scholarly journals EFFECTS OF PHOSPHOTUNGSTATE NEGATIVE STAINING ON THE MORPHOLOGY OF THE ISOLATED GOLGI APPARATUS

1974 ◽  
Vol 62 (2) ◽  
pp. 491-504 ◽  
Author(s):  
William P. Cunningham ◽  
L. Andrew Staehelin ◽  
Robert W. Rubin ◽  
Ross Wilkins ◽  
Mary Bonneville
Keyword(s):  
Membrane Proteins ◽  
Golgi Apparatus ◽  
Protein Extraction ◽  
Membrane Lipids ◽  
Morphological Changes ◽  
Negative Staining ◽  
Thin Sections ◽  
Whole Cells ◽  
Negative Stain

Isolated Golgi complexes can be recognized in phosphotungstate (PTA) negative stain as stacks of membranous plates surrounded by a complex anastomosing network of tubules and vesicles. The extent of this tubular network is, however, much greater than can be observed in thin sections of whole cells. To determine which of the steps leading to the final negatively stained image may produce the observed changes, we have monitored each of the steps by other electron microscope and biochemical methods. The first damage to the membranes seems to occur during the initial isolation procedure as judged by the appearance of smooth patches on the freeze-fractured membrane faces that are normally covered with particles. Subsequent suspension of the Golgi fraction in water, to dilute the sucrose for negative staining, leads to the disappearnce of the stacking, to some tubulation and some vesiculation of the membranes as judged by thin section and freeze-cleave microscopy. The latter technique also reveals an increase in smooth-cleaving membrane faces. Application of the negative stain to the water-washed Golgi fraction, finally, produces extensive tubular arrays and a simultaneous decrease in the remaining large membranous vesicles. The freeze-cleaved tubular membranes appear essentially smooth except for small patches of aggregated particles. Parallel gel electrophoresis studies of the membranes and of the water and negative stain wash extracts indicate that protein extraction is involved in these morphological changes. PTA seems to be a particularly effective solvent for certain membrane proteins that are not removed by the water wash. These observations suggest that removal of membrane proteins alters structural restraints on the membrane lipids so that they behave semiautonomously like myelinics and form new artificial structures. This does not eliminate the possibility, however, that some tubules also exist in the Golgi apparatus in vivo.

scholarly journals STRUCTURE OF ISOLATED PLANT GOLGI APPARATUS REVEALED BY NEGATIVE STAINING

1966 ◽  
Vol 28 (2) ◽  
pp. 169-179 ◽  
Author(s):  
William P. Cunningham ◽  
D. James Morré ◽  
H. H. Mollenhauer
Keyword(s):  
Heavy Metal ◽  
Golgi Apparatus ◽  
Sucrose Gradient ◽  
Phosphotungstic Acid ◽  
Morphological Characteristics ◽  
Negative Staining ◽  
The Other ◽  
Functional Specialization ◽  
Thin Sections ◽  
Texture Size

Sucrose-gradient-purified dictyosomes of plant Golgi apparatus appear, after glutaraldehyde stabilization, as stacks of highly fenestrate and tubate cisternae when negatively stained with phosphotungstic acid, shadowed with heavy metal, or OsO4-stained in thin section. The tubular proliferations (diameter 200 to 400 A) extend for several microns from the central region and are united at intervals into an anastomosing network. Associated with the tubules are two kinds of vesicles which are distinguishable on the basis of texture, size, shape, and staining characteristics. One vesicle type is rough-surfaced, nearly spherical, and of uniform dimensions (diameter approximately 600 A). Metal shadowing shows that these vesicles remain spherical after drying. The other vesicle type is smooth-surfaced and varies in both size and shape. Intercisternal elements are revealed, by negative staining, on the surface of internal cisternae after fragmentation of the dictyosome. The progressive differentiation of cisternae from the forming face to the maturing face is observed in thin sections of these isolated preparations. The morphological characteristics observed in negatively stained dictyosomes indicate regions of functional specialization within the dictyosome cisternae and reveal a dictyosome structure more extensive than that envisioned from sections.

