Association of axonally transported heparan sulfate with isolated synaptic plasma membrane

Studies on isolated synaptic plasma membranes (SPM) have detected little if any heparan sulfate or other glycosaminoglycans (GAGs), while more recent studies employing proteoglycan antibodies have localized heparan sulfate proteoglycan in presynaptic plasma membrane of intact tissue. To further address the issue of proteoglycans in synaptic plasma membrane of intact tissue. To further address the issue of proteoglycans in synaptic plasma membrane, we have investigated the possible presence of axonally transported GAGs in SPM isolated from the goldfish optic tectum. SPMs isolated from tecta following rapid axonal transport of³⁵SO₄ labeled molecules down the optic nerve, showed specific radioactivity approximately two-fold higher than the starting homogenate. Treatment of the transport labeled SPM with the enzyme heparitinase liberated 21% of the radioactivity, indicating the presence of a significant fraction of trnasported label in heparan sulfate. In a separate series of experiments a GAG fraction was isolated from transport labeled SPM and was found to consist of heparan sulfate containing 28% of transported radioactivity. Chondroitin (4 or 6) sulfate, which undergoes axonal transport in the goldfish optic system, was not found associated with SPM. Taken together the results support immunological evidence for the presence of heparan sulfate proteoglycans in presynaptic plasma membrane.To whom to address reprint request..     

Negative staining of freshwater bacterioneuston sampled directly with electron microscope specimen support grids

A technique for observation of surface microlayer bacteria (bacterioneuston) is described, utilizing direct sampling of the air-water interface with carbon-stabilized electron microscope specimen support grids, followed by negative staining and transmission electron microscopy. The method resulted in excellent preservation of forms of microcolonial association, regular surface arrays, surface appendages, and prosthecae in the bacterioneuston of a freshwater pond.     

Development of an atmospheric plasma jet device for versatile treatment of electron microscope sample grids

Atmospheric-pressure plasmas have been widely applied for surface modification and biomedical treatment because of their ability to generate highly reactive radicals and charged particles. In negative-stain electron microscopy (Neg-EM) and cryogenic electron microscopy (cryo-EM), plasmas have been used to generate hydrophilic surfaces and eliminate surface contaminants to embed specimens onto grids. In addition, plasma treatment is a prerequisite for negative-stain and Quantifoil grids, whose surfaces are coated with hydrophobic amorphous carbon. Although the conventional glow discharge system has been used successfully in this purpose, there has been no further effort to take an advantage from the recent progress in the plasma field. Here, we developed a nonthermal atmospheric plasma jet system as an alternative tool for treatment of surfaces. The low-temperature plasma is a nonequilibrium system that has been widely used in biomedical area. Unlike conventional glow discharge systems, the plasma jet system successfully cleans and introduces hydrophilicity on the grid surface in the ambient environment without a vacuum. Therefore, we anticipate that the plasma jet system will have numerous benefits, such as convenience and versatility, as well as having potential applications in surface modification for both negative-stain and cryo-EM grid treatment.     

Use of the analytical electron microscope (AEM) in cytochemical studies of the central nervous system.

Central nervous tissues (median eminence and arcuate nucleus) were studied by means of energy dispersive x-ray analysis using electron optical systems (analytical electron microscopy). These studies were conducted after the tissue had been treated specifically for localized biogenic amines (BA). The results indicate that not only is the BA cytochemical reaction highly specific, and that BAs can be localized intraneuronally in areas not previously identified, but also that the analytical electron microscope is a very valuable and potentially powerful tool in the studies of inclusion bodies and organelles in the central nervous system and other tissues. Thus, the nonspecific density production by osmium tetroxide can be elucidated from the specific BA reaction plus other areas of the nervous system containing density producing heavy metals, i.e., iron are readily identifiable.     

Cryo-electron microscopy of insect flight muscle thick filaments. An approach to dynamic electron microscope studies

Suspensions of isolated insect flight muscle thick filaments were embedded in layers of vitreous ice and visualized in the electron microscope under liquid nitrogen conditions. The unfixed, unstained, unsupported and fully hydrated filaments were observed under various biochemical conditions. We demonstrate here the first successful application of this method to thick filaments, and show that this is a possible approach to following dynamic processes by rapid freezing and electron microscopy.     

Localization of actin filaments in internodal cells of characean algae. A scanning and transmission electron microscope study

New methods of visualizing subcortical actin filament bundles, or fibrils, in Characean internodes confirm that they are associated with chloroplasts at the surface facing the streaming endoplasm, and reveal that they are continuous over long distances. With the scanning electron microscope, an average of four to six fibrils are seen bridging a file of chloroplasts. The same configuration appears in negatively stained preparations of large blocks of chloroplast files connected by actin fibrils. Few branches of the subcortical fibrils are evident. These findings are discussed with respect to the mechanism of cytoplasmic streaming in Characeae.     

