Electron spin resonance study of rapidly frozen solution of iron-glutathione

Rapidly mixed anaerobic solutions (at pH 2.7) of FeCl₃ and glutathione were quickly frozen at various times after mixing. EPR spectra of these frozen solutions showed the progressive reduction of the iron(III) with time and the transient presence of a g = 2 radical signal. This signal is discussed in terms of an intermediate in the reduction pathway containing a high spin iron(II) centre weakly coupled to a sulphur radicalSimilar experiments were carried out at pH 9 in the presence of oxygen.     

Measurement of grain growth in the recrystallization of rapidly frozen solutions of antifreeze glycoproteins

A quantitative estimate of the activation energy for grain growth has been obtained by analyzing ice recrystallization experiments from water and from solutions with small amounts (< 1.0 μg/mL) of antifreeze glycoprotein (AFGP). Rates of grain growth are measured as changes of grain diameter in time, with the supercooled holding temperature aVid glycoprotein concentration as parameters. Arrhenius plots of these rates vs (1/T) yielded slopes proportional to the activation energies for the particular species. The values of activation energy are almost independent of solution concentration or the species of AFGP. Averaged activation energy value for the AFGP-4 species is Q_g = (6.61 ± 1.02) × 10⁵ J/mole. The “less active” AFGP-8 yielded an average Q_g = (5.71 ± 2.39) × 10⁵ J/mole, quite similar to the AFGP-4 species. The activation energy for recrystallization in a pure ice-water system was estimated from two temperature points, T = ?5.4 and ?7.5°C. The best value is 2.39 × 10⁵ J/mole, nearly twice that obtained by M. N. Martino and N. E. Zaritsky [(1989) Cryobiology, Vol. 26, p. 138] in a recrystallization experiment using salt solution, but much smaller than the values derived from the AFGP solutions. Results further show that activation entropy is at least a factor of 2 larger for the AFGP species than that of pure ice-water system under the same growth conditions. These results suggest significant roles, both energetically and entropically, for AFGP molecules in their ability to inhibit grain growth of ice. © 1994 John Wiley & Sons, Inc.     

Electron microscopy of lung rapidly frozen under controlled physiological conditions

Light microscopy of lung rapidly frozen under controlled physiological conditions has been very successful in correlating pulmonary structure and function. However, to study some aspects of pulmonary capillary morphology, the higher resolution of electron microscopy (EM) is necessary. To date, most EM of lung has involed the instillation of a fixative through the airways or vascular system, techniques that probably alter the normal pressure relationships of the capillaries and therefore their morphology. We describe here a technique for rapidly freezing lung to a depth of 1--2 mm below the pleural surface and preparing sections for EM. Lungs from open-chest rats were frozen at various transpulmonary pressures with cold (--80 degrees C) 70% ethylene glycol. Small pieces were then fixed with a solution containing glutaraldehyde and paraformaldehyde for 24 h at --50 degrees C. Staining was with osmium tetroxide and uranyl acetate. Lung frozen at high volumes showed marked stretching of the alveolar septa with severe deformation of the capillaries. Lung frozen at low inflation pressures revealed open capillaries containing numerous red blood cells; in addition, infolding of the alveolar wall was frequently seen. We conclude that this technique gives a level of preservation of rapidly frozen lung suitable for electron microscopy.     

Electron microscope study ofLeptospira

A leptospira consists of the cytoplasmic cylinder (contained within its own wall) which winds helically anticlockwise around the axistyle; both these components are enclosed by the outer covering membrane enveloping the whole organism.     

