Showing papers in "Journal of Cell Biology in 1958"
TL;DR: Certain hitherto unobserved details are revealed and some sort of specificity exists, although the factors involved are not yet understood.
Abstract: Heavy metals may be incorporated from solution into tissue sections for electron microscopy The resulting increase in density of the tissue provides greatly enhanced contrast with minimal distortion Relative densities of various structures are found to depend on the heavy metal ions present and on the conditions of staining Certain hitherto unobserved details are revealed and some sort of specificity exists, although the factors involved are not yet understood
4,040 citations
TL;DR: The nucleoids of Escherichia coli are shown to be preserved as a fine-stranded fibrillar nucleoplasm by an OsO4 fixation under defined conditions: acetate-veronal buffer pH 6, presence of Ca++ and amino acids, stabilization with uranyl-acetate before dehydration.
Abstract: The nucleoids of Escherichia coli, independently of the physiological state of the bacteria, are shown to be preserved as a fine-stranded fibrillar nucleoplasm by an OsO(4) fixation under defined conditions: acetate-veronal buffer pH 6, presence of Ca(++) and amino acids, stabilization with uranyl-acetate before dehydration. The same fixation procedure applied to the DNA of vegetative phage reveals a pool of homogeneous fibrillar structure very similar to the nucleoplasm. The "versene test," which produces a coarse coagulation of these plasms, emphasizes the similar behaviour of the pool and the nucleoids. The heads of mature phage are preserved in their true polyhedral shape by the standard fixation procedure, although they may be badly distorted when fixed under different conditions. Lanthanum nitrate and uranyl-acetate are shown to increase markedly the contrast of both phage and cytoplasm. The consequences of the fibrillar structure of the genetic material are discussed in relation to the probable division process.
1,181 citations
TL;DR: A mixture of araldite epoxy resins has been developed which is soluble in ethanol, and which yields a block of the required hardness for thin sectioning, which improves the preservation of the fine structure of a variety of specimens.
Abstract: Epoxy resins are suitable media for embedding for electron microscopy, as they set uniformly with virtually no shrinkage. A mixture of araldite epoxy resins has been developed which is soluble in ethanol, and which yields a block of the required hardness for thin sectioning. The critical modifications to the conventional mixtures are the choice of a plasticized resin in conjunction with an aliphatic anhydride as the hardener. The hardness of the final block can be varied by incorporating additional plasticizer, and the rate of setting can be controlled by the use of an amine accelerator. The properties of the araldite mixture can be varied quite widely by adjusting the proportions of the various constituents. The procedure for embedding biological specimens is similar to that employed with methacrylates, although longer soaking times are recommended to ensure the complete penetration of the more viscous epoxy resin. An improvement in the preservation of the fine structure of a variety of specimens has already been reported, and a typical electron microgram illustrates the present paper.
560 citations
TL;DR: Lead hydroxide stains rather selectively two types of particles in liver: those associated with the endoplasmic reticulum and containing ribonucleic acid and other somewhat larger particles.
Abstract: Descriptions of three heavy metal stains and methods of application to tissue sections for electron microscopy are presented. Lead hydroxide stains rather selectively two types of particles in liver: those associated with the endoplasmic reticulum and containing ribonucleic acid and other somewhat larger particles. Barium hydroxide emphasizes certain bodies within vesicles of the Golgi region of hepatic cells. Alkalized lead acetate is useful as a general stain, as are also lead and barium hydroxides.
520 citations
TL;DR: The present investigation concerning the histochemical demonstration of DPN diaphorase follows the development of a new reagent, Nitro-BT, which has already been used successfully for the cytochemical localization of the succinic dehydrogenase system.
Abstract: The present investigation concerning the histochemical demonstration of DPN diaphorase follows the development of a new reagent, Nitro-BT, which has already been used successfully for the cytochemical localization of the succinic dehydrogenase system. The most consistently favorable results were obtained with the lactate-lactic dehydrogenase system buffered at pH 7.4. Using sections of rat kidney and stomach, it was found that the intensity of stain was optimal after 15 minutes incubation at 37°C., conducted aerobically. By appropriate variations in the substrate mixture it was possible to selectively demonstrate the histochemical distribution of certain DPN-linked dehydrogenases in addition to DPN diaphorase. This was made possible by the special distribution of some of these dehydrogenases which distinguished them from one another. Of the dehydrogenases studied the distribution pattern of β-hydroxybutyric dehydrogenase was the most singular. In the gastric mucosa β-hydroxybutyric dehydrogenase was restricted to the cells of the mucous lining epithelium and the gland necks; and in the kidney the enzyme was limited to the cells of the proximal convoluted tubule and thick limbs of Henle's loop. In contrast, lactic dehydrogenase like DPN diaphorase was demonstrable in almost all cytologic elements of both the stomach and the kidney.
