Preparation For Electron Microscopy Research Articles

Amyloid myopathy: a therapeutic trial for the rare and underdiagnosed myopathy with bortezomib

A 69-year-old woman was referred to our clinic because of a 6-month history of progressive muscle weakness. Five years previously, when she was diagnosed as having nephrotic syndrome, the renal biopsy showed deposition of AL-amyloid. Subsequent investigations revealed multiple myeloma with k-type Bence-Jones protein at stage 3 according to the international staging system. A course of vincristine, doxorubicin and dexamethasone (VAD) and an additional course of cyclophosphamide were administered, followed by another course of melphalan (L-PAM) at age 67. The proteinuria slightly improved but persisted for 5 years, when proximal weakness developed. Neurological examination revealed proximally dominant weakness with MRC scale 4 in the deltoid and 3 in the iliopsoas muscles and she was unable to stand up from a full squat. Serum creatine kinase (CK) elevated to 3,399 U/L. Electromyography showed a myogenic pattern with small motor unit potentials, fibrillation potentials and early recruitment pattern. Muscle biopsy revealed a variation of fiber size, little infiltration of inflammatory cells, and homogeneous accumulations beneath the muscle membrane (Fig. 1a). Congo red-positive materials were noted both in vessel walls and in muscle fibers (Fig. 1b, c, d) with fluorescence for rhodamine, consistent with amyloid deposition, the hallmark of amyloid myopathy. We were not able to identify whether the deposits were located beneath the basal lamina or the plasmalemma due to lack of electron microscopic preparation. Five years ago, the serum CK value was 110 U/L (Fig. 2). Two years later, when her nephrotic syndrome deteriorated, the CK elevated to 2,336 IU/L without overt weakness. Interestingly, after she received L-PAM, the CK decreased to 120 U/L just 1 month after the treatment. When girdle weakness developed 6 months ago, the CK again soared to 3,378 U/L. The fluctuation of CK and the amyloid deposition in the muscle led us to the diagnosis of amyloid myopathy due to multiple myeloma. We then administered two consecutive courses of bortezomib (1.3 mg/m on days 1, 4, 8, and 11 for the first course and 1.0 mg/m on days 1, 4, 8, and 11 for the second course). The treatment reduced the proportion of myeloma cells from 16.8 to 1.2%. The CK started to fall a few weeks after the first course and eventually fell to 187 U/L 1 month after the second course, when she was able to rise from a sitting position more easily. However, multiple myeloma deteriorated in the following months and the CK again elevated to 694 U/L. She was not able to receive additional treatment because of her poor general status. Amyloid myopathy, a progressive myopathy in a proximally dominant fashion, is caused by a variety of primary amyloidoses, such as familial amyloidosis, AL-amyloidosis and multiple myeloma [1–3]. Postulated mechanisms include impaired muscle metabolism or failed electrical conduction by amyloid deposition beneath the muscle membrane [4, 5]. Therapeutic trials have included plasmapheresis, administration of corticosteroid, and high-dose K. Shiga (&) R. Mizutani R. Isayama T. Tokuda M. Nakagawa Department of Neurology, Kyoto Prefectural University of Medicine, 465 Kajii-cho, Hirokoji-agaru, Kawaramachi-dori, Kamigyo-ku, Kyoto 602-0841, Japan e-mail: kenshiga@koto.kpu-m.ac.jp

GABA and glutamate immunoreactivity in tentacles of the sea anemone Phymactis papillosa (LESSON 1830)

Sea anemones have a structurally simple nervous system that controls behaviors like feeding, locomotion, aggression, and defense. Specific chemical and tactile stimuli are transduced by ectodermal sensory cells and transmitted via a neural network to cnidocytes and epithelio-muscular cells, but the nature of the neurotransmitters operating in these processes is still under discussion. Previous studies demonstrated an important role of peptidergic transmission in cnidarians, but during the last decade the contribution of conventional neurotransmitters became increasingly evident. Here, we used immunohistochemistry on light and electron microscopical preparations to investigate the localization of glutamate and GABA in tentacle cross-sections of the sea anemone Phymactis papillosa. Our results demonstrate strong glutamate immunoreactivity in the nerve plexus, while GABA labeling was most prominent in the underlying epithelio-muscular layer. Immunoreactivity for both molecules was also found in glandular epithelial cells, and putative sensory cells were GABA positive. Under electron microscopy, both glutamate and GABA immunogold labeling was found in putative neural processes within the neural plexus. These data support a function of glutamate and GABA as signaling molecules in the nervous system of sea anemones.

