“Chromosome exo-skeleton” in plant cells visualized by scanning electron microscopy

Summary Cytoskeletons surrounding the chromosomes of the root tip cells ofPisum sativum and the generative cells ofAllamanda schottii were visualized using Triton X-100 extraction and scanning electron microscopy. The cytoskeleton surrounding the chromosome consisted of a reticulate network of fibres. This is the first report showing the existence of a chromosome exo-skeleton in plant cells.Abbreviations EGTA ethyleneglycol-bis-(-aminoethylether) N,N,N,N-tetraacetic acid - PIPES piperazine-N,N-bis-(2-ethanesulfonic acid)     

Improved transmission electron microscopy (TEM) of cultured cells through a “floating sheet” method

Cultured cells provide isolated systems for both biochemical and morphological studies. Previous methods of processing cell culture specimens for electron microscopy (EM) have been limited to sectioning either a monolayer or centrifuged cell suspensions which are not morphologically intact. In our improved method, N-butylglycidyl ether is added to cell cultures (2–5 min with agitation) following in situ fixation (3.0% glutaraldehyde in 0.1 M Pipes, pH 7.2, for 20 min, osmium tetraoxide 4% for 20 min). A thin pliable “sheet” of cells floats free from the plastic culture device and can be manipulated (centrifuged or folded) to obtain a vast number of morphologically intact cells for examination. We have examined several cell types (vascular smooth muscle, lung, liver, and endothelial cells) grown in two types of plastic culture flasks (Nunc and Falcon). This new method provides excellent EM morphology, maximizes the number of cells examined, and allows determination of cell orientation since a remnant of the dissolved flask remains loosely bound to the bottom of the cells.     

Absence of α-adrenergic inhibition of lipolysis in swine adipose tissue

1. Adipose tissue slices from young and older pigs and genetically obese pigs were incubated to demonstrate α-adrenergic inhibition of lipolysis as found by other investigators in dog, guinea-pig, hamster, human and rabbit adipose tissue.2. Purported α-adrenergic agonists (amidephrine, clonidine, methoxamine, phenylephrine) did not inhibit basal or catecholamine-stimulated lipolysis.3. Purported α-adrenergic antagonists (dihydroergotamine, phenoxybenzamine, phentolamine, prazosin, yohimbine) did not enhance basal or stimulated lipolysis.4. Adipose tissue from pigs is different from that of most species but similar to that of rats with no α-adrenergic inhibition of lipolysis.     

“Thiocyanate gold”: small (2–3 nm) colloidal gold for affinity cytochemical labeling in electron microscopy

Summary Reduction of HAuCl₄ by NaSCN or KSCN produces colloidal gold particles of 2.6 nm in diameter and homogeneous in size (coefficient of variation 15%). The Au_SCN sol forms protein-gold complexes. The amount of protein required to form an Au_SCN-protein complex is best determined in the electron microscope, where serial dilutions of protein with gold sol are inspected for the presence of aggregates.By immuno-electron microscopy SCN-gold complexed to protein A is active and visible as is shown by revealing -amylase in rat pancreatic acinar cells.     

Localization of α-galactomannan on the surface of Schizosaccharomyces pombe cells by scanning electron microscopy

Galactomannan was localized by scanning and transmission electron microscopy on the cells and cell walls of Schizosaccharomyces pombe. The markers were prepared from colloidal gold granules labelled with an -galactopyranosyl-binding lectin isolated from the seeds of Bandeiraea simplicifolia. Part or all of this -galactomannan was present in the outer layer of the cell wall and was uniformly distributed even on the fission scars.Non-Standard Abbreviations Au lectin-labelled colloid - SEM scanning electron microscopy - TEM transmission electron microscopy     

Environmental scanning electron microscopy (ESEM)—a versatile tool in studying plants

Environmental scanning electron microscopy (ESEM) enables the investigation of hydrated and uncoated plant samples and the in situ observation of dynamic processes. Water vapor in the microscope chamber takes part in secondary electron detection and charge prevention. Two ESEM modes are available and offer a broad spectrum of applications. The environmental or wet mode prevents sample dehydration by the combination of sample cooling (5°C) and a vapor pressure of 4–6 Torr. In the low vacuum mode, the maximum chamber pressure is limited to 1 Torr (corresponding to about 5% relative humidity in the chamber) and allows the simultaneous use of a backscattered electron detector for imaging material contrast. A selection of characteristic plant samples and various applications are presented as a guide to ESEM for plant scientists. Leaf surfaces, trichomes, epicuticular waxes, and inorganic surface layers represent samples being comparatively resistant to dehydration, whereas callus cells and stigmatic tissue are examples for dehydration- and beam-sensitive samples. The potential of investigating dynamic processes in situ is demonstrated by studying anther opening, by tensile testing of leaves, and by performing hydration/dehydration experiments by changing the vapor pressure. Additionally, automated block-face imaging and serial sectioning using in situ ultramicrotomy is presented. The strengths and weaknesses of ESEM are discussed and it is shown that ESEM is a versatile tool in plant science.     

