Freeze-substitution of rye grass and ragweed pollen grains

1990 ◽  
Vol 48 (3) ◽  
pp. 686-687
Author(s):  
Liza B. Martinez ◽  
Susan M. Wick
Keyword(s):  
Cell Function ◽  
Pollen Grains ◽  
Freeze Substitution ◽  
Cell Types ◽  
Rapid Freezing ◽  
Rye Grass ◽  
Acrylic Resins ◽  
Allergenic Proteins ◽  
Cold Embedding ◽  
Structural Preservation

Rapid freezing and freeze-substitution have been employed as alternatives to chemical fixation because of the improved structural preservation obtained in various cell types. This has been attributed to biomolecular immobilization derived from the extremely rapid arrest of cell function. These methods allow the elimination of conventionally used fixatives, which may have denaturing or “masking” effects on proteins. Thus, this makes them ideal techniques for immunocytochemistry, in which preservation of both ultrastructure and antigenicity are important. These procedures are also compatible with cold embedding acrylic resins which are known to increase sensitivity in immunolabelling.This study reveals how rapid freezing and freeze-substitution may prove to be useful in the study of the mobile allergenic proteins of rye grass and ragweed. Most studies have relied on the use of osmium tetroxide to achieve the necessary ultrastructural detail in pollen whereas those that omitted it have had to contend with poor overall preservation.

Three Dimensional Immuno-Localization of Green Fluorescent Protein Chimeras Using Rapid Freezing and Freeze-Substitution

2000 ◽  
Vol 6 (S2) ◽  
pp. 298-299
Author(s):  
Mary Morphew ◽  
David Mastronarde ◽  
Eileen O'Toole ◽  
Mark Ladinsky ◽  
Brad Marsh ◽  
...  
Keyword(s):  
Low Temperature ◽  
Fluorescent Protein ◽  
Three Dimensional ◽  
Freeze Substitution ◽  
Cell Types ◽  
Uranyl Acetate ◽  
Rapid Freezing ◽  
Thin Sections ◽  
Green Fluorescent ◽  
Structural Preservation

All microscopy is limited by the quality of the specimen under study. Three-dimensional (3-D) visualization of antigen localization using the electron microscope (EM) is particularly challenging due to the need to maintain the activity of some epitopes while preserving cellular ultrastructure. We have used rapid freezing to immobilize all cellular constituents almost instantaneously. Freeze-substitution of the frozen samples was used to stabilize the specimen and to accomplish low-temperature dehydration, minimizing perturbation of cellular structure. We have found that high pressure freezing, double jet freezing and plunge freezing are all useful for achieving high quality structural preservation for some cell types or for particular applications. For immunolocalization, we have had most success freeze-substituting into acetone containing 0.2% glutaraldehyde and 0.1 % uranyl acetate. We have utilized low-temperature acrylic embedding resins, Lowicryl HM20 and LRGold, to further maintain structure and decrease protein insolubility. Both of these resins have proven suitable for cutting serial thin sections.

scholarly journals Ultrastructural characteristics ofBetula pendulatapetum and pollen grains after rapid freezing and freeze-substitution

Grana ◽  
1999 ◽  
Vol 38 (4) ◽  
pp. 194-202 ◽  
Author(s):  
Mauro Cresti ◽  
Gamal El-Ghazaly ◽  
Claudio Milanesi ◽  
Björn Walles
Keyword(s):  
Pollen Grains ◽  
Freeze Substitution ◽  
Rapid Freezing ◽  
Ultrastructural Characteristics

High-Resolution Scanning Electron Microscopy of Biological Specimens

1988 ◽  
Vol 46 ◽  
pp. 186-187
Author(s):  
M. Müller ◽  
R. Hermann
Keyword(s):  
High Resolution ◽  
Freeze Drying ◽  
Room Temperature ◽  
Structural Information ◽  
Freeze Substitution ◽  
Critical Point Drying ◽  
Sem Study ◽  
Major Factors ◽  
Structural Preservation ◽  
Preparation Techniques

Three major factors must be concomitantly assessed in order to extract relevant structural information from the surface of biological material at high resolution (2-3nm).Procedures based on chemical fixation and dehydration in graded solvent series seem inappropriate when aiming for TEM-like resolution. Cells inevitably shrink up to 30-70% of their initial volume during gehydration; important surface components e.g. glycoproteins may be lost. These problems may be circumvented by preparation techniques based on cryofixation. Freezedrying and freeze-substitution followed by critical point drying yields improved structural preservation in TEM. An appropriate preservation of dimensional integrity may be achieved by freeze-drying at - 85° C. The sample shrinks and may partially collapse as it is warmed to room temperature for subsequent SEM study. Observations at low temperatures are therefore a necessary prerequisite for high fidelity SEM. Compromises however have been unavoidable up until now. Aldehyde prefixation is frequently needed prior to freeze drying, rendering the sample resistant to treatment with distilled water.