Identification and partial sequencing of a crocodile poxvirus associated with deeply penetrating skin lesions in farmed Nile crocodiles, <i>Crocodylus niloticus</i>

When large numbers of crocodile skins were downgraded because of the presence of small pin prick-like holes, collapsed epidermal cysts were found deep in the dermis of juvenile crocodiles while forming cysts were observed in hatchlings. Histopathology of these forming cysts showed the presence of intracytoplasmic inclusions in proliferating and ballooning epidermal cells. Pox virions were seen in electron microscope preparations made from the scabs of such early lesions. The partial sequencing of virus material from scrapings of these lesions and comparison of it with the published sequence of crocodile poxvirus showed the virus associated with the deep lesions to be closely related, but different. To differentiate between the two forms of crocodile pox infection it is suggested that the previously known form should be called "classical crocodile pox" and the newly discovered form "atypical crocodile pox". The application of strict hygiene measures brought about a decline in the percentage of downgraded skins.

Nuclear behaviour during meiosis and ascus formation in Penicillium striatum

Meiosis and ascus formation in Penicillium striatum were examined by aid of light- and electron microscopy. The vegetative mycelium is haploid. In early stages of ascus formation two nuclei may partly fuse to a double haploid nucleus (n + n) with a membrane between them. When the intranuclear membrane breaks down, meiosis begins. After nuclear fusion prophase seems similar to that in Neurospora. Four to five bivalent associations occur arranged in a ring at first metaphase. Before anaphase, the bivalents arrange on line. The chromosome separation is almost synchronous. Some sort of interconnecting material occurs between the chromosomes especially during the second and third meiotic division. Typical centrioles did not occur in either the light- or in the electronmicroscopical preparations. Centrioles in vegetative nuclei lack microtubuli, thus differing considerably from the usual type. A typical nuclear membrane for eucaryote nuclei occurs. The central body is usually located near the nuclear membrane and changes shape during nuclear division. In sections it takes the form of a ring which disappears before metaphase. Ascospores are first delimited by a double membrane and the spore-wall seems to increase in thickness by deposition of material between the membranes.

Isolation and characterization of <i>tobacco necrosis virus</i> detected on some vegetable species

* Corresponding author. E-mail: irena.zitikaite@botanika.lt A virus disease agent was isolated from greenhouse-grown cucumber (Cucumis sativus L.), field-grown common bean (Phaseolus vulgaris L.) and white cabbage (Brassica oleracea var. capitata (L.) Alef) crops in Lithuania. The diseased vegetable plants expressed various spotting or mottling symptoms on leaves or fruits and necrotic symptoms on older leaves. The objective of this study was to identify the causal agent of the virus-like disease of these plant species. For the identification of the causal virus, the infection from cucumber, common bean and white cabbage leaves or fruits was transmitted to main test plant species which reacted by only local lesions. In electron microscopic preparations from infected plants, only icosahedral virus particles about 26 nm in diameter were detected. According to particle morphology, host range and symptoms, the causal virus was identified as tobacco necrosis virus (TNV). This finding was supported by the reverse transcription–polymerase chain reaction (RT–PCR) technique which confirmed that the virus isolates from the Lithuanian cucumber, common bean and white cabbage crops were identical with the TNV strain A “Nebraska” isolate.

Phylogeny of Dicranophoridae (Rotifera: Monogononta) - a maximum parsimony analysis based on morphological characters

This study presents the first phylogenetic analysis of Dicranophoridae (Rotifera: Monogononta), a species rich rotifer family of about 230 species currently recognized. It is based on a maximum parsimony analysis including 77 selected ingroup and three outgroup taxa and a total of 59 phylogenetically informative morphological characters. Character coding is based on personal investigation of material collected by the authors and an extensive survey of the literature. Apart from covering general body organization, character coding primarily relies on scanning electron microscopic preparations of the mastax jaw elements. Our study suggests monophyly of Dicranophoridae with a clade of Dicranophorus and Dorria as the sister taxon of all other dicranophorid species. Monophyly of Encentrum, the most species rich genus within Dicranophoridae, cannot be demonstrated. Within Dicranophoridae our study identifies the monophyletic taxa Caudosubbasifenestrata, Intramalleata, Praeuncinata and Proventriculata, each based on unambiguous character transformations evolved in their stem lineages. However, resolution within Praeuncinata and Proventriculata is very limited. Although some terminal clades within Praeuncinata and Proventriculata are recognized, basal splits remain obscure. Probably, other characters such as DNA sequence data are needed to further our understanding of phylogenetic relationships within these poorly resolved taxa.

