with mAb 4C9, demonstrating identity between the p32 and mMPSA of T. parva. Using immunoblot analysis and immunoelectron microscopy with mAb 4C9 it was shown that the mMPSA is a major antigen of the merozoite and piroplasm at the cell surface, while lower levels of antigen are expressed in the sporozoite and schizont stages. Upregulation of the mMPSA occurs at merogony and can be induced by culturing schizont-infected lymphocytes at 42 °C. Recombinant mMPSA of T. parva induced high titres of specific antibodies in cattle but failed to confer protection against a T. parva sporozoite stabilate challenge. The pre-challenge sera also failed to neutralize infectivity of sporozoites in an in vitro assay. Possible reasons for the lack of parasite neutralization in vivo and in vitro are discussed.

scholarly journals New methods for monitoring mitochondrial biogenesis and mitophagy in vitro and in vivo

2017 ◽  
Vol 242 (8) ◽  
pp. 781-787 ◽  
Author(s):  
Jessica A Williams ◽  
Katrina Zhao ◽  
Shengkan Jin ◽  
Wen-Xing Ding
Keyword(s):  
Cell Differentiation ◽  
Blood Cell ◽  
Tissue Injury ◽  
Animal Tissues ◽  
Drug Induced ◽  
Cellular Homeostasis ◽  
New Methods ◽  
Biochemical Mechanisms

Removal of damaged mitochondria through mitophagy is critical for maintaining cellular homeostasis and functions. Increasing evidence implicates mitophagy in red blood cell differentiation, neurodegeneration, macrophage-mediated inflammation, ischemia, adipogenesis, drug-induced tissue injury, and cancer. Considerable progress has been made toward understanding the biochemical mechanisms involved in mitophagy regulation. However, few reliable assays to monitor and quantify mitophagy have been developed, particularly in vivo. In this review, we summarize the recent development of three assays, MitoTimer, mt-Keima and mito-QC, for monitoring and quantifying mitophagy in cells and in animal tissues. We also discuss the advantages and limitations of these three assays when using them to monitor and quantify mitophagy. Impact statement Removal of damaged mitochondria through mitophagy is critical for maintaining cellular homeostasis and functions. However, reliable quantitative assays to monitor mitophagy, particularly in vivo, are just emerging. This review will summarize the current novel quantitative assays to monitor mitophagy in vivo.

scholarly journals Gamma-tubulin distribution in the neuron: implications for the origins of neuritic microtubules.

1992 ◽  
Vol 119 (1) ◽  
pp. 171-178 ◽  
Author(s):  
P W Baas ◽  
H C Joshi
Keyword(s):  
Immunoelectron Microscopy ◽  
Neuronal Development ◽  
Sympathetic Neurons ◽  
Critical Role ◽  
Explant Cultures ◽  
Gamma Tubulin ◽  
Cultured Sympathetic Neurons ◽  
High Degree ◽  
Tubulin Content

Axons and dendrites contain dense microtubule (MT) assays that are not attached to a traditional MT nucleating structure such as the centrosome. Nevertheless, the MTs within these neurites are highly organized with respect to their polarity, and consist of a regular 13-protofilament lattice, the two known characteristics of MTs nucleated at the centrosome. These observations suggest either that axonal and dendritic MTs arise at the centrosome, or that they are nucleated locally, following a redistribution of MT nucleating material from the centrosome during neuronal development. To begin distinguishing between these possibilities, we have determined the distribution of gamma-tubulin within cultured sympathetic neurons. gamma-tubulin, a newly discovered protein which is specifically localized to the pericentriolar region of nonneuronal cells (Zheng, Y., M. K. Jung, and B. R. Oakley. 1991. Cell. 65:817-823; Stearns, T., L. Evans, and M. Kirschner. 1991. Cell. 65:825-836), has been shown to play a critical role in MT nucleation in vivo (Joshi, H. C., M. J. Palacios, L. McNamara, and D. W. Cleveland. 1992. Nature (Lond.). 356:80-83). Because the gamma-tubulin content of individual cells is extremely low, we relied principally on the high degree of resolution and sensitivity afforded by immunoelectron microscopy. Our studies reveal that, like the situation in nonneuronal cells, gamma-tubulin is restricted to the pericentriolar region of the neuron. Furthermore, serial reconstruction analyses indicate that the minus ends of MTs in both axons and dendrites are free of gamma-tubulin immunoreactivity. The absence of gamma-tubulin from the axon was confirmed by immunoblot analyses of pure axonal fractions obtained from explant cultures. The observation that gamma-tubulin is restricted to the pericentriolar region of the neuron provides compelling support for the notion that MTs destined for axons and dendrites are nucleated at the centrosome, and subsequently released for translocation into these neurites.

scholarly journals FructationIn Vivo: Detrimental and Protective Effects of Fructose

2013 ◽  
Vol 2013 ◽  
pp. 1-9 ◽  
Author(s):  
H. M. Semchyshyn
Keyword(s):  
Oxidative Stress ◽  
Side Effects ◽  
Experimental Models ◽  
Oxygen Species ◽  
Protective Effects ◽  
Compelling Evidence ◽  
Animal Tissues

There is compelling evidence that long-term intake of excessive fructose can have deleterious side effects in different experimental models. However, the role of fructosein vivoremains controversial, since acute temporary application of fructose is found to protect yeast as well as animal tissues against exogenous oxidative stress. This review suggests the involvement of reactive carbonyl and oxygen species in both the cytotoxic and defensive effects of fructose. Potential mechanisms of the generation of reactive species by fructose in the nonenzymatic reactions, their implication in the detrimental and protective effects of fructose are discussed.

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