scholarly journals The selection of aptamers specific for membrane molecular targets

2011 ◽  
Vol 16 (1) ◽  
Author(s):  
Teresa Janas ◽  
Tadeusz Janas
Keyword(s):  
Membrane Proteins ◽  
Rna Structure ◽  
Membrane Lipids ◽  
Molecular Targets ◽  
Experimental System ◽  
Lipid Vesicles ◽  
Rna Aptamers ◽  
Protein Fragments ◽  
Whole Cells ◽  
Selection Of

AbstractA growing number of RNA aptamers have been selected experimentally using the SELEX combinatorial approach, and these aptamers have several advantages over monoclonal protein antibodies or peptides with respect to their applications in medicine and nanobiotechnology. Relatively few successful selections have been reported for membrane molecular targets, in contrast to the situation with non-membrane molecular targets. This review compares the procedures and techniques used in selections against membrane proteins and membrane lipids. In the case of membrane proteins, the selections were performed against soluble protein fragments, detergent-membrane protein mixed micelles, whole cells, vesicles derived from cellular membranes, and enveloped viruses. Liposomes were used as an experimental system for the selection of aptamers against membrane lipids. RNA structure-dependent aptamer binding for rafts in lipid vesicles was reported. Based on the selected aptamers against DOPC and the amino acid tryptophan, a specific passive membrane transporter composed of RNA was constructed. The determination of the selectivity of aptamers appears to be a crucial step in a selection, but has rarely been fully investigated. The selections, which use whole cells or vesicles derived from membranes, can yield aptamers not only against proteins but also against membrane lipids.

scholarly journals Mitotic disassembly of the Golgi apparatus in vivo

1995 ◽  
Vol 108 (7) ◽  
pp. 2715-2727 ◽  
Author(s):  
T. Misteli ◽  
G. Warren
Keyword(s):  
Golgi Apparatus ◽  
Morphological Changes ◽  
Temperature Sensitive ◽  
Cross Sectional ◽  
Mitotic Kinase ◽  
Transport Vesicles ◽  
A Cell ◽  
Cell Cycle Block ◽  
Conventional Electron Microscopy

Populations enriched in prophase cells were obtained either by using a cell line with a temperature-sensitive mutation in the mitotic kinase, p34cdc2, or by treating cells with olomoucine, an inhibitor of this kinase. Both methods resulted in efficient and reversible block of the cells at the G2/M boundary. After cells were released from the cell cycle block, the morphological changes to the Golgi apparatus were characterised using both quantitative conventional electron microscopy and immuno-gold microscopy. The early mitotic phases were divided into six stages (G2 to pro-metaphase) based on the morphology of the nucleus. During prophase the cross-sectional length of Golgi stacks decreased prior to unstacking. At the same time, small vesicular profiles, typically 50–70 nm in diameter, accumulated in the vicinity of the stacks. The disappearance of Golgi stacks was accompanied by the transient appearance of tubular networks. By the time cells entered prometaphase, the stacks had completely disassembled and only clusters consisting of Golgi vesicles and short tubular elements were left. When cells were released from the G2/M boundary and pulsed briefly with [AlF4]- to prevent uncoating of transport vesicles, vesicular profiles with a morphology reminiscent of COP-coated vesicles appeared. These vesicular profiles were either associated with Golgi stacks or, at later stages, with clusters, but were formed at all stages of disassembly. Together these results provide further support for our model that continued budding of vesicles from the rims of Golgi cisternae is at least partly responsible for the disassembly of the Golgi apparatus.

