Characteristics of Phytochrome in Glutaraldehyde-Treated Maize Coleoptiles

1975 ◽  
Vol 2 (3) ◽  
pp. 281 ◽  
Author(s):  
R Yu
Keyword(s):  
Soluble Fraction ◽  
Red Light ◽  
Intracellular Distribution ◽  
Subcellular Fractions ◽  
Particulate Fraction ◽  
Cell Fractionation

Glutaraldehyde treatment was used to stabilize the in situ distribution of phytochrome at intervals during the course of phytochrome dark reactions. Total cellular phytochrome decreased in maize coleoptiles when they were returned to darkness and incubated after red irradiation. Photoreversibility was lost in both the soluble and particulate fractions, being faster in the particulate fraction. Glutaraldehyde treatment of coleoptiles immediately after irradiation inhibited loss of particulate phytochrome in the dark. When coleoptiles were irradiated with R/FR, i.e, red light (R, 660 nm) followed by far-red light (FR, 737 nm), and then incubated in the dark, the loss of particulate phytochrome was compensated for by an increase of phytochrome in the soluble fraction, resulting in negligible loss of total phytochrome. The phytochrome dark reactions in subcellular fractions of coleoptiles irradiated in vivo with R and R/FR and extracted in Mg2+-containing buffer were similar to those in corresponding subcellular fractions of coleoptiles treated with glutaraldehyde before cell fractionation. If the pattern of phytochrome dark reactions in subcellular fractions of R- and R/FR-irradiated coleoptiles treated with glutaraldehyde before cell fractionation truly reffects the in situ situation, this similarity suggests that the phytochrome distribution in subcellular fractions obtained by extraction in Mg2+-containing buffer of coleoptiles irradiated in vivo (without ghtaraldehyde treatment) also represents the intracellular distribution. This conclusion however cannot be extended to the distribution obtained following irradiation of Mg2+-containing non-cellular extracts.

scholarly journals Preparation of nuclear matrices from cultured cells: subfractionation of nuclei in situ.

1984 ◽  
Vol 98 (5) ◽  
pp. 1886-1894 ◽  
Author(s):  
M Staufenbiel ◽  
W Deppert
Keyword(s):  
Cultured Cells ◽  
Cellular Structures ◽  
Structural Systems ◽  
Cell Fractionation ◽  
Cross Contamination ◽  
Extraction Step ◽  
Before And After ◽  
Cytoskeletal Elements

Analyses of the different structural systems of the nucleus and the proteins associated with them pose many problems. Because these systems are largely overlapping, in situ localization studies that preserve the in vivo location of proteins and cellular structures often are not satisfactory. In contrast, biochemical cell fractionation may provide artifactual results due to cross-contamination of extracts and structures. To overcome these problems, we have developed a method that combines biochemical cell fractionation and in situ localization and leads to the preparation of a residual cellular skeleton (nuclear matrix and cytoskeletal elements) from cultured cells. This method's main feature is that cell fractionation is performed in situ. Therefore, structures not solubilized in a particular extraction step remain attached to the substrate and retain their morphology. Before and after each extraction step they can be analyzed for the presence and location of the protein under study by using immunological or cytochemical techniques. Thereby the in vivo origin of a protein solubilized in a particular extraction step is determined. The solubilized protein then may be further characterized biochemically. In addition, to allow analyses of proteins associated with the residual cellular skeleton, we have developed conditions for its solubilization that do not interfere with enzymatic and immunological studies.

scholarly journals Immunogold electron microscopy of phytochrome in Avena: identification of intracellular sites responsible for phytochrome sequestering and enhanced pelletability.

1986 ◽  
Vol 103 (6) ◽  
pp. 2541-2550 ◽  
Author(s):  
D W McCurdy ◽  
L H Pratt
Keyword(s):  
Electron Microscopy ◽  
Red Light ◽  
Freeze Substitution ◽  
Immunogold Labeling ◽  
Immunogold Electron Microscopy ◽  
Subcellular Component ◽  
Discrete Structures

Using monoclonal antibodies to the plant photoreceptor, phytochrome, we have investigated by immunogold electron microscopy the rapid, red light-induced, intracellular redistribution (termed "sequestering") of phytochrome in dark-grown Avena coleoptiles. Pre-embedding immunolabeling of 5-micron-thick cryosections reveals that sequestered phytochrome is associated with numerous, discrete structures of similar morphology. Specific labeling of these structures was also achieved by post-embedding ("on-grid") immunostaining of LR-White-embedded tissue, regardless of whether the tissue had been fixed chemically or by freeze substitution. The phytochrome-associated structures are globular to oval in shape, 200-400 nm in size, and are composed of amorphous, granular material. No morphologically identifiable membranes are present either surrounding or within these structures, which are often present as apparent aggregates that approach several micrometers in size. An immunogold labeling procedure has also been developed to identify the particulate, subcellular component with which phytochrome is associated in vitro as a consequence of irradiation of Avena coleoptiles before their homogenization. Structures with appearance similar to those identified in situ are the only components of the pelletable material that are specifically labeled with gold. We conclude that the association of phytochrome with these structures in Avena represents the underlying molecular event that ultimately is expressed both as red light-induced sequestering in vivo and enhanced pelletability of phytochrome detected in vitro.

