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PubMed1977

Embryologic development of a mouse sympathetic ganglion in vivo and in vitro.

The morphologic and biochemical development of the embryonic mouse superior cervical ganglion was characterized in vivo and in tissue culture. From 13 days of gestation, when the superior cervical ganglion was first visible, to birth at 19 days, tyrosine hydroxylase [tyrosine 3-monooxygenase; L-tyrosine, tetrahydropteridine:oxygen oxidoreductase (3-hydroxylating); EC 1.14.16.2] activity increased 100-fold in vivo. Explants of ganglia from 14-day embryos exhibited abundant neurite outgrowth in basal medium without added nerve growth factor (NGF), and increases in tyrosine hydroxylase activity paralleled that observed in vivo. Ganglia from 14-day embryos elaborated neurites and exhibited 3-fold increases in enzyme activity in vitro in the presence of antiserum to NGF (anti-NGF) or NGF + anti-NGF. In direct contrast, ganglia from 18-day fetuses failed to grow without added NGF or in medium containing anti-NGF or NGF + anti-NGF: virtually no axon outgrowth occurred and tyrosine hydroxylase activity decreased by half. These observations suggest that developmental regulatory mechanisms change radically during embryologic and fetal life of mammalian superior cervical ganglion.

Animals↗
PubMed1979

Dark field imaging of biological macromolecules with the scanning transmission electron microscope.

A scanning transmission electron microscope (STEM) equipped with a field emission gun has been employed for the examination of biological macromolecules at high resolution. The quality of micrographs obtained with the STEM is dependent upon the quality of the substrate used to support biological objects because the image contrast in dark field is proportional to the mass density of the specimen. In order to reduce deleterious effects of the substrates on the image quality, we have developed a method of fabricating substrates consisting of very thin, very clean carbon films supported on very clean fenestrated plastic films. These films are approximately 15 A thick. Well-known biological macromolecules such as glutamine synthetase and tobacco mosaic virus (both stained) and low-density lipoprotein and ferritin (both unstained were placed on these substrates and examined with the STEM by using various modes of contrast. The micrographs obtained by using the dark field mode of contrast employing an annular detector were free from phase contrast, as expected. Using this contrast mode, we have been able to directly observe (in-focus) 2.5- to 4.4-A lattice spacings in the ferritin core. The effect of electron radiation damage on the helical structure of tobacco mosaic virus was also examined. Micrographs as well as corresponding optical diffraction patterns obtained with moderately low doses showed very clear helical structure from both sides of the virus. In addition, the (11.5 A)(-1) layer lines indicated the effective resolution attained on these particles.

Ferritins↗
PubMed1975

In vitro polymerization of microtubules into asters and spindles in homogenates of surf clam eggs.

The eggs of the surf clam Spisula solidissima were artificially activated, homogenized at various times in cold 0.5 M MES buffer, 1mM EGTA at pH 6.5, and microtubule polymerization was induced by raising the temperature to 28 degrees C. In homogenates of unactivated eggs few microtubules form and no asters are observed. By 2.5 min after activation microtubules polymerize in association with a dense central cylinder, resulting in the formation of small asterlike structures. By 4.5 min after activation the asters formed in vitro contain a distinct centriole, and microtubules now radiate from a larger volume of granular material which surrounds the centriole. By 15 min (metaphase I) the granular material is more disperse and only loosely associated with the centriole. Microtubules are occasionally observed which appear to radiate directly from one end of the centriole. The organizing center can be partially isolated by centrifugation of homogenates of metaphase eggs and will induce aster formation if mixed with tubulin from either activated or unactivated eggs. Pretreatment of the eggs with colchicine does not prevent the formation of a functional organizing center. Complete spindles can also be obtained under polymerizing conditions by either homogenizing the eggs directly into warm buffer or by adding a warm high-speed supernate to spindles which have been isolated in a microtubule stabilizing medium. Extensive addition of new tubulin occurs onto the isolated spindles, resulting primarily in growth of astral fibers, although there occasionally appears to be growth of chromosomal fibers and of pole-to-pole fibers. Negatively stained aster microtubules have a strong tendency to associate side by side, and under some conditions distinct cross bridges can be observed. However, under other conditions large numbers of 300-400-A particles surround the microtubules; the presence of stain between particles can give the appearance of cross bridges.

Animals↗
PubMed1979

Developmental and ultrastructural observations on two stalked marine choanoflagellates, Acanthoecopsis spiculifera Norris and Acanthoeca spectabilis Ellis.

Acanthoecopsis spiculifera and Acanthoeca spectabilis are stalked, loricate choanoflagellates found in littoral sea water pools. The two taxa are distinguished from each other by the arrangement of costae forming the lorica chamber. In addition, Acanthoecopsis spiculifera usually has a longer stalk and may be colonial, consisting of two or more attached individuals. Division in Acanthoeca results in the production of a juvenile, flagellated, protoplast without a lorica. After separation, the juvenile protoplast swims away, settles down and produces an accumulation of costal strips. When sufficient strips have been produced the lorica is rapidly assembled.