Light and electron microscope observations on Nephroselmis astigmatica sp. nov. (Prasinophyceae)

Nephroselmis astigmatica sp. nov. is described based on light and electron microscope observations of cultured material, originally collected and isolated from the Natal South Coast, Republic of South Africa. It is characterized by (1) large cell size, (2) absence of a stigma, (3) markedly differentiated anterior part of the cell, (4) possessing two types of flagellar scales in addition to hair scales, (5) possessing four types of body scales and (6) the presence of characteristic pit scales in the flagellar pit. Scale morphology was compared with previously described species, and the morphology of spiny (or stellate) body scales thought to be one of the most useful diagnostic characters in delineating species within the genus. The origin of pit scales is discussed and a similar origin for the third layer of flagellar scales of the type species, N. olivacea Stein is suggested. N. astigmatica shares many ultrastructural features with the type species, including the microtubular flagellar root system consisting of three different roots, one of which is multilayered. The validity of this root system as a generic character is suggested.     

Examination of peripheral nerves with the scanning electron microscope.

The theoretical applications and advantages of the scanning microscope in peripheral nerve research are presented. The internal anatomy of the peripheral nerve can be distinctly examined, and long segments of axons can be examined without the necessity of tedious study of multiple sections. The SEM should make it possible to more readily study the migration of axon sprouts across a repair site. New concepts in teaching and research may develop from the use of this excellent tool.     

Interaction of an ion beam with the target in electron microscope applications

When based on the power-potential law of Lindhard et al. (Mat. Fys. Dan. Vid. Selsk, 33: 1–42, 1963) for ionic impact phenomena on the surfaces of a target, the universal curves of nuclear and electronic energy loss-energy, their resulting yield-energy relationships of sputtering and secondary electron emission yield-energy and range-energy have consistently been derived.According to the results obtained from the above experimental data, a diffusion model of an ion beam penetrating a target is proposed, which takes place throughout a hemisphere with its centre located at half the diffusion depth, and which is found to agree well with the empirical data of ion beam penetration, energy-dissipation profiles and the backscattering coefficient as a function of the reduced depth.Owing to the diffusion model's data, the total secondary electron emission yield due to both primary and backscattering ions is obtained. More importantly, radiation damage in ion beam applications is consistently evaluated as a function of the reduced energy ratio.     

In vitro incorporation of (3H)threonine and (3H)glucose by the mucous and serous cells of the human bronchial submucosal gland. A quantitative electron microscope study

Incorporation of [3H]threonine and [3H]glucose by the mucous and serous cells of the human bronchial submucosal gland has been studied over 8 h using, for the first time in vitro pulse labeling and electron microscope autoradiography. In assessing the autoradiographs, two methods were compared, the circle analysis and the recently described hypothetical grain analysis. Preliminary studies showed formaldehyde to be the most suitable fixative. Chemical analysis of tissue revealed that [3H]threonine was incorporated into the polypeptide moiety of the bronchial gland product and that metabolites of [3H]-glucose were incorporated into the carbohydrate. Tritiated threonine was first localized in the endoplasmic reticulum of both mucous and serous cells and later migrated to the Golgi apparatus, while metabolites of [3H]glucose localized first mainly in the Golgi apparatus. From here, both radioactive precursors were next identified in vacuoles and, finally, in secretory granules. The mucous cell incorporated strikingly more of both radioactive precursors than the serous cell. Thus, it seems that oligosaccharides of mucous and serous cell glycoproteins are synthesized mainly in the Golgi apparatus and added there to the polypeptide core which is synthesized in the endoplasmic reticulum. The relationship of the mucous cell to the serous cell is discussed. It seems that under "normal" conditions each cell represents a different line but that injury may transform a serous cell into a mucous cell.     

The distribution of desmin (100 A) filaments in primary cultures of embryonic chick cardiac cells.

Using antibodies to desmin, the major component of the 100Å-filaments from smooth muscle cells, we studied by indirect immunofluorescence the distribution of this protein in primary cultures of embryonic chick cardiac cells. We show that desmin is a component of cytoplasmic filamentous structures which comprise a network distinct from actin filament bundles and microtubules. Exposure of these cells to colcemid results in a rapid disaggregation of microtubules, and a slow aggregation of the desmin-containing filaments towards the nuclear area with the ultimate formation of a perinuclear ring. In differentiated skeletal or cardiac muscle cells, in addition to its cytoplasmic filamentous distribution, desmin is found intimately associated with the Z lines of sarcomeres. We further show that approx. 50% of the cells in these primary cardiac cultures are unreactive with desmin antibodies. Similarly the majority of the cells in a number of established cell lines from various species grown in tissue culture, are unreactive to desmin antibodies in indirect immunofluorescence, despite the fact that these cells are known to contain cytoplasmic 100Å-filaments. These results indicate that desmin occurs in at least two distinct cytoplasmic distribution in cardiac cells. They also demonstrate the existence of immunological and biochemical differences in the major component of 100Å-filaments between muscle and non-muscle cells as evidenced by the failure of non-muscle cells to react with antibodies to chick smooth muscle desmin.