Evaluation of Electron Microscopic Sample Preparation Methods and Imaging Techniques for Characterization of Cell-Mineral Aggregates

Scanning Electron Microscopy (SEM) is used to image geomicrobiological samples, typically containing interfaces between “hard and soft materials” such as minerals and cells, which represent challenges for artifact-free preparation for high-resolution imaging. We used cell-mineral aggregates produced during microbial Fe(II) oxidation and Fe(III) reduction to evaluate different sample preparation and imaging techniques. Both rapid freezing and standard critical point drying (CPD) preserve structures of geomicrobiological samples, at least the ones obtained for Fe(II)-oxidizing and Fe(III)-reducing bacteria, without artifacts. We recommend a SEM sample preparation scheme for geomicrobiological specimens and discuss critical parameters like fixation, dehydration, coating, and acceleration voltages.

Preparation and ultrastructure of spermatozoa from Green Poison Frogs, Dendrobates auratus, following hormonal induced spermiation (Amphibia, Anura, Dendrobatidae)

Few ultrastructural studies have been performed on members of the Dendrobatidae, although such investigations can be useful for the understanding of reproductive patterns, as a diagnostic method for males in breeding programs for endangered amphibians and for phylogenetic analysis. The sperm ultrastructure of the Green Poison Frog, Dendrobates auratus, from Panama is described following induced spermiation in living animals. To date only testicular spermatozoa in other dendrobatid frogs have been analysed. Moreover, an electron microscopic preparation method (transmission and scanning electron microscopy) for dendrobatid sperm cells in low concentration is presented. Sperm cells from stimulated frogs (100 IU human chorionic gonadotropin, hCG, twice at an interval of 1 h) were recovered via cloaca lavage using 600 μl isotonic phosphate-free amphibian saline (IPS). Centrifuged flushings (5 min, 173 × g) were deposited on microscopic slides. Adherent spermatozoa were treated with Karnovsky fixative (overnight, 4 °C). After postfixation (2 h, 1% osmium tetroxide), samples were dehydrated in series of ascending acetones (30–100%). For transmission electron microscopy sperm cells were encapsulated using Epon and 1.5% 2,4,6-tris(dimethylaminomethyl)phenol (DMP 30). Ultrathin sections (70 nm) were cut and stained with uranyl acetate (30 min) and lead citrate (5 min). Sperm cells are filiform with a 21.1 ± 2.7 μm long and arcuated head and a single tail (35.0 ± 4.2 μm length). Their acrosomal complex is located at the anterior portion of the head and consists of the acrosomal vesicle which has low electron density, and the subjacent electron-dense subacrosomal cone. In transverse section, the nucleus is circular (1.9 ± 0.2 μm diameter) and conical in longitudinal section. It is surrounded by several groups of mitochondria. The chromatin is highly condensed and electron-dense but shows numerous electron-lucent inclusions. A short midpiece has a mitochondrial collar with a proximal and a distal centriole. The latter gives rise to the axoneme which alone forms the flagellum. The sperm ultrastructure of D. auratus differs from that of other Dendrobatidae because of the absence of a nuclear space and the absence of the undulating membrane associated with an axial fibre. This tail conformation is found in the Ranoidea but not in the Bufonoidea. These results show that the spermatozoa of D. auratus are the first within the Dendrobatidae without accessory tail structures. Methods of using sperm samples from hormonal treated frogs for ultrastructural studies is not only reasonable to examine e.g. amphibian phylogeny without killing frogs threatened with extinction but allows investigations in the field of assisted reproduction and male fertility for example in conservation programs for endangered amphibians.