450 citations
TL;DR: In the present investigation an analysis has been made of the fine structure of the interrelationships of cells in human forearm epidermis by means of the electron microscope, and it is shown that dense oval thickenings appear in apposed areas of adjacent epidermal cell membranes.
Abstract: In the present investigation an analysis has been made of the fine structure of the interrelationships of cells in human forearm epidermis by means of the electron microscope. The "intercellular bridges," here called attachment zones, are more complex than has previously been recognized. It is shown that dense oval thickenings, called attachment plaques, appear in apposed areas of adjacent epidermal cell membranes. The tonofibrils terminate at the internal face of the attachment plaque and do not traverse the 300 A distance between apposed plaques. Seven intervening layers of unidentified substance occupy the space between attachment plaques. The attachment zones appear in all of the classical histological layers of the epidermis. The portions of epidermal cell membrane not involved in intercellular attachments have extensive surface area resulting from plication of the membrane, and its further modification to form microvilli. The possible functional significance of these observations is discussed. Prior observations concerning the basement membrane of epidermis are confirmed. Identification of epidermal melanocytes is achieved, the finer morphology of their dendritic processes is described, and their relationship to epidermal cells is discussed.
383 citations
TL;DR: Although diaphorase systems are implicated in the reduction of tetrazolium salts, specific dehydrogenases are solely responsible for the distinct distribution pattern obtained in tissues with various substrates.
Abstract: Methods are presented for the intramitochondrial localization of various diphosphopyridine nucleotide and triphosphopyridine nucleotide-linked dehydrogenases in tissue sections. The cytochemical reactions studied involve the oxidation of the substrates by a specific pyridino-protein. The electron transfer of tetrazolium salt is mediated by the diaphorase system associated with the dehydrogenase. The final electron acceptor was either p-nitrophenyl substituted ditetrazole (nitro-BT) or N-thiazol-2-yl monotetrazole (MTT), the latter giving rise to metal formazan in the presence of cobaltous ions. Mitochondrial localization of the formazan precipitate could be achieved by using hypertonic incubating media containing high concentrations of substrate and co-enzyme. A fast reduction of tetrazolium salt was obtained by chemically blocking the respiratory chain enzymes beyond the flavoproteins. Although diaphorase systems are implicated in the reduction of tetrazolium salts, specific dehydrogenases are solely responsible for the distinct distribution pattern obtained in tissues with various substrates. The present findings in tissue sections are discussed in conjunction with existing biochemical evidence from differential centrifugation experiments.
371 citations
TL;DR: In this paper, metal chelation of the formazan of an N-thiazol-2-yl tetrazolium salt is described for the localization of diphosphopyridine nucleotide diaphorase (DPND) and triphosphoprinidyl tetramerase (TPND) in mitochondria.
Abstract: Cytochemical methods involving metal chelation of the formazan of an N-thiazol-2-yl tetrazolium salt are described for the localization of diphosphopyridine nucleotide diaphorase (DPND) and triphosphopyridine nucleotide diaphorase (TPND) in mitochondria. These methods utilize the reduced coenzymes DPNH or TPNH as substrate. The reaction involves a direct transfer of electrons from reduced coenzyme to the respective diaphorase which in turn transfers the electrons to tetrazolium salt, reducing it to the insoluble formazan. Competition for electrons by preferential acceptors in the respiratory chain was prevented by various inhibitors. In the presence of respiratory inhibitors the rate of tetrazolium reduction was markedly increased. The greatest reduction was observed when amytal was used. Sites of diaphorase activity appeared as deposits of blue-black metal formazan chelate measuring 0.2 to 0.3 µ in diameter. Small mitochondria contained 2 deposits, while larger ones contained up to 6. Considerable differences were observed in the rate of tetrazolium reduction and cellular localization of diaphorase activity when DPNH was used as substrate as compared to TPNH. In each instance DPNH was oxidized more rapidly by tissues than TPNH. These findings support the concept that the oxidation of coenzymes I and II is mediated through separate diaphorases.
343 citations
TL;DR: The thicknesses of sections of n-butyl methacrylate polymer were determined by ellipsometry, and correlated with the color shown in reflected light, and agree well with optical theory and with previous published data for thin films.
