Ultrastructure of double minutes from a human tumor cell line

Double minutes (dm) have been isolated from human tumor cells by zonal centrifugation and by differential pelleting of chromosome suspsension. These methods allowed collection of dm in sufficient quantity and purity for visualization with electron microscopy. Ultrastructurally, the chromatin fibers in dm resemble thrance fragments was found. When the two isolation protocols were compared, differential pelleting was shown to increase purity twofold to 85% dm by mass. The differential pelleting procedure enables easy collection of dm in sufficient quantity and purity for chemical analysis.     

A scanning electron microscopy study of vascular casts from senile human brains.

A scanning electron microscope study was performed of vascular casts from 26 senile human brains. In 15 of these, three types of arterial deformities (glomerular loop formations, vascular wickerworks and bundles) were frequently encountered. They were compared with the appearance in microangiograms and histological sections.     

A combined light and scanning electron microscopy study

The uppermost Eocene Florissant Formation, Rocky Mountains, Colorado, has yielded numerous insect, vertebrate and plant fossils. Three previous comprehensive palynological studies investigated sections of lacustrine deposits of the Florissant Formation and documented the response of plant communities to volcanic eruptive phases but overall found little change in plant composition throughout the investigated sections. These studies reported up to 150 pollen and spore phenotypes. In the present paper, we used a taxonomic approach to the investigation of dispersed pollen and spores of the Florissant Formation. Sediment samples from the shale units containing macrofossils were investigated using light microscopy (LM) and scanning electron microscopy (SEM). The general picture of the palynoflora is in agreement with previous studies. However, the combined LM and SEM investigation provides important complementary information to previous LM studies. While a fairly large amount of previous pollen determinations could be confirmed, the purported taxonomic affinities of several pollen phenotypes need to be revised. For example, pollen referred to as Podocarpus or Podocarpidites sp. belongs to the Pinaceae Cathaya, Malus/Pyrus actually belongs to Dryadoideae, pollen of the form genus Boehlensipollis referred to as Proteaceae/Sapindaceae/Elaeagnaceae or Cardiospermum belongs to Sapindaceae but not to Cardiospermum, and pollen of Persicarioipollis sp. B with previously assumed affinities to Polygonaceae actually belongs to Thymelaeaceae. Pandaniidites and one type of Malvacipollis cannot be linked with Pandanaceae and Malvaceae. A few taxa are new records for Florissant (Ebenaceae: Diospyros; Mernispermaceae; Trochodendraceae: Tetracentron). In general, SEM investigations complement the LM palynological studies and improve the identification of dispersed pollen and spores and enable integration of data from dispersed fossil pollen into a wide range of comparative morphological, taxonomic, evolutionary, biogeographic and phylogenetic studies.     

Sugarcane somatic embryogenesis: a scanning electron microscopy study

Spindles of CUBA 87-51 sugarcane were cultured in Murashige and Skoog (MS) basal medium and supplemented with different nutrients. Embryogenic and non-embryogenic callus obtained were comparatively studied by scanning electron microscopy (SEM). Samples of embryogenic callus cultured in regeneration medium (MS without 2.4 dichlorophenoxyacetic acid) were taken at different times for analyzing the sequential process. Distinctive features of two types of callus are shown by SEM: cells organized in embryos are noted in embryogenic callus; while elongated, disorganized cells can be seen in non-embryogenic callus. The characteristics of the embryos during plant regeneration are described. Sugarcane embryoid stages are: globular, globular with lateral notch and scutellum. In this process also appear shoot meristems, leaf and root primordia and finally, true leaves and roots. It is concluded that callus plant regeneration from young leaf segments of sugarcane mainly occur via somatic embryogenesis.     

In vitro excystation of Giardia from humans: a scanning electron microscopy study

The in vitro excystation of Giardia lamblia on cysts isolated from human feces was studied. After purification by sucrose gradient, cysts were incubated in a pepsin-acid solution, then placed in a modified HSP3 medium where excystation occurred within a few minutes. The excystation procedure was studied by continuous observations by light microscopy and sequential observations by scanning electron microscopy (SEM). The in vitro excystation was stopped at timed intervals during incubation by addition of a large amount of 1% glutaraldehyde. The excystation process began by the cyst wall opening at one pole. Flagella protruded rapidly, the parasite emerged progressively from the cyst envelope, posterior end first, the empty cyst collapsed and shrank. Although flagella emerging from the organism were distinguishable, the cell body had not yet shown all the morphological features of the G. lamblia trophozoite. A radical rearrangement of the organism occurred gradually: initially oval in shape, the parasite became round, then elongated, flattened, and underwent cytokinesis. The daughter trophozoites acquired their typical morphological features: the shape, the adhesive disc with the C-shaped structure distinctly visible on the ventral surface, and the definite placement of the flagella. These observations obtained on G. lamblia by SEM were comparable to those obtained with G. muris.     