Animals↗
PubMed1979

The lamina splendens of articular cartilage is an artefact of phase contrast microscopy.

The so-called lamina splendens of articular cartilage is shown to be a characteristic of phase contrast microscopy; this technique provides no evidence for an anatomically distinct surface layer. Fresnel diffraction occurs at edges separating regions of different refractive indices. These diffraction effects, when viewed under phase contrast, lead to the appearance of a bright line along the edge.

Adolescent↗
PubMed1977

Chromosome distribution: experiments on cell hybrids and in vitro.

Ostergren (1951) provided a simple explanation for both chromosome distribution in mitosis and chromosome segregation in meiosis, and more recently a molecular extension of his hypothesis has been possible. This report focuses on experimental tests of these ideas. Micromanipulation experiments on cell hybrids containing both meiotic and mitotic spindles demonstrate that differences in meiotic and mitotic chromosome behavior are determined by something intrinsic to the chromosome: meiotic chromosomes transferred to a mitotic spindle (or vice versa) behave just as they normally would. The molecular explanation postulates polarized growth or binding of microtubules at kinetochores. This has just been tested in vitro by McGill & Brinkley (1975) and by Telzer, Moses & Rosenbaum (1975), and their results are reviewed. In addition, a novel method for in vitro studies is described - mechanical demembranation of cells which is compatible with quite normal chromosome movement in anaphase. After addition of microtubule subunits to a demembranated prophase cell, chromosome orientation and movement toward an aster was observed for the first time in vitro. It is concluded that important aspects of chromosome distribution are probably understood at both the cellular and molecular levels, but final tests are still required. The outlook is hopeful indeed because the gaps in our knowledge are well known - the necessity of observations on prophase is a recurrent theme - and the means of filling the gaps are in hand.

Animals↗
PubMed1976

The transport of S-adenosyl-L-methionine in isolated yeast vacuoles and spheroplasts.

1. The properties of S-adenosyl-L-methionine accumulating system for both vacuoles and spheroplasts are described. Yeast vacuoles were obtained by a modified metabolic lysis procedure from spheroplasts of Saccharomyces cerevisiae. 2. Isolated vacuoles accumulate S-adenosyl-L-methionine by means of a highly specific transport system as indicated by competition experiments with structural analogs of S-adenosyl-L-methionine. The S-adenosyl-L-methionine transport system shows saturation kinetics with an apparent Km of 68 muM in vacuoles and 11 muM in spheroplasts. 3. S-Adenosyl-L-methionine accumulation into vacuoles does not require glucose, phosphoenolpyruvic acid, ATP, ADP nor any other tri- or di-phosphorylated nucleotides. It is insensitive to azide and 2,4-dinitrophenol which strongly inhibit the glucose-dependent accumulation of S-adenosyl-L-methionine in spheroplasts. 4. The transport of S-adenosyl-L-methionine into vacuoles is optimal at pH 7.4 and is insensitive to nystatin while the uptake of S-adenosyl-L-methionine into spheroplasts is optimal at pH 5.0 and is strongly sensitive to nystatin. On this basis it has thus been possible to measure both the intracytoplasmic and the intravacuolar pool of S-adenosyl-L-methionine. 5. Our results indicate the existence of a highly specific S-adenosyl-L-methionine transport system in the vacuolar membrane which is clearly different from the one present in the plasma membrane of yeast cells.

Biological Transport, Active↗
PubMed1975

Attachment and ingestion of gonococci human neutrophils.

Previous studies have indirectly shown that type 1 gonococci are more resistant to phagocytosis by human neutrophils (PMN) than type 3 gonococci. Using phase contrast, fluorescent, and light microscopy, we directly quantitated PMN-gonococcal interaction, with emphasis on separating ingestion from attachment. PMN monolayers were incubated on slides with type 1 or type 3 gonococcal fluorescent antibody (FA). After methanol fixation, the FA-stained gonococci associated with PMN were cointed. Since the live PMN excludes FA, the FA-stained gonococci represent only extracellular gonococci. Methylene blue was then added to the smae slide to stain both ingested and surface attached gonococci. Using these methods, intracellular and extracellular cell-associated gonococci were quantitated under varying conditions. The numbers of methylene blue-stained cell-associated gonococci that were ingested were: with normal serum, 3.7 plus or minus 4.1 per cent for type 1 and 56.2 plus or minus 3.7 percent for type 3 (P smaller than 0.001); with heat-inactivated serum, 1.0 plus or minus 3.0 per cent for type 1 and 52.6 plus or minus 3.7 per cent for type 3 (P smaller than 0.001); with higher-titer anti-gonococcal antibody serum, 4.8 plus or minus 4.3 percent for type 1 and 64.0 plus or minus 1.6 per cent for type 3 (P smaller than 0.001). Thus, most type 3 organisms were ingested, but most type 1 gonococci were bound on the PMN surface.