Abstract: Knowledge of the thickness of sections is important for proper interpretation of electron micrographs Therefore, the thicknesses of sections of n-butyl methacrylate polymer were determined by ellipsometry, and correlated with the color shown in reflected light The results are: gray, thinner than 60 mµ; silver, 60 to 90 mµ; gold, 90 to 150 mµ; purple, 150 to 190 mµ; blue, 190 to 240 mµ; green, 240 to 280 mµ; and yellow, 280 to 320 mµ These results agree well with optical theory and with previous published data for thin films Sections, after cutting, are 30 to 40 per cent shorter than the face of the block from which they were cut Only a small improvement results from allowing the sections to remain in the collecting trough at room temperature Heating above room temperature, however, reduces this shortening, with a corresponding improvement in dimensions and spatial relationships in the sections When the thickness of the section is considered in interpreting electron micrographs instead of considering the section to be two-dimensional, a more accurate interpretation is possible The consideration of electron micrographs as arising from projections of many profiles from throughout the whole thickness of the section explains the apparent lack of continuity often observed in serial sections It is believed that serial sections are actually continuous, but that the change in size of structure through the thickness of one section and the consideration of only the largest profile shown in the micrograph can account for the lack of continuity previously observed
309 citations
TL;DR: In sections of KMnO4-fixed, developing mouse sciatic nerves, the central gap of mesaxons in myelinating fibers is normally closed with close apposition of the outside ∼20 A dense strata of the two ∼75 A Schwann cell membranes.
Abstract: In sections of KMnO4-fixed, developing mouse sciatic nerves, the central gap of mesaxons in myelinating fibers is normally closed with close apposition of the outside ∼20 A dense strata of the two ∼75 A Schwann cell membranes. The two combined outside strata make the intraperiod line bisecting each myelin lamella. The ∼150 A mesaxon is elaborated spirally around the axon in either a right hand or left hand spiral, and its inside (cytoplasmic) ∼20 A strata in apposition form the major dense lines of myelin. In hypotonic solutions the lamellae of adult frog sciatic myelinated fibers split apart along the outside membrane strata apposed at the intraperiod line throughout the spiral. Under similar conditions the inside (cytoplasmic) strata of the membranes, in apposition at the major dense lines, do not separate. The ∼150 A membranous structure resulting from this is called an "internal compound membrane." The double membranes of normal and control frog sciatic unmyelinated fibers have a central gap ∼100 to 150 A wide. After soaking in 4 to 10 times normal strength Ringer solution or 10 N sucrose-Ringer solution, this gap closes and a membranous structure ∼150 A wide resembling developing mouse mesaxons results. This is designated by the term "external compound membrane." The latter membranes resemble internal compound membranes, but their central dense zones, each consisting of two apposed outside membrane strata, are less dense.
272 citations
TL;DR: New-born rat and kitten cerebellum may be maintained for prolonged periods (over 5 months) in the Maximow assembly if explanted on to a coverslip previously coated with a thin gel of reconstituted rat tail collagen and fed a glucose-enriched "natural" medium.
Abstract: New-born rat and kitten cerebellum may be maintained for prolonged periods (over 5 months) in the Maximow assembly if explanted on to a coverslip previously coated with a thin gel of reconstituted rat tail collagen and fed a glucose-enriched "natural" medium. After a 2 week period of adjustment and early outgrowth, most cultures exhibit myelin formation. Axons located within the surrounding neuroglial sheet of the explant area myelinate. The sheaths are first evident as long, unsegmented, smooth, parallel, refractile lines. Simultaneously, neuronal nuclei tend to assume central positions and powdery granules of Nissl substance and lipoid materials begin to accumulate within the cytoplasm. During prolonged maintenance, axons may increase in width and the myelin may thicken. Some exhibit transient irregularities and swellings. Degeneration of some axons occurs manifested either by (a) progressive swellings and distortions of the myelin sheath and thinning of intervening portions of the axons which finally yield, leaving the swellings as myelin bodies, or by (b) small aneurysm-like distortions of myelin sheaths on thinning axons which become dull, irregular, and thread-like filaments beaded by the former herniations. The observations are compared with previous studies of in vitro and in vivo myelin formation with particular reference to neuronal-neuroglial relationships.
TL;DR: The general fine structure of the atrial musculature of the turtle heart is described, including the nature of the sarcolemma; the cross-banded structure ofThe myofibrils; the character of the sarcoplasm, and the form and disposition of its organelles.