High resolution scanning electron microscopy of the cell

The scanning electron microscope (SEM) has become a powerful tool for ultrastructural research with improvement of the instrument's resolution and progress in specimen preparation techniques. With regard to resolution, it has been improved step-by-step in this decade and, in 1985, an ultra-high resolution SEM (UHS-T1) was developed, with a resolution of 0.5 nm. Concerning specimen preparation, the osmium-DMSO-osmium method, which is effective for revealing intracellular structures, has come to be widely used. Techniques for observing smaller objects, such as bacteriophages, viruses, and biological macromolecules, have also been devised in recent years. As a result of these preparation techniques and the availability of the ultra-high resolution SEM, the application of SEM in biology is expanding rapidly. In this paper, an outline of the ultra-high resolution SEM, techniques for specimen preparation, findings of some biological materials by these techniques, and guidelines to making the specimens, are described.     

DNA content and structure of (double) minutes of a methotrexate-resistant cell line

We have determined the DNA content of intact double minutes (DMs) and of single minutes (SMs) by fluorometry of the individual chromatin bodies in metaphase spreads after staining with Feulgen-Schiff pararosaniline. We find that the intact DMs and SMs of the methotrexate-resistant mouse cell line 3T6R50 contain 4.4 megabase pairs (Mb) and 2.6 Mb DNA respectively, using the DNA content of E. coli (4.7 Mb) as a reference. As the pulsed field gradient gel electrophoresis experiments by van der Bliek et al. (1988) have indicated that the minutes of 3T6R50 cells contain a homogeneous population of 2.5 Mb DNA circles, we conclude that a SM contains one circular double strand DNA molecule of approximately 2.5 Mb, whereas DMs contain two.     

DNA content and structure of (double) minutes of a methotrexate-resistant cell line

Summary We have determined the DNA content of intact double minutes (DMs) and of single minutes (SMs) by fluorometry of the individual chromatin bodies in metaphase spreads after staining with Feulgen-Schiff pararosaniline. We find that the intact DMs and SMs of the methotrexate-resistant mouse cell line 3T6R50 contain 4.4 megabase pairs (Mb) and 2.6 Mb DNA respectively, using the DNA content of E. coli (4.7 Mb) as a reference. As the pulsed field gradient gel electrophoresis experiments by van der Bliek et al. (1988) have indicated that the minutes of 3T6R50 cells contain a homogeneous population of 2.5 Mb DNA circles, we conclude that a SM contains one circular double strand DNA molecule of approximately 2.5 Mb, whereas DMs contain two.This study was supported in part by grant NKI 84-20 of the Queen Wilhelmina Fund to P.B.     

A structural basis for R- and T-banding: a scanning electron microscopy study

The structure of reverse (R)-banded and telomeric (T)-banded chromosomes was studied by examination of the same chromosomes first in the light microscope (LM) followed by the scanning electron microscope (SEM). This procedure demonstrated a structural basis to both the R- and T-banding techniques. A direct correlation was shown between the LM staining patterns and the structural patterns observed in the SEM. In the R-banded chromosomes the positively stained R-bands, viewed by LM, corresponded to highly fibrous three-dimensional regions in the SEM. The negatively stained R-interbands corresponded to flatter regions from which material appeared to have been extracted. These structural observations strongly support the suggestion that chromosomal material is preferentially lost from the R-interbands with aggregation of fibres in the R-bands. T-banded chromosomes showed a similar structure to the R-banded chromosomes. The positively stained T-bands located at the telomeres corresponded to regions of highly aggregated fibres. The remainder of the chromosome, corresponding to the negatively stained area, had a flattened and extracted appearance. These similarities in morphology between the T- and R-banded chromosomes support the view that T-bands result from a progressive breakdown of the R-banded chromosome structure.     

A scanning electron microscopy study of micro-arterial damage and repair.

The effects of microvascular clamps on the femoral vessels of rats were studied, using the SEM. The early changes observed were (1) local fusiform dilatation of the area secondary to necrosis of the muscular wall, (2) flattening of the longitudinal ridges in the endothelial (3) loss of laminar flow, (4) endothelial sloughing, (5) platelet aggregation, and (6) leukocyte adherence and diapedesis. The repair of the endothelium occurred by an early replication of the adjacent undamaged endothelial cells -- with their subsequent migration across a platelet bed. The coverage was complete in one week, although reorientation of the neo-endothelial cells took longer. On the basis of this study and our clinical experience, we think the ideal microvascular clamp would possess the following characteristics: small size, light weight, mechanical simplicity, flat jaws (one to two mm in diameter) coated with a non-slip surface, and calibrated to produce a pressure less than 30 gm per mm2. In addition the clamp should be unaffected by blood, autoclaving, or repeated use. No such clamp is commercially available now, but we hope that one will be available in the near future.     

