The structure of cytoplasm in directly frozen cultured cells. I. Filamentous meshworks and the cytoplasmic ground substance

Cultured fibroblasts or epithelial cells derived from Xenopus laevis embryos were directly frozen, freeze-substituted by an improved method, and then either critical-point-dried and viewed as whole mounts, or embedded and thin sectioned. In thin regions of these cells, where ice crystal artifacts are absent, the cytoplasm consisted of a dense, highly interconnected meshwork of filaments, embedded in a finely granular ground substance. The meshwork in directly frozen, intact cells was compared with that in cells that were lysed (physically, with detergents, or with filipin), or fixed with glutaraldehyde before freezing. Although filaments tended to be less numerous in lysed cells, their overall organization was the same as that in intact cells. However, fixation with glutaraldehyde before freezing distorted the meshwork to variable degrees depending on the osmolarity of the fixation buffer, and also obscured the granular ground substance which is obvious in directly frozen cells. With optimal preparative methods, the cytoplasm of these directly frozen cells is shown to consist of a cytoskeleton composed of discrete interwoven filaments interconnected by numerous finer filaments and a readily extractable granular matrix which presumably represents aggregations of cytoplasmic proteins.     

Effects of cytoplasmic acidification on clathrin lattice morphology

Reducing the internal pH of cultured cells by several different protocols that block endocytosis is found to alter the structure of clathrin lattices on the inside of the plasma membrane. Lattices curve inward until they become almost spherical yet remain stubbornly attached to the membrane. Also, the lattices bloom empty "microcages" of clathrin around their edges. Correspondingly, broken-open cells bathed in acidified media demonstrate similar changes in clathrin lattices. Acidification accentuates the normal tendency of lattices to round up in vitro and also stimulates them to nucleate microcage formation from pure solutions of clathrin. On the other hand, several conditions that also inhibit endocytosis have been found to create, instead of unusually curved clathrin lattices with extraneous microcages, a preponderance of unusually flat lattices. These treatments include pH-"clamping" cells at neutrality with nigericin, swelling cells with hypotonic media, and sticking cells to the surface of a culture dish with soluble polylysine. Again, the unusually flat lattices in such cells display a tendency to round up and to nucleate clathrin microcage formation during subsequent in vitro acidification. This indicates that regardless of the initial curvature of clathrin lattices, they all display an ability to grow and increase their curvature in vitro, and this is enhanced by lowering ambient pH. Possibly, clathrin lattice growth and curvature in vivo may also be stimulated by a local drop in pH around clusters of membrane receptors.     

Histochemistry of glycosaminoglycans in cartilage ground substance

Summary The critical-electrolyte-concentration staining method using Alcian blue (AB) was applied to etched semithin Epon-embedded sections. The distribution of various glycosaminoglycans (GAGs) was studied in hyaline, elastic, cellular and fibrous cartilage obtained from humans and rodents. The staining patterns in semithin sections were found to correspond to those obtained using paraffin-embedded material. Lectin histochemistry was performed on consecutive sections. The following peroxidase-labelled lectins were used: Ricinus communis A I, Arachis hypogaea, Ulex curopaeus A I, Triticum vulgaris, Helix pomatia, Limax flavus, and concanavalin A. The lectin-binding capacity of cartilaginous ground substance was found to be low, as was expected on account of the few free sugar residues of GAGs. Chondroitin sulphate, the most widely distributed GAG, did not exhibit lectin staining. The lectin-binding site (positive staining for all lectins tested except H. pomatia) observed corresponded to areas positive forkeratan sulphate, as shown by AB staining in preceding or following sections. The pronounced lectin binding seen in cellular structures and the inner territorial matrix regions is considered to be due to higher concentrations of oligosaccharides involved in the metabolism of GAGs.Supported by the Hochschuljubiläumsstiftung der Stadt Wien     

Transformations in the structure of the cytoplasmic ground substance in erythrophores during pigment aggregation and dispersion. I. A study using whole-cell preparations in stereo high voltage electron microscopy

Pigment migration in cultured erythrophores of the squirrel fish Holocentrus ascensionis, after manipulation with K+, epinephrine, 3',5'- dibutyryl cyclic adenosine monophosphate, theophylline, and caffeine, is essentially identical to that observed in this chromatophore in situ. For such observations, the erythrophores are dissociated from the scales with hyaluronidase and collagenase, and allowed to spread on an amorphous collagen substrate, where they resemble the discoid erythrophore in situ. In this state, they are readily fixed by glutaraldehyde and osmium tetroxide, and are then critical-point dried for whole-cell viewing in the high voltage electron microscope. The organization and fine structure of the erythrophore cytoplast was stereoscopically examined after fixation of the pigment granules in four experimental states: pigment dispersed, pigment aggregated, pigment aggregating, and pigment dispersing. In the dispersed cell, granules are contained in an extensive three-dimensional lattice composed of radially oriented microtubules and a network of fine filaments 3-6 nm in diameter (microtrabeculae), whereas in the aggregated cell, the microtrabecular system is absent, and the majority of the microtubules appear displaced into the cortices on the cytoplasmic surface of the plasma membrane. In cells fixed while aggregating, few microtrabeculae are observed, although formless thickenings are observed in the cortices, on granules, and between clumped granules. In dispersing cells, the microtrabecular system is reformed from material stored in the cortices and with the granules in the centrosphere. These observations suggest that the granules are suspended in a dynamic microtrabecular system that withdraws during pigment aggregation and is restructured during pigment dispersion. The microtubules guide linear granule motion not by defining physical channels, but by a recognizable affinity of microtubules, microtrabeculae, and granules for one another.     