Abstract: The general fine structure of the atrial musculature of the turtle heart is described, including; the nature of the sarcolemma; the cross-banded structure of the myofibrils; the character of the sarcoplasm, and the form and disposition of its organelles. An abundant granular component of the sarcoplasm in this species is tentatively identified as a particulate form of glycogen. The myocardium is composed of individual cells joined end to end at primitive intercalated discs, and side to side at sites of cohesion that resemble the desmosomes of epithelia. Transitional forms are found between desmosomes and intercalated discs. Both consist of a thickened area of the cell membrane with an accumulation of dense material in the subjacent cytoplasm. This dense amorphous component is often continuous with the Z substance of the myofibrils and may be of the same composition. The observations reported reemphasize the basic similarity between desmosomes and terminal bars of epithelia and intercalated discs of cardiac muscle. Numerous unmyelinated nerves are found beneath the endocardium. Some of these occupy recesses in the surface of Schwann cells; others are naked axons. No specialized nerve endings are found. Axons passing near the sarcolemma contain synaptic vesicles, and it is believed that this degree of proximity is sufficient to constitute a functioning myoneural junction.
TL;DR: The findings have reaffirmed the epithelioid character of brown adipose tissue, and provided additional information on the relation of its cells to each other and to the rich capillary bed, but observations on the mitochondria and their relation to fat droplets have led to different conclusions concerning the role of these organelles in lipogenesis.
Abstract: Interscapular fat from newborn rats and mice was fixed in buffered 1 per cent osmium tetroxide and thin sections of the methacrylate-embedded tissue were studied with the electron microscope. The findings have reaffirmed the epithelioid character of brown adipose tissue, and have provided additional information on the relation of its cells to each other and to the rich capillary bed. For the most part, the earlier description of the fine structure of brown adipose cells by Lever, has been confirmed, but our observations on the mitochondria and their relation to fat droplets have led us to different conclusions concerning the role of these organelles in lipogenesis. Mitochondria were often found to be very closely associated with lipide inclusions, but no actual communication between the two was observed and no evidence was found to support the hypothesis that mitochondria are transformed into lipide droplets. Large dense bodies which showed a highly ordered fine structure suggesting a crystalline protein were seen in the matrix of some mitochondria. The cytoplasm of the adipose cells contained fine granules that seemed to be of two kinds: particles of uniform size (∼150 A) and appreciable density that are believed to be ribonucleoprotein, and granules of lower density and more variable size that are tentatively interpreted as a form of glycogen. The Golgi complex of the adipose cells was small and the endoplasmic reticulum almost entirely absent. The significance of the poor development of these organelles is discussed in relation to current concepts of their function.
TL;DR: Although the diaphorase alone is responsible for the passage of electrons from TPNH to the tetrazole, it has been found that sites of activity of different TPN-linked dehydrogenases can be visualized in tissue sections, and characteristic loci for each enzyme may be observed.
Abstract: A histochemical method is described for the localization of triphosphopyridine nucleotide diaphorase using a recently synthesized tetrazolium salt (Nitro-BT). By virtue of the favorable histochemical properties of this reagent, it has been possible to demonstrate that whereas DPN diaphorase is usually restricted to the mitochondria, the TPN diaphorase activity of corresponding cells was distributed throughout the cytoplasm in granules too fine to be considered mitochondria. Furthermore, although the diaphorase alone is responsible for the passage of electrons from TPNH to the tetrazole, it has been found that sites of activity of different TPN-linked dehydrogenases can be visualized in tissue sections, and characteristic loci for each enzyme may be observed. For example, whereas TPN diaphorase and isocitric dehydrogenase have an extensive distribution in the kidney cortex, 6-phosphogluconic dehydrogenase is limited to the cells of the macula densa.
TL;DR: During investigations on the fixation of the bacterial nucleus and on the intracellular development of bacterial viruses, morphological observations are made which may contribute to the understanding of integument organization in the gram-negative bacterium Escherichia coli.
Abstract: The existence of two envelopes in bacteria has been postulated to account for numerous observations related to the presence of an osmotic barrier with differential permeability (1, 2), the occurrence of free, metabolizing protoplasts (3), and the possibility of producing plasmolysis (1, 4). All of these observations are easily explained by assuming both a cytoplasmic membrane as the sdective osmotic barrier, and a rigM cell wall conferring mechanical strength and defined form to the bacterial cell. In ultrathin sections of bacteria, systems of layered envelopes have actually been observed (5-10). These have been interpreted either as a double-structured cell wall, or as wall and cytoplasmic membrane. I t has not been possible to clarify further these differences in interpretation either by ultrathin sectioning of free protoplasts (11) or by the study of isolated cytoplasmic membranes, which, in contrast to the cell walls, are very difficult to isolate and purify (12). Since the existence of a visible cytoplasmic membrane was not evident, it could be postulated that the functionally defined cytoplasmic membrane was constituted of a so called pseudomembrane. Such a membrane might be formed at the interface of cytoplasm with surrounding medium, by the polarized arrangement of a common constituent of the cytoplasm, in a way comparable to that of detergents a t the liquid gas interface. During investigations on the fixation of the bacterial nucleus and on the intracellular development of bacterial viruses (13, 14), we had the opportunity to make some morphological observations which may contribute to the understanding of integument organization in the gram-negative bacterium Escherichia coli.