Effects of phosphatidylethanolamine glycation on lipid–protein interactions and membrane protein thermal stability

2008 ◽  
Vol 416 (1) ◽  
pp. 145-152 ◽  
Author(s):  
Valeria Levi ◽  
Ana M. Villamil Giraldo ◽  
Pablo R. Castello ◽  
Juan P. F. C. Rossi ◽  
F. Luis González Flecha
Keyword(s):  
Membrane Proteins ◽  
Protein Interactions ◽  
Membrane Lipids ◽  
Structural Rearrangement ◽  
Normal Functioning ◽  
Complex Organization ◽  
Lipid Glycation ◽  
Lipid Protein Interactions ◽  
The Stability

Non-enzymatic glycation of biomolecules has been implicated in the pathophysiology of aging and diabetes. Among the potential targets for glycation are biological membranes, characterized by a complex organization of lipids and proteins interacting and forming domains of different size and stability. In the present study, we analyse the effects of glycation on the interactions between membrane proteins and lipids. The phospholipid affinity for the transmembrane surface of the PMCA (plasma-membrane Ca2+-ATPase) was determined after incubating the protein or the phospholipids with glucose. Results show that the affinity between PMCA and the surrounding phospholipids decreases significantly after phosphospholipid glycation, but remains unmodified after glycation of the protein. Furthermore, phosphatidylethanolamine glycation decreases by ∼30% the stability of PMCA against thermal denaturation, suggesting that glycated aminophospholipids induce a structural rearrangement in the protein that makes it more sensitive to thermal unfolding. We also verified that lipid glycation decreases the affinity of lipids for two other membrane proteins, suggesting that this effect might be common to membrane proteins. Extending these results to the in vivo situation, we can hypothesize that, under hyperglycaemic conditions, glycation of membrane lipids may cause a significant change in the structure and stability of membrane proteins, which may affect the normal functioning of membranes and therefore of cells.

Studies on Platelet Storage Biomarkers: Effect of Different Protein Extraction Buffers on Platelet Gelsolin and B-Actin Profiling

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 4075-4075
Author(s):  
Sandhya Kulkarni ◽  
Meganathan Kannan ◽  
Chintamani D Atreya
Keyword(s):  
Protein Extraction ◽  
Morphological Changes ◽  
Surrogate Marker ◽  
Buffer System ◽  
Protein Levels ◽  
Platelet Protein ◽  
Platelet Storage ◽  
Platelet Proteins

Abstract Platelet concentrates (PCs) are one of the blood cell components that play a major role in transfusion medicine. The shelf-life of PC is 5–7 days at 22°C. During storage PCs undergo certain morphological changes as well as physiological changes such as loosing of its surface glycoproteins, and reduced aggregation response to its agonists, which are collectively referred to as “storage lesions”. To date there is no in vitro “gold standard” that unequivocally serves as a surrogate marker to predict PC quality in vivo following transfusion. A comprehensive survey of reports in this field clearly indicate that there is some confusion with regards to the determination of which platelet protein or a panel of proteins could serve as potential markers associated with PC storage. Following a careful review of all available information, we recognized that perhaps one core issue to the observed discrepancies could be the buffer system that is being utilized by individual laboratories in extracting proteins in profiling protein alterations in PCs during storage. To address this, in the present study we analyzed two platelet proteins, b-actin and gelsolin-related 40 kDa protein. Profiles of these two proteins were previously reported by others to be altered during PC storage and hence implicated to be useful as biomarkers of platelet storage. By employing 3 different protein extraction buffer systems to isolate proteins from platelets at three storage intervals (Table 1), our results indicate that b-actin and gelsolin 40 kDa protein levels do not appear to change with time in platelet storage and therefore may not be useful as platelet storage markers and Buffer systems do make a difference in extracting different platelet proteins for analysis, for example 40 kDa gelsolin was not extractable in buffer 3 (Table 1), suggesting that one should be careful in selecting a buffer system for platelet proteomics to have meaningful results. Same level of 40 kDa Gelsolin Same level of b-actin Buffer system Day 0 Day 5 Day 16 Day 0 Day 5 Day 16 Buffer 1 Not identified Yes Yes Yes Yes Yes Buffer 2 Not identified Yes Yes Yes Yes Yes Buffer 3 Not identified Not identified Not identified Yes Yes Yes The findings and conclusions in this abstract have not been formally disseminated by the Food and Drug Administration and should not be construed to represent any Agency determination or policy.

