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.

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.

The cytoskeletal elements of human retinal pigment epithelium: in vitro and in vivo

1988 ◽  
Vol 91 (2) ◽  
pp. 303-312
Author(s):  
N.M. McKechnie ◽  
M. Boulton ◽  
H.L. Robey ◽  
F.J. Savage ◽  
I. Grierson
Keyword(s):  
Retinal Pigment Epithelium ◽  
Pigment Epithelium ◽  
Retinal Pigment ◽  
Cytokeratin 18 ◽  
Rpe Cells ◽  
Human Retinal Pigment Epithelium ◽  
Cytoskeletal Elements

The cytoskeletal elements of normal (in situ) and cultured human retinal pigment epithelium (RPE) were studied by a variety of immunocytochemical techniques. Primary antibodies to vimentin and cytokeratins were used. Positive immunoreactivity for vimentin was obtained with in situ and cultured material. The pattern of reactivity obtained with antisera and monoclonals to cytokeratins was more complex. Cytokeratin immunoreactivity could be demonstrated in situ and in cultured cells. The pattern of cytokeratin expression was similar to that of simple or glandular epithelia. A monoclonal antibody that specifically recognizes cytokeratin 18 identified a population of cultured RPE cells that had particularly well-defined filamentous networks within their cytoplasm. Freshly isolated RPE was cytokeratin 18 negative by immunofluorescence, but upon culture cytokeratin 18 positive cells were identifiable. Cytokeratin 18 positive cells were identified in all RPE cultures (other than early primaries), regardless of passage number, age or sex of the donor. In post-confluent cultures cytokeratin 18 cells were identified growing over cytokeratin 18 negative cells, suggesting an association of cytokeratin 18 immunoreactivity with cell proliferation. Immunofluorescence studies of retinal scar tissue from two individuals revealed the presence of numerous cytokeratin 18 positive cells. These findings indicate that RPE cells can be identified by their cytokeratin immunoreactivity and that the overt expression of cytokeratin 18 may be associated with proliferation of human RPE both in vitro and in vivo.

scholarly journals Apical polarization of N-CAM in retinal pigment epithelium is dependent on contact with the neural retina.

1993 ◽  
Vol 121 (2) ◽  
pp. 335-343 ◽  
Author(s):  
D Gundersen ◽  
S K Powell ◽  
E Rodriguez-Boulan
Keyword(s):  
Retinal Pigment Epithelium ◽  
Cultured Cells ◽  
Pigment Epithelium ◽  
Retinal Pigment ◽  
Neural Retina ◽  
Apical Surface ◽  
Neural Cell Adhesion ◽  
Adhesion Role

The retinal pigment epithelium (RPE) is unique among epithelia in that its apical surface does not face a lumen, but, instead, is specialized for interaction with the neural retina. The molecules involved in the interaction of the RPE with the neural retina are not known. We show here that the neural cell adhesion molecule (N-CAM) is found both on the apical surface of RPE in situ and on the outer segments of photoreceptors, fulfilling an important requisite for an adhesion role between both structures. Strikingly, culture of RPE results in rapid redistribution of N-CAM to the basolateral surface. This is not due to an isoform shift, since the N-CAM expressed by cultured cells (140 kD) is the same as that expressed by RPE in vivo. Rather, the reversed polarity of N-CAM appears to result from the disruption of the contact between the RPE and the photoreceptors of the neural retina. We suggest that N-CAM in RPE and photoreceptors participate in these interactions.

scholarly journals Noninvasive Orthogonal Polarization Spectral Imaging as Applied to Microvascular Studies in Mice

2004 ◽  
Vol 5 (3) ◽  
pp. 211-217 ◽  
Author(s):  
P. Nivoit ◽  
A. M. Chevrier ◽  
M. Lagarde ◽  
C. Renaudin ◽  
N. Wiernsperger
Keyword(s):  
Spectral Imaging ◽  
Diabetic Microangiopathy ◽  
Orthogonal Polarization ◽  
High Contrast ◽  
Hormonal Stimulation ◽  
Orthogonal Polarization Spectral ◽  
Before And After ◽  
Topical Applications