TL;DR: A new technique for the cultivation of living tissues in the multipurpose culture chamber that employs strips of cellophane as the agent for anchoring tissue explants to the coverslip walls of the chamber and disposes of the time-honored plasma-clot technique.
Abstract: A new technique for the cultivation of living tissues in the multipurpose culture chamber is described. This procedure employs strips of cellophane as the agent for anchoring tissue explants to the coverslip walls of the chamber and disposes of the time-honored plasma-clot technique. The primary advance embodied in this procedure lies in the fact that cells emigrating from so-cultured explants manifest themselves in a highly differentiated manner comparable to the cells of origin, whereas the outgrowth from the same types of tissue in plasma clots results in a more undifferentiated type of growth. Comparisons of outgrowths from embryonic thyroid, bone, and muscle (chicken) are photographically documented, and attention is called to certain cytochemical methods which further corroborate the differentiated quality obtained with the cellophane-strip technique.
TL;DR: The final supernatant had properties similar to those of the trypsin inhibitor described by Kunitz and Northrop, and had specific activities 2 to 4 times greater than the crude zymogen fractions.
Abstract: DL-leucine-1-C14 was administered by intracardiac injection to guinea pigs and its in vivo incorporation into the proteins of various pancreatic cell fractions followed over a period of 2 hours. The pancreas was homogenized in 0.88 M sucrose and fractionated by differential centrifugation to give nuclear, zymogen, mitochondrial, microsomal, postmicrosomal, and final supernatant fractions. The proteins of these fractions, obtained by precipitation with trichloroacetic acid followed by washing, were counted. The proteins of the microsomal fraction showed the highest early specific activity and were followed by those of the zymogen and mitochondrial fractions. The microsomal fraction was broken up into two subfractions: one consisting of detached RNP particles, the other representing mainly the microsomal content and membranes. The incorporation of labelled leucine into the proteins of microsomal subtractions and in those of postmicrosomal fractions was studied comparatively in the pancreas of fasted and fed guinea pigs as well as in the liver and pancreas of fasted animals. A tentative cytological picture of protein synthesis and transport based on these findings is presented.
TL;DR: The sites of reaction product resulting from ATPase and 5-nucleotidase activities remaining in parenchymatous cells of osmium-fixed rat liver were studied by electron microscopy of thin sections to indicate that both ATP enzyme activities are localized in the plasma membrane where it folds to form the microvilli of the bile canaliculus.
Abstract: The sites of reaction product resulting from ATPase and 5-nucleotidase activities remaining in parenchymatous cells of osmium-fixed rat liver were studied by electron microscopy of thin sections. These indicate that both ATPase and 5-nucleotidase activities are localized in the plasma membrane where it folds to form the microvilli of the bile canaliculus, and that 5-nucleotidase activity is also present in the microvilli at the sinusoidal aspects of the cells. It is suggested that these enzymes, particularly ATPase, may play a role in molecular transport or in some kind of membrane activity at the cell surface. Of special interest is the apparent differential localization of these enzymes at the absorptive and secretory regions of the plasma membrane of the cell. It may be of interest to study changes in these enzyme localizations in pathologic states, as a sign of changed cell function. Some of the difficulties in the interpretation of enzyme reaction products seen in electron micrographs are discussed.
TL;DR: Evidence was obtained that the chemical agent fixes tissue at low temperatures: new chemical cross-linkages, which contain the fixing agent, join tissue constituents together.
Abstract: Freeze-substitution is based on rapid freezing of tissues followed by solution ("substitution") of ice at temperatures well below O degrees C. A 1 to 3 mm. specimen was thrown into 3:1 propane-isopentane cooled by liquid nitrogen to -175 degrees C. (with precautions). The frozen tissue was placed in substituting fluid at -70 degrees C. for 1 week to dissolve ice slowly without distorting tissue structure. Excess substituting agent was washed out, and the specimen was embedded, sectioned, and stained conventionally. For best morphological and histochemical preservation, substituting fluids should in general contain both chemical fixing agent and solvent for ice, e.g., 1 per cent solutions of osmium tetroxide in acetone, mercuric chloride in ethanol, and picric acid in ethanol. Preservation of structure was poorer after substitution in solvent alone. Evidence was obtained that the chemical agent fixes tissue at low temperatures. The chemical mechanisms of fixation are probably similar to those operating at room temperature: new chemical cross-linkages, which contain the fixing agent, join tissue constituents together. This process is distinguished from denaturation by pure solvents. Freeze-substitution has many advantages, particularly the preservation of structure to the limit of resolution with the light microscope, and the accurate localization of many soluble and labile substances.