Probing the ultrastructure of the mitotic and meiotic spindle in eggs: An expeditious approach based on semi-thin (0.25 μm) serial sections

1983 ◽  
Vol 41 ◽  
pp. 552-555
Author(s):  
Conly L. Rieder ◽  
S. Bowser ◽  
R. Nowogrodzki ◽  
K. Ross ◽  
G. Sluder
Keyword(s):  
Small Sample Size ◽  
Small Sample ◽  
Meiotic Spindle ◽  
Serial Sections ◽  
Mitotic Apparatus ◽  
Thin Sections ◽  
Cell Cycles ◽  
Ultrastructural Studies

Eggs have long been a favorite material for studying the mechanism of karyokinesis in-vivo and in-vitro. They can be obtained in great numbers and, when fertilized, divide synchronously over many cell cycles. However, they are not considered to be a practical system for ultrastructural studies on the mitotic apparatus (MA) for several reasons, the most obvious of which is that sectioning them is a formidable task: over 1000 ultra-thin sections need to be cut from a single 80-100 μm diameter egg and of these sections only a small percentage will contain the area or structure of interest. Thus it is difficult and time consuming to obtain reliable ultrastructural data concerning the MA of eggs; and when it is obtained it is necessarily based on a small sample size.We have recently developed a procedure which will facilitate many studies concerned with the ultrastructure of the MA in eggs. It is based on the availability of biological HVEM's and on the observation that 0.25 μm thick serial sections can be screened at high resolution for content (after mounting on slot grids and staining with uranyl and lead) by phase contrast light microscopy (LM; Figs 1-2).

An Ultra-Structural Study of Human Melanoma in Vivo and Vitro

1976 ◽  
Vol 34 ◽  
pp. 258-259
Author(s):  
James R. Gaylor ◽  
Fredda Schafer ◽  
Robert E. Nordquist
Keyword(s):  
Endoplasmic Reticulum ◽  
Golgi Apparatus ◽  
Protein Aggregation ◽  
Structural Study ◽  
Human Melanoma ◽  
Protein Matrix ◽  
Early Form ◽  
Endoplasmic Reticulum Protein ◽  
Mitochondrial Origin

Several theories on the origin of the melanosome exist. These include the Golgi origin theory, in which a tyrosinase-rich protein is "packaged" by the Golgi apparatus, thus forming the early form of the melanosome. A second theory postulates a mitochondrial origin of melanosomes. Its author contends that the melanosome is a modified mitochondria which acquires melanin during its development. A third theory states that a pre-melanosome is formed in the smooth or rough endoplasmic reticulum. Protein aggregation is suggested by one author as a possible source of the melanosome. This fourth theory postulates that the melanosome originates when the protein products of several genetic loci aggregate in the cytoplasm of the melanocyte. It is this protein matrix on which the melanin is deposited. It was with these theories in mind that this project was undertaken.

Ultrastructural Study of a differentiating human colon carcinoma cell line

1990 ◽  
Vol 48 (3) ◽  
pp. 208-209
Author(s):  
Sylvie Polak-Charcon ◽  
Mehrdad Hekmati ◽  
Yehuda Ben Shaul
Keyword(s):  
Cell Line ◽  
Morphological Changes ◽  
Secretory Granules ◽  
Human Colon ◽  
Cell Types ◽  
Culture Conditions ◽  
Morphological Evidence ◽  
Human Colon Carcinoma Cell ◽  
Colon Cell

The epithelium of normal human colon mucosa “in vivo” exhibits a gradual pattern of differentiation as undifferentiated stem cells from the base of the crypt of “lieberkuhn” rapidly divide, differentiate and migrate toward the free surface. The major differentiated cell type of the intestine observed are: absorptive cells displaying brush border, goblet cells containing mucous granules, Paneth and endocrine cells containing dense secretory granules. These different cell types are also found in the intestine of the 13-14 week old embryo.We present here morphological evidence showing that HT29, an adenocarcinoma of the human colon cell line, can differentiate into various cell types by changing the growth and culture conditions and mimic morphological changes found during development of the intestine in the human embryo.HT29 cells grown in tissue-culture dishes in DMEM and 10% FCS form at late confluence a multilayer of morphologically undifferentiated cell culture covered with irregular microvilli, and devoid of tight junctions (Figs 1-3).