Chronically Administered Risperidone Did Not Change the Number of Hepatocytes in Rats: A Stereological and Histopathological Study

Risperidone, an antipsychotic agent, has been used in the therapy of patients with acute and chronic schizophrenia. The strategy in the choice of antipsychotic agent should take into account the hepatic tolerance. The aim of the present study was to analyse whether or not the chronic administration of risperidone has a toxic effect on rat livers. Rats were divided into three groups. Low dose (n = 5) and high dose (n = 5) groups that were given 0.5 and 1 mg/kg intraperitoneal doses of risperidone daily for 6 weeks, respectively. The control group received distilled water as a vehicle. At the end of the experiment, the rats were killed, and livers were dissected out immediately and transferred into the fixation solution. Both conventional light and transmission electron microscopic tissue preparation procedures were applied to liver tissues and they were evaluated using stereological and histopathological methods; in this respect, this is the first study using stereological methods for searching the effects of risperidone on liver. There were no significant differences about the total hepatocyte number and their numerical density of rat liver between groups (P > 0.05). Stereological results were also confirmed by the histopathological findings that come from both structural and ultrastructural levels examination. The results of the current study indicate that neither low nor high doses of risperidone resulted in damage to the rat livers at cellular level. In conclusion, we did not find any evidence for hepatotoxicity of risperidone rats. Larger-scale, prospective studies are needed in order to confirm these findings.

Scanning electron microscopic examination of the nasal mucosa epithelium in atrophic rhinitis

Objective: In this study, we aimed to examine the ultrastructural characteristics of nasal mucosa in patients with atrophic rhinitis and reported our findings. Methods: The biopsy specimens were obtained from two children and three adult patients, who had some or all of the classical manifestations of atrophic rhinitis. The tissue samples were prepared according to routine histological scanning electron microscopic tissue preparation procedure and examined using scanning electron microscope. The study was carried out in the electron microscopy laboratory of the Anatomy Department of Hacettepe University, Ankara, Turkey, between February 2001 and March 2003. Results: Squamous epithelium showing keratinization was observed in large areas. Additionally, desquamation of the horny cells of the nasal mucosa epithelium was found. The findings obtained in this study were in accordance with preview studies. Besides all these, we also observed accumulation of bacteria, especially the cocci in some focal regions. This accumulation of the bacteria may play a very important role in the etiology of the disease. Conclusion: Depending upon to the difficulty of finding patients with atrophic rhinitis, even the few number of cases observed in this study will have an importance for the ultrastructural characteristics of the disease.

Ultrastructural changes in embryoic neuroepithelial cells caused by passive smoking in golden hamsters at different periods of pregnancy: A randomized controlled trial*

Ultrastructural changes in embryoic neuroepithelial cells caused by passive smoking in golden hamsters at different periods of pregnancy: A randomized controlled trial*

Visualization and quantitative analysis of nanoparticles in the respiratory tract by transmission electron microscopy.

Nanotechnology in its widest sense seeks to exploit the special biophysical and chemical properties of materials at the nanoscale. While the potential technological, diagnostic or therapeutic applications are promising there is a growing body of evidence that the special technological features of nanoparticulate material are associated with biological effects formerly not attributed to the same materials at a larger particle scale. Therefore, studies that address the potential hazards of nanoparticles on biological systems including human health are required. Due to its large surface area the lung is one of the major sites of interaction with inhaled nanoparticles. One of the great challenges of studying particle-lung interactions is the microscopic visualization of nanoparticles within tissues or single cells both in vivo and in vitro. Once a certain type of nanoparticle can be identified unambiguously using microscopic methods it is desirable to quantify the particle distribution within a cell, an organ or the whole organism. Transmission electron microscopy provides an ideal tool to perform qualitative and quantitative analyses of particle-related structural changes of the respiratory tract, to reveal the localization of nanoparticles within tissues and cells and to investigate the 3D nature of nanoparticle-lung interactions.This article provides information on the applicability, advantages and disadvantages of electron microscopic preparation techniques and several advanced transmission electron microscopic methods including conventional, immuno and energy-filtered electron microscopy as well as electron tomography for the visualization of both model nanoparticles (e.g. polystyrene) and technologically relevant nanoparticles (e.g. titanium dioxide). Furthermore, we highlight possibilities to combine light and electron microscopic techniques in a correlative approach. Finally, we demonstrate a formal quantitative, i.e. stereological approach to analyze the distributions of nanoparticles in tissues and cells.This comprehensive article aims to provide a basis for scientists in nanoparticle research to integrate electron microscopic analyses into their study design and to select the appropriate microscopic strategy.