TL;DR: Five ribonucleoprotein fractions isolated from the postmitochondrial supernatant of the pancreas of the guinea pig showed different RNA/protein ratios and also different RNA turnover patterns, but little discernible differences could be detected in the nucleotide composition of the RNA moieties of these RNP fractions.
Abstract: Microsomes were isolated from the pancreas of starved and fed guinea pigs. In the first case, the gland was removed from animals starved for 48 hours; in the second, the pancreas was excised 1 hour after the beginning of a meal that ended a fast of 48 hours. These are referred to below as fed animals. In both cases the tissue was homogenized in 0.88 M sucrose and the microsomes obtained by centrifuging the mitochondrial supernatant at 105,000 g for 60 minutes. In starved animals the content of the endoplasmic reticulum of the exocrine cells and the content of the microsomes were found to be of low or moderate density. In fed guinea pigs the cavities of the reticulum frequently contained dense intracisternal granules and the microsomes were distinguished by a content of high density sometimes in the form of recognizable intracisternal granules. In starved animals, the microsomes were found to account for 5 to 20 per cent of the trypsin-activatable proteolytic activity and ribonuclease activity of the whole cell, whereas in fed animals they contained uniformly almost 30 per cent of these activities. In fed animals the dense, cohesive content of the microsomes (intracisternal granules) could be isolated by breaking up the microsomes with dilute (0.1 per cent) deoxycholate solutions and separating microsomal subfractions by differential centrifugation. The specific enzymatic activities of a heavy microsomal subfraction rich in intracisternal granules were almost equal to those of isolated purified zymogen granules. The ribonucleoprotein particles attached to the microsomal membranes could be isolated by the same technique and found also to exhibit some of the same enzymatic activities. Corresponding subfractions isolated from the microsomes of starved animals were considerably less active. The relevance of these findings for the synthesis and intracellular transport of protein in the exocrine cell of the pancreas is discussed.
TL;DR: The fine structure of the rod-bipolar synapse is described and illustrated and it is shown that minute cytoplasmic vesicles found in the cy toplasm of both the rod spherule and the bipolar terminal are most abundant near the rod synaptic lamella.
Abstract: The fine structure of the rod-bipolar synapse is described and illustrated. Each rod spherule possesses a large, single, oval or elongate mitochondrion approximately 0.5 x 2.0 microns. Surrounding the mitochondrion are elements of agranular endoplasmic reticulum. The bipolar dendrite projects into the lower pole of the spherule and usually terminates in two lobes separated by a cleft. The plasma membranes appear dense and thicker in the region of the synapse. In the rod spherule cytoplasm, contiguous with the plasma membrane is a dense, slightly concave arciform structure, the rod arciform density, extending from the base of the bipolar bifid process through the cleft to an equivalent point on the opposite side. Also within the spherule, and external (towards the sclera) to the rod arciform density, is a parallel, dense, thin lamella, the rod synaptic lamella. This is approximately 25 mµ in thickness and 400 mµ in width at its widest extent. This halfmoon-shaped plate straddles the cleft between the two lobes of the bipolar process. The lamella appears to consist of short regular rodlets or cylinders 5 to 7 mµ in diameter, oriented with their long axes perpendicular to the plane of the lamella. Minute cytoplasmic vesicles found in the cytoplasm of both the rod spherule and the bipolar terminal are most abundant near the rod synaptic lamella.
TL;DR: It is shown that the destruction of color is a characteristic of metachromasy and that water molecules intercalated between approximated dye ions are responsible for the loss and change of color.
Abstract: Non-chromotropic substances such as fibrin and gelatin and most tissue and cellular structures stain orthochromatically with internal dye concentrations of such metachromatic dyes as methylene blue and toluidine blue which, if in solution, would be metachromatic. Therefore, at ordinary levels of staining these substances depress the natural tendency of these dyes to change color. However, at elevated levels of dye-binding metachromasy eventually occurs. This phenomenon is explained on the basis of the distribution of dye-binding sites. In these substrates, by contrast with chromotropic substances, many binding sites are too far removed for dye interaction, consequently the interaction frequency can become high enough to produce a color change only as saturation of the available sites is approached. It is also shown that the destruction of color is a characteristic of metachromasy and that water molecules intercalated between approximated dye ions are responsible for the loss and change of color. A concept of metachromasy is proposed in which the interaction between water molecules and suitably approximated dye ions plays an essential role. The experimental studies are described against a background of the history and evolution of ideas on metachromasy. The literature is reviewed and reassessed particularly from the physicochemical viewpoint.