Cell Adhesion↗
PubMed1975

Observations on phagocytosis of urate crystals by polymorphonuclear leucocytes.

The sequence of events that may initiate the inflammatory reaction in acute gout has been investigated with specific reference to phagocytosis of urate crystals by polymorphonuclear leucocytes and the results have shown (1) that neutrophil leucocytes avidly ingest microcrystals of sodium monourate, (2) that this causes the rapid degranulation and disintegration of the leucocytes, (3) that fresh leucocytes ingest the debris and crystals liberated by the dead cells, and in their turn degranulate and die, thus possibly establishing a vicious circle in the system.

Animals↗
PubMed1975

A new program for investigating adult human skeletal muscle grown aneurally in tissue culture.

With our new "explant-reexplanting" technique, abundant growth of mature human muscle in long-term tissue culture was achieved,and with the "sandwich" technique several histochemical reactions were obtained on serial cross sections of the cultured fibers. An advanced degree of maturation but lack of differentiation into reciprocally staining fiber-types was demonstrated. For electron-microscopic and electronmicroscopic-histochemical study, a method was developed in which the embedded fibers of greatest potential interest were identified by light microscopy and punched out by our specially-designed hollow drill. This selection procedure is critically important when the goal is to study in cultured diseased human muscle: (1) successive stages of development and (2) certain structural changes that often occur only in some fibers and only in certain regions of those fibers. The electronmicroscopic-histochemical appearance of developing cultured muscle fibers correlated well with the fresh-frozen light microscopic histochemical cross-sections and longitudinal whole preparations of similar fibers.

Adenosine Triphosphatases↗
PubMed1975

Use of peroxidatic-enzyme staining to enhance resolution of cultured mammalian cells under phase microscopy.

Staining of glutaraldehyde-fixed mammalian cells with peroxidatic enzymes (horseradish peroxidase or horse heart cytochrome c) greatly enhances resolution of their structure under phase microscopy. The topography of cell processes and regions of intercellular contact and overlapping is resolved precisely, even in dense cultures mounted in media which ordinarily do not permit clear demonstration of these areas. The technique is therefore a useful aid to the study of cultured cells with phase optics. Labeling depends on introducing free aldehydes into cells through the use of bifunctional fixatives such as glutaraldehyde. Acetone or formaldehyde fixation prevents staining, and labeling intensity is greatly diminished by pretreatment with spermine, a polyamine that reacts with glutaraldehyde. Electron microscopy reveals that peroxidase tags membranes preferentially; some areas are labeled smoothly, others in a punctate manner. Ribosomes are sharply contrasted, but nuclei remain unstained. Cytochrome c labels condensed nuclear chromatin intensely, and also stains ribosomes and portions of the cytoplasmic ground substance; membranes are mostly unmarked.

Animals↗
PubMed1975

The use of Herr four-and-a-half clearing fluid for the rapid microscopic examination of thick sections of normal and neoplastic tissues.

We have demonstrated that Herr's 4 1/2 clearing fluid, developed for use with plant tissues, can be successfully used for the microscopic examination of thick sections of normal and neoplastic mammalian tissues. Rat Novikoff hepatoma, rat liver, and human colon and skin samples were fixed in Bouin's, stained with iron hematoxylin, treated with Herr's 4 1/2 clearing fluid and examined by phase contrast microscopy. Tissue architecture and cytological detail were easily observed by focusing through tissue sections as thick as 70 mu. The method permits rapid microscopic examination of mammalian tissues and enables the investigator to detect readily morphological abnormalities within a tissue.

Animals↗
PubMed1976

Aggregation of platelets and inert particles induced by thrombin.

Thrombin-induced platelet aggregation and release were investigated in washed platelet suspensions and in suspensions of inert particles in order to evaluate the role of fibrinogen-fibrin transformation in aggregometer tracings. Thrombin (0.25-2.0 U/ml) produced two waves of light transmission increase (LTI) in both platelet and inert particle suspensions containing fibrinogen, and concomittantly aggregates were observed under phase microscopy. Without fibrinogen, thrombin induced rapid release of platelet ADP but failed to cause second wave of LTI. The kinetics of LTI in platelet and inert particle systems were related to both thrombin and fibrinogen concentrations. A rapid second wave of LTI could be produced by direct interaction of thrombin-treated platelets or inert particles with polymerizing fibrin, and was inhibited by sodium sulfite and low pH of 5.1 which prevent fibrin monomer polymerization. No fibrin strands were noted in platelet aggregates fixed at the completion of the second wave of LTI. Apyrase and PGE1 inhibited the rate of first but not that of second wave LTI. The results suggest that the release of platelet ADP induced by thrombin primarily affects the first phase aggregation, and the second phase may result from interaction of thrombin-exposed platelets and polymerizing fibrin. Thus, the blood coagulation mechanism may be directly involved in platelet aggregation.

Adenosine Diphosphate↗