THE INTRACELLULAR LOCALIZATION OF OESTROGEN IN RAT TISSUES

1965 ◽  
Vol 32 (1) ◽  
pp. 9-15 ◽  
Author(s):  
R. J. B. KING ◽  
J. GORDON ◽  
D. R. INMAN
Keyword(s):  
Anterior Pituitary ◽  
Soluble Fraction ◽  
Intracellular Localization ◽  
Female Rats ◽  
Radioactive Material ◽  
Rat Tissues ◽  
Nuclear Fraction ◽  
Cell Fractionation ◽  
Rat Pituitary

SUMMARY The radioactive material in rat pituitary, liver and uterus 1 hr. after the s.c. injection of either 0·73 or 7·3 μg. [6,7-3H]oestradiol has been investigated. Oestrone and oestradiol were the only compounds detected in pituitary and uterus whereas at least six compounds were present in liver. Cell fractionation studies have been carried out on anterior pituitary and liver from female rats which had received an injection of either 0·73 or 7·3 μg. [6,7-3H]oestradiol in vivo. Most of the tritium in the anterior pituitary was associated with the nuclear fraction whereas most of the tritium in the liver was in the soluble fraction. The uptake of [6,7-3H]oestradiol by the anterior pituitary reached a maximum ½-1 hr. after injection of the steroid and remained at this level for the next 3 hr. The intracellular distribution of this steroid at different times after injection suggests that the steroid passes into the cytoplasm and then into the nucleus.

TRANSFORMATION OF CORTICOSTERONE IN THE FOETAL AND PLACENTAL COMPARTMENTS OF THE RAT

1972 ◽  
Vol 69 (4) ◽  
pp. 689-700 ◽  
Author(s):  
S. Roy ◽  
J. R. Pasqualini
Keyword(s):  
Quantitative Evaluation ◽  
Liver Tissue ◽  
Subcellular Fractions ◽  
Pure Form ◽  
Radioactive Material ◽  
Corticosterone Metabolites ◽  
Liver Tissues

ABSTRACT 3H-Corticosterone was administered subcutaneously in vivo and in situ to 32 rat foetuses at 16–18 days of gestation. 30 min after the injection, 11% of the administered dose was localized in the liver tissue, 5% in the lungs, 3% in the intestines and 5% in the placenta. The foetal tissues were separated into different subcellular fractions: I) nucleo-fibrillar, II) mitochondrial-microsomal, and III) cytosol. It was observed that corticosterone was extensively metabolized in all three of these subcellular fractions. The following metabolites were identified in a radiochemically pure form: 11-dehydrocorticosterone, 5α-dihydrocorticosterone, tetrahydrocorticosterone, 5α-tetrahydrocorticosterone. Quantitative evaluation of the corticosterone metabolites showed that the greatest part of these steroids consisted of dihydro- and tetrahydrocorticosterone derivatives, principally with a 5α configuration. In the liver tissues, 0.5 % of the radioactive material was found in a conjugated form, mainly as ester sulphates. The radioactive material liberated after solvolysis of these conjugates was tentatively identified as corticosterone, 5α-dihydrocorticosterone and 5α-tetrahydrocorticosterone.

scholarly journals Immunohistochemical Detection of Phosphorylated Rhodopsin in Light-exposed Retina of Living Mouse with In Vivo Cryotechnique

2006 ◽  
Vol 54 (4) ◽  
pp. 479-486 ◽  
Author(s):  
Nobuo Terada ◽  
Nobuhiko Ohno ◽  
Hiroshi Ohguro ◽  
Zilong Li ◽  
Shinichi Ohno
Keyword(s):  
Visible Light ◽  
Light Exposure ◽  
Red Light ◽  
Time Dependent ◽  
Immunohistochemical Detection ◽  
In Vivo Cryotechnique ◽  
Whole Mount ◽  
Living Mouse

The purpose of this study is to analyze the time-dependent molecular states of rhodopsin (Rho) phosphorylation in the specimens originating from eyeballs cryoimmobilized in situ in living animals. Whole eyeballs of living mice under various dark- and light-exposure conditions were quickly frozen using the in vivo cryotechnique with isopentane-propane cryogen cooled down in liquid nitrogen (−196C). The frozen whole-mount eyeballs were freeze substituted in acetone containing paraformaldehyde and embedded in paraffin wax. Deparaffinized sections were immunostained with anti-phosphorylated 334Ser Rho (P-Rho334) antibody. Immunoreactivity of P-Rho334 was specifically recognized in the outer segments of mouse retinas exposed to daylight. In the 12-h dark-adapted retinas, P-Rho334 immunoreactivity was completely eliminated. Moreover, in other retinas dark adapted for 12 or 36 hr and then exposed under the safety red light for 2 min, it was still barely recognized. Even in the eyeballs exposed to strong visible light for 10 sec, it was not detected. However, after 30, 60, and 180 sec of visible light exposure, P-Rho334 immunoreactivity was definitely recovered, similar to that under daylight condition. This is a new immunohistochemical approach to visualize the time-dependent Rho phosphorylation of living mice using the in vivo cryotechnique, in which changes could be detected within seconds following exposure to light.