TL;DR: Axoplasmic changes were studied in the saphenous nerve of the albino rat during the early stages of Wallerian degeneration and the neurofilaments underwent complete disintegration and the axoplasm became filled with finely granular material which later formed irregular clumps surrounded by a structureless matrix.
Abstract: Axoplasmic changes were studied in the saphenous nerve of the albino rat during the early stages of Wallerian degeneration. The axons were examined at 0, 24, and 48 hours after the surgical transection of the nerve. The material was fixed in 2 per cent OsO(4) in phosphate buffer (pH 7.2-7.5) with sucrose (added to a final osmolar concentration of approximately 0.37 M). The earliest changes were seen in the endoplasmic reticulum which became fragmented into rows of small vesicles. Then, between 24 and 48 hours, the neurofilaments underwent complete disintegration and the axoplasm became filled with finely granular material which later formed irregular clumps surrounded by a structureless matrix, probably fluid in vivo. The fragmentation of the neurofilaments was accompanied by pronounced swelling of the mitochondria.
TL;DR: The incorporation of labelled leucine into the proteins of microsomal subtractions and in those of postmicrosomal fractions was studied comparatively in the pancreas of fasted and fed guinea pigs as well as in the liver and pancrea of fasting animals.
Abstract: Pancreatic tissue, (guinea pig) homogenized in 0.88 M sucrose, was fractionated by differential centrifugation into a nuclear, zymogen, mitochondrial, microsomal, and final supernatant fraction. The components of the particulate fractions were identified with well known intracellular structures by electron microscopy. The fractions were analyzed for protein-N and RNA, and were assayed for RNase and trypsin-activatable proteolytic (TAPase) activity. The zymogen fraction accounted for 30 to 40 per cent of the total TAPase and RNase activities, and its specific enzymatic activities were 4 to 10 times higher than those of any other cell fraction. The zymogen fraction was cytologically heterogeneous; zymogen granules and mitochondria represented its main components. More homogeneous zymogen fractions, obtained by successive washing or by separation in a discontinuous density-gradient, had specific activities 2 to 4 times greater than the crude zymogen fractions. Chymotrypsinogen was isolated by column chromatography from pancreas homogenates and derived cell fractions. The largest amount was recovered in the zymogen fraction. The final supernatant had properties similar to those of the trypsin inhibitor described by Kunitz and Northrop.
TL;DR: A set of tables which combined four operations into one has been designed and is reproduced within, based on Patau's formulae, for two-wavelength measurements of chromophore.
Abstract: The calculations required for two-wavelength measurements are time consuming and laborious In order to circumvent this limitation of the method, a set of tables which combined four operations into one has been designed and is reproduced within The tables are based on Patau's formulae The two transmission readings obtained according to the photometric method provide the coordinates which lead directly to a value for the relative absorbance The product of this absorbance and the area of the photometric field gives the relative amount of chromophore in the field The range of transmission values covered in the table corresponds to the effective range of the two-wavelength method
TL;DR: After a period of rapid cell division, the morphology of the blastema cell changes and the differentiating cartilage cell develops an extensive, highly organized endoplasmic reticulum and the Golgi apparatus also appears to become more highly differentiated and more extensive at this time.
Abstract: Regenerating forelimbs of larval salamanders, Amblystoma punctatum, were fixed in OsO4 at various intervals after amputation and were sectioned for study with the electron microscope. The dedifferentiated cells comprising the early blastema were found to have a fine structure similar to that of other undifferentiated cells and to have lost all of the identifying morphological features of their tissues of origin. The cytoplasm of such cells is characterized by numerous free ribonucleoprotein granules and a discontinuous vesicular endoplasmic reticulum. The cells have more abundant cytoplasm and are in closer contact with each other than was previously realized. The layer of condensed ground substance investing most differentiated cell types is lacking. After a period of rapid cell division, the morphology of the blastema cell changes. Cytoplasm is now sparse and contains a high concentration of free ribonucleoprotein granules, but little endoplasmic reticulum. The differentiating cartilage cell, however, develops an extensive, highly organized endoplasmic reticulum and the Golgi apparatus also appears to become more highly differentiated and more extensive at this time. Small vesicles appear throughout the cytoplasm at the time the new cisternae originate and may contribute to their formation. These and other changes in the cytoplasmic organelles are discussed.
TL;DR: The observation that the cisternae are visible in certain cell types under phase contrast optics opens the way for experimental investigations on the behavior of this class of cytoplasmic membranes in living cells.