Spectroscopic imaging of human disease

1996 ◽  
Vol 54 ◽  
pp. 884-885
Author(s):  
D.J. Meyerhoff
Keyword(s):  
Magnetic Field ◽  
Magnetic Resonance ◽  
Field Gradient ◽  
Human Disease ◽  
Morphological Changes ◽  
Magnetic Field Gradient ◽  
Spectroscopic Imaging ◽  
Resonance Spectroscopy ◽  
Tissue Water

Magnetic Resonance Imaging (MRI) observes tissue water in the presence of a magnetic field gradient to study morphological changes such as tissue volume loss and signal hyperintensities in human disease. These changes are mostly non-specific and do not appear to be correlated with the range of severity of a certain disease. In contrast, Magnetic Resonance Spectroscopy (MRS), which measures many different chemicals and tissue metabolites in the millimolar concentration range in the absence of a magnetic field gradient, has been shown to reveal characteristic metabolite patterns which are often correlated with the severity of a disease. In-vivo MRS studies are performed on widely available MRI scanners without any “sample preparation” or invasive procedures and are therefore widely used in clinical research. Hydrogen (H) MRS and MR Spectroscopic Imaging (MRSI, conceptionally a combination of MRI and MRS) measure N-acetylaspartate (a putative marker of neurons), creatine-containing metabolites (involved in energy processes in the cell), choline-containing metabolites (involved in membrane metabolism and, possibly, inflammatory processes),

Facilitated migration of cells from a transected peripheral nerve over collagen fibers: in vivo observations

1989 ◽  
Vol 47 ◽  
pp. 888-889
Author(s):  
Arthur J. Wasserman ◽  
Azam Rizvi ◽  
George Zazanis ◽  
Frederick H. Silver
Keyword(s):  
Sciatic Nerve ◽  
Peripheral Nerve ◽  
Collagen Gel ◽  
Collagen Fibers ◽  
Control Group ◽  
Guide Tube ◽  
Sprague Dawley ◽  
Silicone Tube ◽  
Thin Sections

In cases of peripheral nerve damage the gap between proximal and distal stumps can be closed by suturing the ends together, using a nerve graft, or by nerve tubulization. Suturing allows regeneration but does not prevent formation of painful neuromas which adhere to adjacent tissues. Autografts are not reported to be as good as tubulization and require a second surgical site with additional risks and complications. Tubulization involves implanting a nerve guide tube that will provide a stable environment for axon proliferation while simultaneously preventing formation of fibrous scar tissue. Supplementing tubes with a collagen gel or collagen plus extracellular matrix factors is reported to increase axon proliferation when compared to controls. But there is no information regarding the use of collagen fibers to guide nerve cell migration through a tube. This communication reports ultrastructural observations on rat sciatic nerve regeneration through a silicone nerve stent containing crosslinked collagen fibers.Collagen fibers were prepared as described previously. The fibers were threaded through a silicone tube to form a central plug. One cm segments of sciatic nerve were excised from Sprague Dawley rats. A control group of rats received a silicone tube implant without collagen while an experimental group received the silicone tube containing a collagen fiber plug. At 4 and 6 weeks postoperatively, the implants were removed and fixed in 2.5% glutaraldehyde buffered by 0.1 M cacodylate containing 1.5 mM CaCl2 and balanced by 0.1 M sucrose. The explants were post-fixed in 1% OSO4, block stained in 1% uranyl acetate, dehydrated and embedded in Epon. Axons were counted on montages prepared at a total magnification of 1700x. Montages were viewed through a dissecting microscope. Thin sections were sampled from the proximal, middle and distal regions of regenerating sciatic plugs.

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