To be or not to be? Examples of incorrect identification of autophagic compartments in conventional transmission electron microscopy of mammalian cells

Transmission electron microscopy is one of the most sensitive methods to detect autophagic compartments in mammalian cells. The number of scientists that are experts in this technique has declined during recent years, probably because the method was developed over forty years ago and is not in fashion today. This has lead to a situation where, too many times, authors and reviewers alike are not able to interpret electron microscopy pictures correctly. In this commentary I present some common mistakes in identification of autophagic compartments in conventional transmission electron microscopy of mammalian cells.     

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

A novel ”in vivo cryotechnique” with replica immunoelectron microscopy was developed for detecting caveolin localization on replica membranes prepared directly from living smooth muscle cells. After quick-freezing mouse duodenal walls by our ”in vivo cryotechnique”, the specimens were prepared for freeze-fracture and deep-etch replica membranes. Then they were treated with 5% SDS and 0.5% collagenase to keep some antigens on the replica membranes. The immunogold method could be used to clarify the localization of the caveolin antigen in relation to three-dimensional ultrastructures of living smooth muscle cells. Our new cryotechnique can provide native organization of functional molecules in living cells. Accepted: 7 October 1999     

Improved methods for detection of β-galactosidase (lacZ) activity in hard tissue

The β-galactosidase gene (lacZ) of Escherichia coli is widely used as a reporter gene. The expression of lacZ can be detected by enzyme-based histochemical staining using chromogenic substrates such as 5-bromo-4-chloro-3-indolyl-β-D: -galactoside (X-gal). Because the enzymatic activity of lacZ is vulnerable to high temperatures and acid treatment for demineralization, detection of lacZ on paraffinized sections is difficult, especially for hard tissues, which require demineralization before sectioning in paraffin. To circumvent this problem, whole-mount X-gal staining before sectioning is performed. However, detection of lacZ activity in the center of larger portions of hard whole adult tissues is challenging. In this study, focusing on fixation procedures, we determined the conditions conducive to improved detection of lacZ activity in deeper areas of whole tissues. We used an annexin a5 (Anxa5)-lacZ reporter mouse model in which the Anxa5 expression in hard tissue is indicated by lacZ activity. We found that lacZ activity could be detected throughout the periodontal ligament of adult mice when fixed in 100% acetone, whereas it was not detected in the periodontal ligament around the root apex fixed in glutaraldehyde and paraformaldehyde. This staining could not be detected in wild-type mice. Acetone maintains the lacZ activity within 48 h of fixation at both 4°C and at room temperature. In conclusion, acetone is the optimal fixative to improve permeability for staining of lacZ activity in large volumes of adult hard tissues.     

Bright field electron microscopy of biological specimens—VI Signal-to-noise ratio in specimens prepared on amorphous carbon and graphite crystal supports

Three specimen supports (1.3nm and 15nm thick evaporated carbon films and 3–4nm thick graphite crystals) have been used in order to evaluate the gain in signal-to-noise (S/N) ratio in electron micrographs of negatively stained Tobacco Mosaic Virus (TMV) protein. An attempt was also made to obtain images of atoms of selectively stained organic molecules by brigth field electron microscopy. Microdensitometry and optical Fourier analysis were used. For the enhancement of contrast and resolution below 5nm we concluded that the 15nm thick carbon film is not good enough when supporting biological specimens similar to the one used in this investigation. The S/N ratio increased when the TMV protein was supported on 1.3nm thick carbon films. A further improvement was observed when the specimens were prepared on ‘low noise’ graphite supports. If atomic phase contrast is wanted in selectively stained objects, crystal supports must be used. Electron micrographs of a mellitic acid-uranyl acetate complex mixed in the right stoichiometric ratio revaled equilateral projections of triangularly oriented black spots 1.0nm apart. This atomic configuration was difficult to identify amid the defocus dependent granulation of evaporated carbon supports.     