Abstract: In freshly isolated cells of the guinea pig germinal epithelium examined with phase contrast, dark contours are seen in the cytoplasm that appear to be optical sections of the cisternae of the endoplasmic reticulum. These increase in contrast, in number, and in linear extent with increasing time up to 4 hours after isolation of the cells from the testis. During this period, cisternae originally present in the cells are extended and new ones appear to be formed by coalescence of tubular and vesicular elements of the reticulum. The cisternae become associated in parallel array and ultimately form elaborate concentric systems resembling structures that have often been interpreted as intracellular "myelin figures." Until now our knowledge of the endoplasmic reticulum has been based largely upon electron micrographs. The observation that the cisternae are visible in certain cell types under phase contrast optics opens the way for experimental investigations on the behavior of this class of cytoplasmic membranes in living cells.
TL;DR: The abdominal nerve cord of Periplaneta americana was studied utilizing light and electron microscopes and delicate granules, similar to those probably responsible for cytoplasmic basophilia, are evenly distributed in "dark" cells and clumped in "light" cells.
Abstract: The abdominal nerve cord of Periplaneta americana was studied utilizing light and electron microscopes. In the nerve cells, delicate granules, similar to those probably responsible for cytoplasmic basophilia, are evenly distributed in "dark" cells and clumped in "light" cells. Neuroglial cells are stained metachromatically by cresyl violet. The neuroglial cells have many processes which ramify extensively and are enmeshed to form overlapping layers. These imbricated processes ensheath the nerve cells; the inner layer of the sheath penetrates into the neuron and is responsible for the appearance of the trophospongium of Holmgren. Nerve fibers are embedded within glial cells and surrounded by extensions of the plasma membrane similar to mesaxons. Depending on their size, two or several nerve fibers may share a single glial cell. Nerve fibers near their terminations on other nerve fibers contain particles and numerous, large mitochondria. The ganglion is ensheathed by a thick feltwork of connective tissue and perilemmal cells. The abdominal connective has a thinner connective tissue sheath which is without perilemmal cells. The nerve fibers and sheaths in the connective become thinner as they pass through ganglia.
TL;DR: Two structural features extending throughout the length of the animal have been identified per cortical stripe in the body wall of Stentor; two structural features lying just beneath the pellicle are identical with the birefringent fibres observed in the living animal.
Abstract: The fine structure of the ciliate Stentor has been studied by means of the electron microscope and the results have been correlated with observations made on the living organism by means of light microscopy; special reference has been made to structural features which may be responsible for contraction and extension in Stentor. Descriptions have been given of the structure of the macronucleus, the vacuolated cytoplasm, mitochondria and the pellicle; a detailed study has also been made of the adoral membranelles. About 250 membranelles encircle the peristomal cap and each is composed of 3 rows of cilia, with 20 to 25 cilia in each row; a fibrillar root system connected with the membranelles depends into the endoplasm for about 20 µ and each is essentially in the shape of a fan, the terminal ends of each root bifurcating to connect to neighbouring roots. The membranelles thus form a cohesive unit and this morphological arrangement may have a bearing on the motion and coordination of the whole system. Two structural features extending throughout the length of the animal have been identified per cortical stripe in the body wall of Stentor; first, km fibres lying just beneath the pellicle are composed of stacks of fibrillar sheets and are identical with the birefringent fibres observed in the living animal. The individual fibrils of the sheets are in turn connected to the kinetosomes of the body cilia; thus the km fibres are homologous to kinetodesmata. Secondly, M bands lie beneath the km fibres and form an interconnected system in contact with the surrounding vacuolated cytoplasm; the thickness of the M bands is greatest at the base of a contracted animal. The contractile and extensile properties of these organelles have been discussed in the light of experimental results and theoretical considerations.
TL;DR: Histochemical procedures for succinic dehydrogenase, lipides, adenosinetriphosphatase, esterase, and glycogen were utilized to differentiate muscle fibers, and these methods facilitated the study of the distribution of fiber types within whole muscle.
Abstract: The distribution and characterization of the fibers of normal and denervated red and white muscles of the albino rat are reported in this study. Histochemical procedures for succinic dehydrogenase, lipides, adenosinetriphosphatase, esterase, and glycogen were utilized to differentiate muscle fibers, and these methods facilitated the study of the distribution of fiber types within whole muscle. Muscle fibers of the granular type (dark or red fibers) can be clearly distinguished from those with clearer sarcoplasm (light or white fibers) by methods for demonstrating succinic dehydrogenase, lipides, and esterase. The method for adenosine-triphosphatase reveals differences only under the special conditions described in the text. Additional fiber types are described in the cat9s diaphragm and in the extrinsic ocular muscles of the rat. Succinic dehydrogenase and adenosinetriphosphatase activities of the soleus and biceps femoris were studied 14 days after denervation of these muscles. The histochemical findings are discussed principally in the light of current biochemical knowledge of these enzymes.