In vivo observations of the mouse microcirculation can hardly be performed due to technical difficulties, limiting the knowledge that could be obtained from gene manipulated mice models. The aim of the present study was to check the applicability of a novel optical system, the orthogonal polarization spectral technology, to study the mouse microcirculation. In anaesthetized mice, the spinotrapezius muscle microcirculation was observed in situ. The diameter of precapillary arterioles was measured before and after a pharmacological or hormonal stimulation. High-contrast images of the muscle microcirculation were obtained and significant vasodilatation of arterioles was observed after topical applications of acetylcholine, sodium nitroprusside, and insulin. As compared to conventional techniques, orthogonal polarization spectral imaging makes it possible to assess and study microvascular beds in mice, which were inaccessible until now, allowing the use of gene manipulated mice to investigate, for example, the mechanisms involved in the development of diabetic microangiopathy.

scholarly journals Tetracycline-regulated expression enables purification and functional analysis of recombinant connexin channels from mammalian cells

2004 ◽  
Vol 383 (1) ◽  
pp. 111-119 ◽  
Author(s):  
Irina V. KOREEN ◽  
Wafaa A. ELSAYED ◽  
Yu J. LIU ◽  
Andrew L. HARRIS
Keyword(s):  
Mammalian Cells ◽  
Cultured Cells ◽  
Expression System ◽  
Physiological Processes ◽  
Messenger Molecules ◽  
One Step ◽  
Cellular Control

Intercellular coupling mediated by gap junction channels composed of connexin protein underlies numerous physiological processes, such as cellular differentiation, tissue synchronization and metabolic homoeostasis. The distinct molecular permeability of junctional channels composed of different connexin isoforms allows cellular control of coupling via regulation of isoform expression. However, the permeability properties of most connexin isoforms have not been well characterized due to the difficulty of manipulating and measuring the diffusible concentrations of cytoplasmic messenger molecules and metabolites, and to a lack of control over channel isoform composition, in vivo. Here we present a method to express and purify active connexin hemichannels of a single isoform or a consistent ratio of two isoforms from cultured cells using the Tet-On inducible expression system and one-step anti-haemagglutinin immunoaffinity purification. The procedure yields 10–20 μg of pure connexin protein from 2.5×108 HeLa cells. The purified channels are shown to be useful for in vitro permeability analysis using well established techniques. This method has substantial advantages over existing methods for heterologous connexin expression, such as the ease of co-expression of two isoforms at a constant ratio, consistently high expression levels over many passages, and the ability to study channel properties in situ as well as in purified form. Furthermore, the generic cloning site of the new pBI-GT vector and the commercial availability of anti-haemagglutinin (clone HA-7)–agarose make this affinity tagging and purification procedure easily applicable to other proteins.

Cool Storage of Human Tissue Engineered Bone for Bone Regeneration Therapy

2006 ◽  
Vol 309-311 ◽  
pp. 1005-1008
Author(s):  
N. Satoh ◽  
Takafumi Yoshikawa ◽  
Kazuhide Miyazaki ◽  
Hideki Shigematsu ◽  
Y. Ueda ◽  
...  
Keyword(s):  
Bone Tissue ◽  
Cultured Cells ◽  
Storage Conditions ◽  
Bone Diseases ◽  
Significant Activity ◽  
Cool Storage ◽  
Before And After ◽  
Time Required

Availability, storage and transportation of engineered bone tissue fabricated in vitro are major practical problems associated with adequate use of bone replacement grafts for the treatment of bone diseases. The ability to maintain viable engineered bone tissue would facilitate future clinical applications. In the present study, we investigated time required for transportation of engineered bone removed from cool storage, from the culture room to the operating room; and examined effects of cool storage on survival of engineered bone tissue. Bone marrowcells were obtained from the iliac bone of a 60-year-old male affected with lumbar spondylosis, and then incubated in standard medium. After two weeks in primary culture, cultured cells were trypsinized, and a concentrated cell suspension was incubated with a porous beta-TCP block. After 3 weeks of subculture with the osteogenic medium containing dexamethasone etc., engineered bone tissue was collected, stored for 0, 6, 12, 24 hours at 4 °C, and was subcutaneously implanted into the back of nude mice. Six weeks after implantation, implants were harvested. Before and after implantation, significant activity could be detected in all animals. In in vitro and in vivo situations, osteogenic activity of engineered bone tissue could be maintained even after 24 hours. These results provided information on appropriate storage conditions for engineered bone tissue.