Blood serum proteins and the mineralization of bone ground substance

Summary Direct tracing experiments with fluorochrome-labeled homologous blood serum show that certain serum components are taken up by the bone substance of young and adult rats. These proteins are concentrated and incorporated into the organic matrix while it is being formed. They retain their fluorescent label for long periods of time and withstand histological fixation and decalcification. In experimental rickets, no labeled serum protein is seen to be incorporated into the uncalcified rachitic osteoid. Its uptake begins, however, concomitantly with the onset of mineralization during the healing period. The results of experiments are interpreted in terms of a calcium-carrying serum protein being complexed and precipitated by osteoblast products to form an essential component of apposition zones which, possibly, initiates nucleation and the subsequent steps of calcification. Fractionation experiments to define the serum component(s) involved in this process, will be continued. So far, they have resulted in a fraction containing albumin, ₁-macroglobulin, transferrin, and haptoglobin.

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Zusammenfassung Gewisse Anteile von Fluorochrom-markiertem homologem Blutserum lassen sich nach parenteraler Verabreichung in der Knochensubstanz junger und erwachsener Ratten direkt nachweisen. Diese Serumproteine werden in hoher Konzentration während der Knochenbildung in die organische Matrix eingebaut. Sie behalten ihre Markierungsfluoreszenz für lange Zeit und widerstehen der Fixierung und der Entkalkung. Während experimenteller Rachitis ist eine Aufnahme dieser markierten Serumproteine in das unverkalkte rachitische Osteoid nicht festzustellen. Ihre Inkorporation beginnt jedoch wieder gleichzeitig mit dem Einsetzen der Verkalkung, wenn die Rachitis zur Ausheilung gebracht wird. Die experimentellen Resultate deuten darauf hin, daß ein calciumtragendes Serumprotein mit gewissen Produkten der Osteoblasten Komplexe bildet, dabei präzipitiert und zu einem Bestandteil der Appositionssäume wird, der möglicher Weise die Verkalkung der organischen Knochengrundsubstanz in Gang setzt. Fraktionierungsexperimente zur Bestimmung der an diesem Vorgang beteiligten Serumkomponente(n) sind noch nicht abgeschlossen. Sie halten derzeit bei einer Fraktion, die Albumin, -1-Makroglobuline, Transferrin und Haptoglobin enthält, und werden weiter fortgesetzt.

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Supported by United States Public Health Service Grant No. AM06705.     

Pro- versus anti-inflammatory cytokines: myth or reality.

Inflammation is characterized by an interplay between pro- and anti-inflammatory cytokines. Cytokines are commonly classified in one or the other category: interleukin-1 (IL-1), tumor necrosis factor (TNF), gamma-interferon (IFN-gamma), IL-12, IL-18 and granulocyte-macrophage colony stimulating factor are well characterized as pro-inflammatory cytokines whereas IL4, IL-10, IL-13, IFN-alpha and transforming growth factor-beta are recognized as anti-inflammatory cytokines. In this review, we point out that this classification is far too simplistic and we provide numerous examples illustrating that a given cytokine may behave as a pro- as well as an anti-inflammatory cytokine. Indeed, the cytokine amount, the nature of the target cell, the nature of the activating signal, the nature of produced cytokines, the timing, the sequence of cytokine action and even the experimental model are parameters which greatly influence cytokine properties.     

Structure of the interstitial space and ground substance of the human articular cartilage

By means of transmissive and scanning electron microscopy (investigation of ultra-replicas) three-dimensional organization of the interstitial (interfibrillar) space of the articular cartilage has been demonstrated; it repeats, to some extent, construction of the fibrous base. By means of mercury porometry quantitative characteristics of various parameters of the interfibrillar space are obtained. Their specific volume is 0.96 cm3/g of the dehydrated cartilage, space with equivalent diameters from 300 up to 5 nm makes 94%. By means of the gas adsorption method it has been stated that the specific internal surface is 23.8 m2 per 1 g of the dehydrated articular cartilage. Transmissive and scanning electron histochemistry has revealed several various forms of structured proteoglycans, demonstrated their spatial organization and interconnection with collagenous fibrils. The methodical complex applied can be used for investigating the connective tissue interstitial spaces in other parts of the human locomotor apparatus.     

Structuro-functional organization of the fibrillar component and ground substance of human rib cartilage

A complex morphological investigation (histology, histochemistry, scanning and transmissive electron microscopy, electron histochemistry) has been performed to study the intercellular substance of the costal hyalinous cartilage. It has been demonstrated that the fibrillar framework of the costal cartilage consists of branching collagenous fibrillae, chaotically scattering. The fibrillae are surrounded with the ground substance; one of its components is the reticular ruthenium-positive structure.