Ultrastructural and immunocytochemical studies of the rat hypothalamo-neurohypophysial system processed by rapid freezing and freeze-substitution fixation

1990 ◽  
Vol 48 (3) ◽  
pp. 884-885
Author(s):  
Seiji Shioda ◽  
Yasumitsu Nakai ◽  
Atsushi Ichikawa ◽  
Hidehiko Ochiai ◽  
Nobuko Naito
Keyword(s):  
Osmium Tetroxide ◽  
Freeze Substitution ◽  
Ultrastructural Localization ◽  
Wistar Rat ◽  
Neurosecretory Cells ◽  
Rapid Freezing ◽  
Sodium Metaperiodate ◽  
Tissue Sections ◽  
Ultrathin Sections ◽  
Electron Microscopes

The ultrastructure of neurosecretory cells and glia cells in the supraoptic nucleus (SON) of the hypothalamus and the neurohypophysis (PN) was studied after rapid freezing followed by substituion fixation. Also, the ultrastructural localization of vasopressin (VP) or its carrier protein neurophys in II (NPII) in the SON and PN was demonstrated by using a post-embedding immunoco1loidal gold staining method on the tissue sections processed by rapid freezing and freeze-substitution fixation.Adult male Wistar rat hypothalamus and pituitary gland were quenched by smashing against a copper block surface precooled with liquid helium and freeze-substituted in 3% osmium tetroxide-acetone solutions kept at -80°C for 36-48h. After substituion fixation, the tissue blocks were warmed up to room temperature, washed in acetone and then embedded in an Epon-Araldite mixture. Ultrathin sections mounted on 200 mesh nickel grids were immersed in saturated sodium metaperiodate and then incubated in each of the following solutions: 1 % egg albumin in phosphate buffer, VP or NPII (1/1000-1/5000) antiserum 24h at 4°C, 3) colloidal gold solution (1/20) 1h at 20°C. The sections were washed with distilled waterand dried, then stained with uranylacetate and lead citrate and examined with Hitachi HU-12A and H-800 electron microscopes.

scholarly journals Multiple Roles for Cholinergic Signaling from the Perspective of Stem Cell Function

2021 ◽  
Vol 22 (2) ◽  
pp. 666
Author(s):  
Toshio Takahashi
Keyword(s):  
Stem Cells ◽  
Stem Cell ◽  
Cell Function ◽  
Cell Activity ◽  
Embryonic Stem ◽  
Cholinergic Neurons ◽  
Cell Types ◽  
Proliferative Potential ◽  
True Nature ◽  
Cholinergic Signaling

Stem cells have extensive proliferative potential and the ability to differentiate into one or more mature cell types. The mechanisms by which stem cells accomplish self-renewal provide fundamental insight into the origin and design of multicellular organisms. These pathways allow the repair of damage and extend organismal life beyond that of component cells, and they probably preceded the evolution of complex metazoans. Understanding the true nature of stem cells can only come from discovering how they are regulated. The concept that stem cells are controlled by particular microenvironments, also known as niches, has been widely accepted. Technical advances now allow characterization of the zones that maintain and control stem cell activity in several organs, including the brain, skin, and gut. Cholinergic neurons release acetylcholine (ACh) that mediates chemical transmission via ACh receptors such as nicotinic and muscarinic receptors. Although the cholinergic system is composed of organized nerve cells, the system is also involved in mammalian non-neuronal cells, including stem cells, embryonic stem cells, epithelial cells, and endothelial cells. Thus, cholinergic signaling plays a pivotal role in controlling their behaviors. Studies regarding this signal are beginning to unify our understanding of stem cell regulation at the cellular and molecular levels, and they are expected to advance efforts to control stem cells therapeutically. The present article reviews recent findings about cholinergic signaling that is essential to control stem cell function in a cholinergic niche.

scholarly journals Recent advances in transport of water-soluble vitamins in organs of the digestive system: a focus on the colon and the pancreas