Spatiotemporal profile of dendritic outgrowth from newly born granule cells in the adult rat dentate gyrus

Neurogenesis in the adult dentate gyrus occurs in the subgranular zone where newborn neurons (NNs) migrate a short distance into the granule cell layer and extend their rudimentary apical dendritic processes upon a radial glial scaffold. Using doublecortin (DCX) immunocytochemistry, these growing dendrites can be visualized because dendritic growth cones, including filipodia and lamellipodia, are labeled in both light and electron microscopic preparations. To study the rate of dendritic outgrowth of newborn dentate granule cells, single injections of 5-bromo-2-deoxyuridine (BrdU) with different survival times were combined with double immunolabeling for BrdU and DCX. At the earliest time points (4 and 12 h after BrdU injections), a rudimentary process can be observed to emanate from BrdU/DCX double-labeled cells. By 48 h the dendrites first appeared in the molecular layer. By 96 h after BrdU injection, these apical dendrites extended into the middle of the molecular layer where they ramified. The calculated rate of dendritic growth for NNs was about 15 μm per day for the first 3 days, and then a doubling in length occurred at 4 and 5 days that coincided with a retraction of the basal dendrite. In addition, electron microscopy of DCX-labeled apical dendrites showed that they were much thinner (1/4 to 1/3 the size) in diameter than unlabeled, mature apical dendrites and that they had developing synapses on them in the molecular layer.

A plasma membrane‐associated AAA‐ATPase from Glycine max

An AAA-ATPase (ATPases Associated with a Variety of Cellular Activities) localized to the plasma membrane of soybean (Glycine max) was isolated, partially sequenced and cloned (SBPM AAA-ATPase). The protein with an apparent monomer molecular mass of about 97 kDa was isolated using a combination of anion exchange, preparative SDS-PAGE, reverse phase HPLC, and ATP affinity chromatography. The cDNA for the full-length SBPM AAA-ATPase was cloned by screening an expression library using an antibody against the highly conserved Walker B AAA-ATP-binding motif. Northern blot analysis detected one transcript of approximately 2700 bp. The full-length cDNA sequence was that previously obtained (GenBank Database; U20213) encoding a protein with two copies of the conserved AAA-ATP-binding motif and regions of sequence homology with other AAA-ATPases. Electron microscopic preparations of the recombinant SBPM AAA-ATPase revealed hexamers typically formed by these proteins. The cloned and expressed protein was identical to the protein isolated from the soybean plasma membrane as confirmed using antisera raised to a non-conserved region of the derived protein sequence and by N-terminal sequencing of peptides derived from the isolated protein.

Methods for in situ detection and characterization of extracellular polymers in biofilms by electron microscopy

Electron microscopy of biofilms and the localization of extracellular polymers at high resolution require the adaptation of conventional electron microscopic preparation and imaging techniques. A method developed for in situ fixation and embedding of biofilms, imaging of unstained thick sections with electron spectroscopic imaging and the application of lectin or antibody-based marker systems allowed interpretation of extracellular polymer distribution at micrometer scale. By this way, it is possible to discriminate in situ between extracellular polymers produced by different organisms.

Effects of chronic alcohol consumption on myocardial ischemia in rats

The effects of chronic alcohol consumption on myocardial ischemia and gas perfusion with 95% O 2–5% CO 2 were investigated in isolated rat heart. Eighteen adult male Wistar rats were used. Rats were assigned into six groups for each group to contain three rats: normal, alcoholic, normal ischemic, alcoholic ischemic, normal ischemic and 95% O 2–5% CO 2 perfused, alcoholic ischemic and 95% O 2–5% CO 2 perfused, respectively. Alcohol (7.2%, v/v) was given to rats by a modified liquid diet for 21 days. Rats were anaesthetized with ketamine (1–2 mg kg −1). Hearts were quickly isolated. Normal and alcoholic rat hearts were directly sent to the electron microscopic preparation. The other hearts were cut into small pieces and put into Krebs solution. The solution was continuously bubbled using 95% N 2–5% CO 2 20 min for ischemia. After removal of normal ischemic and alcoholic ischemic heart specimens for electron microscopic examination, the remaining hearts of the last two groups were bubbled with 95% O 2–5% CO 2 for another 20 min for the purpose of reperfusion and then were also prepared for electron microscopic examination. The hearts were investigated with a transmission electron microscope (Jeol 100 CXII TEM). Twenty-one days of chronic alcohol consumption was found to have no significant effect on myocardial ischemia determined by transmission electron microscopic examination. Our results suggest that there is no significant relationship between 21 days of alcohol consumption by a liquid diet and myocardial protection.

Suggested Standard Operating Procedures (SOPs) for the preparation of electron microscopy samples for toxicology/pathology studies in a GLP environment.