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.

Potentiation of Bradykinin by Angiotensin-(1-7) on Resistance Vessels of Hypertensive Rats, Studied in Vivo .

Hypertension ◽  
2000 ◽  
Vol 36 (suppl_1) ◽  
pp. 696-696
Author(s):  
Liliam Fernandes ◽  
Zuleica B Fortes ◽  
Dorothy Nigro ◽  
Regina Scivoletto ◽  
Robson A S Santos ◽  
...  
Keyword(s):  
Topical Application ◽  
Ace Inhibition ◽  
Pharmacological Effects ◽  
Hypertensive Rats ◽  
Specific Receptor ◽  
Resistance Vessels ◽  
Before And After ◽  
Arteriolar Diameter

P19 Objective: To verify the Angiotensin-(1-7) [Ang-(1-7)]-activity on Bradykinin (BK)-induced vasodilation in SHR mesenteric arterioles, in vivo-in situ. Methods: Arteriolar diameter was measured by intravital microscopy before and after topical application of BK(1pmol), Acetylcholine(ACh 1.6nmol), Sodium nitroprusside (SNP 38pmol) or Histamine (5.4nmol) in the absence or presence of Ang-(1-7) (100pmol). To investigate the Ang-(1-7)/BK interaction, treatments were employed through topical application of antagonists of BK (HOE140,100pmol), Ang-(1-7)(A779,100pmol)and potassium channel (tetraethylammoniun - TEA,90pmol), with an inhibitor of NOSynthase (L-NAME 10nmol) and after cyclooxygenase blockade (indomethacin 5mg/Kg or diclofenac 2.5mg/Kg). To evaluate the effect of ACE- and/or AT 1 blockade on Ang-(1-7)/BK interaction, rats were treated for 21 days with enalapril, quinapril (10mg/Kg), losartan (15mg/Kg) or enalapril + losartan (10 and 15 mg/Kg, respectively). In those enalapril-treated rats the effect of BK (1pmol) was also analysed in the presence of A779 (100pmol). Results: BK-induced vasodilation, but not ACh, SNP or Histamine responses, was increased in the presence of Ang-(1-7) (4.96±0.7% vs 9.07±1.0% * ).This interaction was abolished by HOE (1.11±0.8% * ), A779 (5.13±0.6% * ), TEA (3.37±0.5% * ), indomethacin (1.73±0.4% * )and diclofenac (3.63±0.5% * ), whereas L-NAME did not modify the Ang-(1-7)-potentiating activity. The BK-potentiation by Ang-(1-7) was also observed after enalapril (10.57±0.5% * ), quinapril (8.9±0.7% * ), losartan (9.93±1.2% * ) and enalapril + losartan (10.59±0.5% * ). Enalapril increased the BK-vasodilation(8.21±0.7% * ), but this effect was reversed in the presence of A779 (4.27±0.5% * ). * p≤0.05 Conclusion: In the SHR microcirculation Ang-(1-7) potentiates BK through a specific receptor, probably releasing prostaglandins and EDHF. Our results indicate that the BK-potentiation by Ang-(1-7) may occur endogenously and contribute to the pharmacological effects of ACE inhibition. HOE 140 and Quinapril were gifts from HOECHST and Warner Lambert, respectively.

scholarly journals In Vivo Mitochondrial p53 Translocation Triggers a Rapid First Wave of Cell Death in Response to DNA Damage That Can Precede p53 Target Gene Activation