2013 ◽  
Vol 305 (9) ◽  
pp. G601-G610 ◽  
Author(s):  
Hamid M. Said
Keyword(s):  
Large Intestine ◽  
Digestive System ◽  
Cell Function ◽  
Strong Support ◽  
Alcohol Exposure ◽  
Cell Types ◽  
Water Soluble ◽  
Recent Advances ◽  
Efficient Uptake ◽  
Metabolic Activities

This review focuses on recent advances in our understanding of the mechanisms and regulation of water-soluble vitamin (WSV) transport in the large intestine and pancreas, two important organs of the digestive system that have only recently received their fair share of attention. WSV, a group of structurally unrelated compounds, are essential for normal cell function and development and, thus, for overall health and survival of the organism. Humans cannot synthesize WSV endogenously; rather, WSV are obtained from exogenous sources via intestinal absorption. The intestine is exposed to two sources of WSV: a dietary source and a bacterial source (i.e., WSV generated by the large intestinal microbiota). Contribution of the latter source to human nutrition/health has been a subject of debate and doubt, mostly based on the absence of specialized systems for efficient uptake of WSV in the large intestine. However, recent studies utilizing a variety of human and animal colon preparations clearly demonstrate that such systems do exist in the large intestine. This has provided strong support for the idea that the microbiota-generated WSV are of nutritional value to the host, and especially to the nutritional needs of the local colonocytes and their health. In the pancreas, WSV are essential for normal metabolic activities of all its cell types and for its exocrine and endocrine functions. Significant progress has also been made in understanding the mechanisms involved in the uptake of WSV and the effect of chronic alcohol exposure on the uptake processes.

Abstract 1440: Endothelial And Cardiomyocyte Toxicity Of Endogenously Produced Nucleoside: 4-pyridone-3-carboxamide-1-β-d-riboside

Circulation ◽  
2007 ◽  
Vol 116 (suppl_16) ◽  
Author(s):  
Ewa M Slominska ◽  
Czeslawa Orlewska ◽  
Cesare M Terracciano ◽  
Ada H Yuen ◽  
Urszula Siedlecka ◽  
...  
Keyword(s):  
Renal Failure ◽  
Endothelial Cells ◽  
Chronic Renal Failure ◽  
Cell Line ◽  
Cell Function ◽  
Cell Types ◽  
Human Erythrocytes ◽  
Rat Hearts ◽  
Prolongation Of Action Potential ◽  
Ribonucleoside Triphosphate

Our recent studies identified hitherto unknown, naturally occurring nucleotide: 4-pyridone-3-carboxamide-1-β-D-ribonucleoside triphosphate (4PYTP). This nucleotide was found to accumulate in the erythrocytes of patients with chronic renal failure, a condition that precipitates heart failure and atherosclerosis. We found also increase in plasma concentration of nucleoside precursor of 4PYTP: 4-pyridone-3-carboxamide-1-β-D-ribonucleoside (4PYR) in patients with renal failure. This study was aimed to evaluate metabolism and potential toxic effect of this nucleoside in cardiomyocytes and endothelial cells. We used human erythrocytes isolated from peripheral blood, human endothelial cell line HMEC-1 cell line and cardiomyocytes isolated from rat hearts. Cells were incubated with chemically synthesized 4PYR. Metabolic assessments were performed with HPLC or liquid chromatography/mass spectrometry. We demonstrated formation of 4PYTP in human erythrocytes during incubation with 4PYR. We noted however, preferential accumulation of monophosphate of 4PYR (4PYMP) over 4PYTP. Concentration of 4PYMP increased in the erythrocytes from below 5 μM to 76.9±7.1, 254.7±13.9 and 674.3±34.3 μM after 6h incubation with 0.1, 0.3 and 1 mM 4PYR. 4PYMP progressively accumulated in the cultured endothelial cells during incubation with 4PYR up to 72 h. Cardiomyocytes were also shown to accumulate 4PYMP. In all cell types formation of 4PYR nucleotides was accompanied by decrease in cellular ATP concentration. Furthermore, treatment with 4PYR and formation of its nucleotides in endothelial cells reduced NO synthase pathway while in cardiac myocytes resulted in prolongation of action potential. We conclude that endogenously formed 4PYR is effectively metabolized to nucleotide derivatives in the erythrocytes, cardiomyocytes and endothelium. Depletion of ATP in these cells accompanying 4PYR metabolism and deleterious effects on cell function suggests that accumulation of 4PYR and its enhanced metabolism to nucleotides in endothelium and cardiomyocyted may contribute to cardiovascular pathology observed in chronic renal failure.

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