Effect of age on protein and ribonucleic acid metabolism in mouse skeletal muscle

1969 ◽  
Vol 47 (3) ◽  
pp. 231-235 ◽  
Author(s):  
Uma Srivastava ◽  
K. D. Chaudhary
Keyword(s):  
Skeletal Muscle ◽  
Protein Content ◽  
Soluble Fraction ◽  
Subcellular Fractions ◽  
Maximum Increase ◽  
Continuous Increase ◽  
Rna Content ◽  
Slow Decline

Protein content, RNA concentration, in vivo and in vitro incorporation of L-leucine-14C (U.L.) into protein and of uridine-2-14C into RNA of the homogenate, and various fractions of muscle of mice were measured at various ages. Protein content showed a continuous increase in the homogenate, nuclei–myofibrillar, and soluble fraction of the muscle of mice undergoing development. Maximum increase was noted in the soluble fraction. RNA content dropped rapidly in the homogenate and in all subcellular fractions of the muscle up to 30 days, followed by a slow decline. Ratio of DNA as well as RNA content to protein decreased continuously in the homogenate and in all subcellular fractions with aging.In vivo incorporation of L-leucine-14C (U.L.) into protein of the homogenate and various subcellular fractions of the muscle decreased with the increasing age of the animal. In vitro incorporation of labelled leucine into protein by the cell-free preparation of skeletal muscle also decreased with aging. Such a decrease in the incorporation was thought to be due to decrease in the content of polysomes in the aging muscle.In vivo incorporation of uridine-2-14C into RNA of the homogenate and subcellular fractions of muscle showed a decrease with age. Similar types of results were obtained in studies in vitro.

In vitro and in vivo polysaccharides assembly: ultrastructural and cytochemical study of growing plant cell wall components.

1978 ◽  
Vol 36 (2) ◽  
pp. 434-435
Author(s):  
D. Reis ◽  
B. Vian ◽  
J. C. Roland
Keyword(s):  
Cell Wall ◽  
Self Assembly ◽  
Plant Cell Wall ◽  
Higher Plants ◽  
Three Dimensional ◽  
Cytochemical Study ◽  
Wall Components

Wall morphogenesis in higher plants is a problem still open to controversy. Until now the possibility of a transmembrane control and the involvement of microtubules were mostly envisaged. Self-assembly processes have been observed in the case of walls of Chlamydomonas and bacteria. Spontaneous gelling interactions between xanthan and galactomannan from Ceratonia have been analyzed very recently. The present work provides indications that some processes of spontaneous aggregation could occur in higher plants during the formation and expansion of cell wall.Observations were performed on hypocotyl of mung bean (Phaseolus aureus) for which growth characteristics and wall composition have been previously defined.In situ, the walls of actively growing cells (primary walls) show an ordered three-dimensional organization (fig. 1). The wall is typically polylamellate with multifibrillar layers alternately transverse and longitudinal. Between these layers intermediate strata exist in which the orientation of microfibrils progressively rotates. Thus a progressive change in the morphogenetic activity occurs.

Three-Dimensional Subcellular Organization of Hepatocytes in Intact Liver: Simultaneous Visualization of Cytoskeletal and Membrane Markers by Confocal Microscopy

1996 ◽  
Vol 54 ◽  
pp. 288-289
Author(s):  
Greg V. Martin ◽  
Ann L. Hubbard
Keyword(s):  
Three Dimensional ◽  
Integral Membrane Proteins ◽  
Polarized Secretion ◽  
Microscope Images ◽  
Computer Aided ◽  
Intracellular Organelles ◽  
Cytoskeletal Elements ◽  
Simultaneous Visualization

The microtubule (MT) cytoskeleton is necessary for many of the polarized functions of hepatocytes. Among the functions dependent on the MT-based cytoskeleton are polarized secretion of proteins, delivery of endocytosed material to lysosomes, and transcytosis of integral plasma membrane (PM) proteins. Although microtubules have been shown to be crucial to the establishment and maintenance of functional and structural polarization in the hepatocyte, little is known about the architecture of the hepatocyte MT cytoskeleton in vivo, particularly with regard to its relationship to PM domains and membranous organelles. Using an in situ extraction technique that preserves both microtubules and cellular membranes, we have developed a protocol for immunofluorescent co-localization of cytoskeletal elements and integral membrane proteins within 20 µm cryosections of fixed rat liver. Computer-aided 3D reconstruction of multi-spectral confocal microscope images was used to visualize the spatial relationships among the MT cytoskeleton, PM domains and intracellular organelles.

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