2004 ◽  
Vol 24 (15) ◽  
pp. 6728-6741 ◽  
Author(s):  
Susan Erster ◽  
Motohiro Mihara ◽  
Roger H. Kim ◽  
Oleksi Petrenko ◽  
Ute M. Moll
Keyword(s):  
Target Gene ◽  
Caspase 3 ◽  
Target Genes ◽  
Cultured Cells ◽  
Gene Activation ◽  
Lag Phase ◽  
Cell Fractionation ◽  
Γ Irradiation ◽  
P53 Target Gene

ABSTRACT p53 promotes apoptosis in response to death stimuli by transactivation of target genes and by transcription-independent mechanisms. We recently showed that wild-type p53 rapidly translocates to mitochondria in response to multiple death stimuli in cultured cells. Mitochondrial p53 physically interacts with antiapoptotic Bcl proteins, induces Bak oligomerization, permeabilizes mitochondrial membranes, and rapidly induces cytochrome c release. Here we characterize the mitochondrial p53 response in vivo. Mice were subjected to γ irradiation or intravenous etoposide administration, followed by cell fractionation and immunofluorescence studies of various organs. Mitochondrial p53 accumulation occurred in radiosensitive organs like thymus, spleen, testis, and brain but not in liver and kidney. Of note, mitochondrial p53 translocation was rapid (detectable at 30 min in thymus and spleen) and triggered an early wave of marked caspase 3 activation and apoptosis. This caspase 3-mediated apoptosis was entirely p53 dependent, as shown by p53 null mice, and preceded p53 target gene activation. The transcriptional p53 program had a longer lag phase than the rapid mitochondrial p53 program. In thymus, the earliest apoptotic target gene products PUMA, Noxa, and Bax appeared at 2, 4, and 8 h, respectively, while Bid, Killer/DR5, and p53DinP1 remained uninduced even after 20 h. Target gene induction then led to further increase in active caspase 3. Similar biphasic kinetics was seen in cultured human cells. Our results suggest that in sensitive organs mitochondrial p53 accumulation in vivo occurs soon after a death stimulus, triggering a rapid first wave of apoptosis that is transcription independent and may precede a second slower wave that is transcription dependent.

scholarly journals Differential Regulation of the Extracellular Cysteine/Cystine Redox State (EhCySS) by Lung Fibroblasts from Young and Old Mice

2016 ◽  
Vol 2016 ◽  
pp. 1-11 ◽  
Author(s):  
Walter H. Watson ◽  
Tom J. Burke ◽  
Igor N. Zelko ◽  
Edilson Torres-González ◽  
Jeffrey D. Ritzenthaler ◽  
...  
Keyword(s):  
Extracellular Matrix ◽  
Redox State ◽  
Redox Regulation ◽  
Cultured Cells ◽  
Glutamate Transporter ◽  
Lung Fibroblasts ◽  
Extracellular Matrix Components ◽  
Before And After ◽  
Oxidative Challenge

Aging is associated with progressive oxidation of plasma cysteine (Cys)/cystine (CySS) redox state, expressed asEhCySS. Cultured cells condition their media to reproduce physiologicalEhCySS, but it is unknown whether aged cells produce a more oxidized extracellular environment reflective of that seen in vivo. In the current study, we isolated primary lung fibroblasts from young and old female mice and measured the mediaEhCySSbefore and after challenge with Cys or CySS. We also measured expression of genes related to redox regulation and fibroblast function. These studies revealed that old fibroblasts produced a more oxidizing extracellularEhCySSthan young fibroblasts and that old fibroblasts had a decreased capacity to recover from an oxidative challenge due to a slower rate of reduction of CySS to Cys. These defects were associated with 10-fold lower expression of the Slc7a11 subunit of the xCT cystine-glutamate transporter. Extracellular superoxide dismutase (Sod3) was the only antioxidant or thiol-disulfide regulating enzyme among 36 examined that was downregulated in old fibroblasts by more than 2-fold, but there were numerous changes in extracellular matrix components. Thus, aging fibroblasts not only contribute to remodeling of the extracellular matrix but also have a profound effect on the extracellular redox environment.

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