We provide a set of Standard Operating Procedures (SOPs) for preparing samples for electron microscopic evaluation that allow storage of samples in the primary fixative for at least 17 years without noticeable degradation, do not compromise the ability to prepare the same samples for standard light microscopic evaluation, and provide tips for orientation of samples that may be necessary for evaluation. Guidelines for proper sample size, buffer composition, and fluid concentrations during processing are given. The impact of these procedures on specimen quality, ability to produce truly comparable samples for drug development studies, and ways to minimize time spent by technicians preparing these samples during necropsies is evaluated. Although many laboratories routinely employ most of these techniques, this compilation will facilitate the simultaneous light and electron microscopic preparation by the pathologist of comparable specimens that can be stored long-term at 4 degrees C in McDowell's and Trump's 4F:1G fixative (4F:1G).

Suitable test structures for submicron ion beam analysis

For the precise determination of the sizes of submicron beam spots test structures with an excellent edge definition are required. For this purpose a semiconductor heterostructure consisting of an 1.62 μm GaInP epi-layer grown on (0 0 1) GaAs has been made, which provides atomically sharp edges for beam spot size measurements. Since the sample has been thinned down by standard transmission electron microscope (TEM) preparation techniques, it can be used for both PIXE and STIM. The sample has been investigated with a TEM and the ion nanoprobe LIPSION. A one-dimensional beam profile in the low current mode was determined by a STIM measurement using 2 MeV protons and yielded a FWHM of (41±4) nm, which is the smallest value reported so far for high energy nuclear micro- and nanoprobes. Furthermore we present nickel nanowhiskers produced at the GSI Darmstadt by electrochemical preparation of etched ion track membranes that have been used to obtain two-dimensional images of the shapes of submicron beam spots. For these measurements a scan over a single nickel nanowhisker having a diameter of 220 nm and a height of about 6 μm was performed.

Transmission electron microscopical studies on some haemolymph proteins from the marine polychaete Nereis virens

The hexagonal bilayer haemoglobin molecule from Nereis virens has been investigated in a comparative study using several different negative stain electron microscopical specimen preparations (i.e. by conventional adsorption to continuous carbon support films, by the negative staining-carbon film technique and by negative staining across the holes of holey carbon support films with air-drying and rapid freezing/cryo-negative staining). The benefits and limitations of these different approaches are indicated, with the overall conclusion that negative staining with ammonium molybdate across holes creates the best possibilities for molecular imaging, and also has the potential for the creation of two-dimensional (2D) crystals/arrays at the fluid–air interface. Of the different negative staining procedures presented, cryo-negative staining reveals the greatest details of N. virens haemoglobin. This is exemplified by the direct visualisation of the central linker-assembly within the haemoglobin molecule, a structural feature less clearly defined by the other negative staining techniques. A discoidal lipoprotein molecule (diameter 30–60 nm; thickness ca 8 nm) has been detected in N. virens, which represents the first documented account of an annelid haemolymph lipoprotein. The biological implications of this lipoprotein for lipid transport remain to be established. The presence of a low concentration of ferritin molecules in N. virens haemolymph is also shown, assisted by the formation of small 2D ferritin arrays in negatively stained specimens prepared across holes.

Variation in Chromatin Structure of the rDNA Transcribed Region in Human Peripheral Blood Lymphocytes

The rDNA transcribed region (TR) was tested for its accessibility for RsaI recognizing 15 TR sites, DNase I, and photoinducible arylazide (N-(4-azido-2-hydroxybenzoyl)-N,N"-diaminoheptane acetate) in isolated nuclei, and for the arylazide in intact cells. Arylazide readily entered the cells and did not appreciably affect the chromatin structure. Its photolysis products efficiently modified DNA in accessible sites. Single-strand breaks made by DNase I were not transformed into two-strand ones in rDNA TR, suggesting the necessity of denaturing electrophoresis for such an analysis. About 70% of all rDNA copies proved poorly accessible for endonucleases and arylazide, the accessibility being higher in their 18S and 5.8S rRNA gene regions than in the regions of the external transcribed spacers (ETSs) and the 28S rRNA gene. Proteinase K disrupted this structure, and the corresponding copies were extracted from nuclei. This explained whyin situ hybridization occasionally fails to reveal rDNA in the nucleolar fibrillar center (FC) on electron microscopic preparations. In other rDNA copies, TR (excluding 5"-ETS) was accessible for nucleases and arylazide. These copies were not extracted from nuclei treated with proteinase K. Some of their RsaI sites were protected by tightly bound proteins. Seven such regions were identified in TR. Possible association of the molecular structure, nucleolar location, and functional state of rDNA is discussed.