Pharmacological evaluation of ocular β-adrenoceptors in rabbit by tissue segment binding method

AimsThis study evaluates ocular (iris, ciliary body and ciliary process) and nonocular (atria and lung) β-adrenoceptors in rabbit to characterize the plasma membrane β-adrenoceptors and binding affinities of β-adrenoceptor antagonists.Main methodsThe tissue segment binding method with a hydrophilic radioligand (?)-4-[3-t-butylamino-2-hydroxypropoxy]-[5,7-³H]benzimidazol-2-one ([³H]-CGP12177) was employed.Key findingsSpecific and saturable binding of [³H]-CGP12177 to intact tissue segments was detected by using (±)-propranolol to define nonspecific binding, showing a single population of plasma membrane binding sites with high affinity. Competition experiments with selective β₁- and β₂-adrenoceptor antagonists revealed a single population of β₂-adrenoceptors in ocular tissues and of β₁-adrenoceptors in atria, but mixed populations of β₁- and β₂-adrenoceptors in 70% and 30%, respectively, in lung. A competition curve for timolol was biphasic in lung and its binding affinity for β₂-adrenoceptors was approximately 158-fold higher than for β₁-adrenoceptors, indicating the β₂-selectivity of timolol. In contrast, competition curves for stereoisomers of befunolol, carteolol, and propranolol were monophasic in all tissues. The (?)-enantiomers of these antagonists were more potent than corresponding (+)-enantiomers in displacing from [³H]-CGP12177 binding, and the isomeric potency ratios of befunolol and carteolol were less than those of propranolol.SignificanceThis study with tissue segment binding method suggests that the binding affinity of (?)-enantiomers of β-adrenoceptor antagonists for plasma membrane β-adrenoceptors (β₁-adrenoceptors of atria, β₂-adrenoceptors of ocular tissues, and mixed β₁-/β₂-adrenoceptors of lung) is higher than that of corresponding (+)-enantiomers and their stereoselectivity is different between β-adrenoceptor antagonists.     

Adipose tissue derived mesenchymal stem cells are better respondents to TGFβ1 for in vitro generation of cardiomyocyte-like cells

Molecular and Cellular Biochemistry - S100A11 as a S100 protein family member has been documented to play dual-direction regulation over cancer cell proliferation. We explored the role of S100A11...     

Some personal and historical notes on the utility of “deep-etch” electron microscopy for making cell structure/function correlations

This brief essay talks up the advantages of metal replicas for electron microscopy and explains why they are still the best way to image frozen cells in the electron microscope. Then it explains our approach to freezing, namely the Van Harreveld trick of “slamming” living cells onto a supercold block of metal sprayed with liquid helium at −269ºC, and further talks up this slamming over the alternative of high-pressure freezing, which is much trickier but enjoys greater favor at the moment. This leads me to bemoan the fact that there are not more young investigators today who want to get their hands on electron microscopes and use our approach to get the most “true to life” views of cells out of them with a minimum of hassle. Finally, it ends with a few perspectives on my own career and concludes that, personally, I''m permanently stuck with the view of the “founding fathers” that cell ultrastructure will ultimately display and explain all of cell function, or as Palade said in his Nobel lecture,electron micrographs are “irresistible and half transparent … their meaning buried under only a few years of work,” and “reasonable working hypotheses are already suggested by the ultrastructural organization itself.”     

Effect of adenosine 3′,5′-(cyclic)-monophosphate on the synthesis of progestational steroids by rabbit ovarian tissue in vitro

1. Adenosine 3',5'-(cyclic)-monophosphate (3',5'-AMP) stimulates the synthesis of progestational steroids by rabbit ovarian tissue in vitro. 2. Other adenosine phosphates fail to increase steroidogenesis. 3. The ratio of 20alpha-hydroxypregn-4-en-3-one to progesterone, the maximal response of the tissue, and the responses of separated corpora lutea and interstitial tissue produced by luteinizing hormone are closely paralleled by 3',5'-AMP. 4. In tissues maximally stimulated by luteinizing hormone, 3',5'-AMP fails to produce an additional response. 5. The addition of theophylline, an inhibitor of phosphodiesterase, potentiates the effects of 3',5'-AMP and also luteinizing hormone. 6. The results obtained suggest that 3',5'-AMP is a mediator of the action of luteinizing hormone on progestational steroid synthesis by rabbit ovarian tissue.