Development of Dictyostelium discoideum: A study by scanning electron microscopy

Stages in the development of the cellular slime mould Dictyostelium discoideum have been examined by scanning electron microscopy using specimens prepared by freeze-drying, infiltration with polyethylene glycol or freezing. Formation of the slime sheath characteristic of D. discoideum was first observed early in aggregation and by the time discrete aggregates had formed, they were completely enveloped in the slime sheath. At the first finger-like stage, the slime sheath appeared to be thicker at the base than at the tip of the aggregate but such variations in thickness of the slime sheath could not be observed in the migrating pseudoplasmodium although it has previously been suggested that this may be important in controlling cell differentiation during this migratory stage.     

A study of extracellular vesicles isolated from blood plasma conducted by low-voltage scanning electron microscopy

Extracellular vesicles secreted by cells represent an almost spherical membrane structures enriched with biological molecules of different types. The number and molecular composition of these structures depend on both the physiological state of an organism and underlying diseases. Despite extracellular vesicles playing an important role in intercellular communication and being potential biomarkers of pathological processes, the mechanisms of their formation, their functions, and their morphological characteristics are poorly studied. Low-voltage scanning electron microscopy is a promising method for studying extracellular vesicles, since it does not need a layer of conductive covering and, consequently, permits morphological details of studied objects to be vizualized at a high resolution in a nanometer range. The results of investigation of the morphology and sizes of objects in blood-plasma fractions by low-voltage scanning electron microscopy are presented in this study.     

Different patterns of collagen-proteoglycan interaction: a scanning electron microscopy and atomic force microscopy study

The extracellular matrix of unfixed, unstained rat corneal stroma, visualized with high-resolution scanning electron microscopy and atomic force microscopy after minimal preliminary treatment, appears composed of straight, parallel, uniform collagen fibrils regularly spaced by a three-dimensional, irregular network of thin, delicate proteoglycan filaments. Rat tail tendon, observed under identical conditions, appears instead made of heterogeneous, closely packed fibrils interwoven with orthogonal proteoglycan filaments. Pre-treatment with cupromeronic blue just thickens the filaments without affecting their spatial layout. Digestion with chondroitinase ABC rids the tendon matrix of all its interconnecting filaments while the corneal stroma architecture remains virtually unaffected, its fibrils always being separated by an evident interfibrillar spacing which is never observed in tendon. Our observations indicate that matrix proteoglycans are responsible for both the highly regular interfibrillar spacing which is distinctive of corneal stroma, and the strong interfibrillar binding observed in tendon. These opposite interaction patterns appear to be distinctive of different proteoglycan species. The molecular details of proteoglycan interactions are still incompletely understood and are the subject of ongoing research.     

Trypsin induces tumour necrosis factor-alpha secretion from a human leukemic mast cell line

Trypsin activating both proteinase-activated receptor (PAR) 2 and PAR4 plays an important role in inflammation. We have investigated the potential of trypsin to induce TNF-alpha secretion from the human leukemic mast cell line (HMC-1). HMC-1 cells co-express both PAR2 and PAR4, and their agonist trypsin signals to HMC-1 cells. Trypsin (100 nm), SLIGKV-NH(2) (100 microm, corresponding to the PAR2 tethered ligand), or GYPGQV-NH(2) (100 microm, corresponding to the PAR4 tethered ligand) induced tumour necrosis factor (TNF)-alpha secretion from HMC-1 cells. TNF-alpha secretion by trypsin was significantly blocked by pretreatment with 50 microm PD098059, MEK-1 inhibitor. Furthermore, trypsin stimulated the activation of extracellular signal-regulated kinase (ERK) in HMC-1 cells without any detectable activation of c-Jun N-terminal kinase (JNK) and p38 MAP kinase homologue. These results show that trypsin may induce TNF-alpha secretion following activation of ERK via both PAR2 and PAR4 on HMC-1 cells.     

A method for performing scanning electron microscopy of colonies growing in a human tumor cloning system

Scanning electron microscopy (SEM) is a valuable tool for examining cell surface morphology and cell-cell interactions. We used SEM to study 38 patients' tumors, representing 16 histological malignancies, growing in soft agar. Using our method, we obtained high quality micrographs without residual agar or preparation artifacts. We present our method for obtaining high quality SEM of tumor colonies growing in soft agar, which provides micrographs free of debris and necrotic host tissue.