Electron microscope study on spermatogenesis

Summary The morphogenesis of chromosomes at early prophase of spermatocytes I is studied in three ortoptherian species: Grillus argentinus (Grillidae), Laplatacris dispar (Acrididae) and Blaptica dubia (Blaptidae).The first chromosome component appearing at the beginning of meiosis is a thread (single elementary thread; S.E.T.) of low electron density measuring about 700 Å to 0.1 width. A group having the same width and integrated by three helically twisted ribbon-like components develops from S.E.T. and is called primary tripartite group, P.T.G. The three components are at first of the same width (about 200 Å) but the lateral arms progressively increase in thickness and in that way the group becomes coated by a layer of dense microfibrils supposed to be chromatin.Late stages of prophase were not thoroughly investigated in this study, but many evidences were however found helping to identify synaptene stage. In accordance with these evidences each homologous chromosome is integrated by an axial component (tripartite groups) coated by chromatin.The medial component of tripartite groups of Blaptica dubia is double and also shows multistranded regions which are called puffy regions.A comparative optical and electron microscope study was made in order to better understand the above described process. This study includes the comparison of the thickness of chromosomes as measured in light and electron micrographs.On the other hand, rubber models were made to illustrate the same process and pohotographs of these models are exhibited in the text.The nuclear structure of early spermatids of the same species is also studied in this paper. It was found that Blaptica and Grillus early spermatids nuclei contain groups similar to those found at the beginning of prophase of spermatocyte I, with the only difference that in many cases composite groups, formed by fusion of the lateral arms of two or more than two single groups, were found.The nuclear structure of late spermatids was also considered in this paper. Notwithstanding the study only aimed to point out that the pattern of organization of the spermatozoon chromatin greatly differs in the species examined.     

Electron microscope study on meiosis

Summary The cycle of the nucleolus and sex chromosome was studied with the electron microscope during the following stages of spermatogenesis and spermiogenesis of Gryllus argentinus: (1) spermatogonia; (2) prophase cyte I, leptotene, part of pachytene, and end of diplotene till breakdown of the nuclear envelope; (3) division I, metaphase and anaphase; (4) cyte II, prometaphase; (5) division II, metaphase, anaphase and telophase; (6) early and late spermatids. Some observations were also carried out in the primary oocyte until beginning of the growth period.It was found that nucleolus and sex chromosome are associated, at first without mixture of their components (leptotene) and afterwards interchanging components (pachytene). The interchange takes place by the passage from one element to another of filamentous units ot low electron density, similar in appearance to those existing in the medial plane of tripartite groups (synaptinemal complexes).At pachytene the primary results of interchange are: (1) the nucleolus contains filaments of chromosomal nature; (2) the nucleolus emits a long rod-like prolongation containing a cylindrical bundle of filaments, and an axial unit of the same nature; two equidistant clear spaces separate the axial unit from the cylindrical bundle and the latter from the dark wall of nucleolar material. At the end of diplotene these components are found organized in two bodies and a prolongation. One of the bodies is formed by a number of alternatively dark and light bands, the other by a pack of tubular units each showing the structure of the former nucleolar prolongation. The prolongation is either formed as in the preceding stage or it is composed of five ribbons, two dark ones outside and three light ones between them. It is supposed that both bodies are united by the prolongation but no definite proof was obtained. It is assumed that the complex thus constituted represents the sex chromosome.The sex chromosome was found at any phase of both divisions as well as at the intermediate stages between them; at the division phase the chromosome is separated from the autosomes and moves independently of them.The element could not be traced at telophase II but it reappears within the reorganized nuclei of spermatids. Amorphous nucleolar-like material and chromosome-like material are found associated at this stage with banded complexes like those seen at the end of prophase I. All these components undergo involution during spermatid maturation. At the final step of maturation no traces of them are found.A similar association of nucleolus and chromosome was found at prophase of primary oocytes of the same species. The associated body is of the same structure as that described for primary spermatocytes. The structures existing in the primary oocytes disorganize at the beginning of growth. At this time the nucleolus has developed into a large body containing masses of chromatin-like material.This investigation was supported in part by U.S. Public Health Service, Research Grant No. GM-08337 from the National Institute of General Medical Sciences.     

Electron microscope study of Cryptostroma corticale

An ultrastructural study of Cryptostroma corticale has been carried out with scanning and transmission electron microscopes. The usual features in fungi, as well as features characteristic of the family, were shown. It was noted that lomasomes and myelin-type structures were demonstrated by the two fixatives used. Their significance is discussed.     

Electron microscope study of lens fibers

The in vitro swelling action of L-thyroxine on rat liver mitochondria as examined photometrically represents an acceleration of a process which the mitochondria are already inherently capable of undergoing spontaneously, as indicated by the identical kinetic characteristics and the extent of thyroxine-induced and spontaneous swelling, the nearly identical pH dependence, and the fact that sucrose has a specific inhibitory action on both types of swelling. However, thyroxine does not appear to be a "catalyst" or coenzyme since it does not decrease the temperature coefficient of spontaneous swelling. The temperature coefficient is very high, approximately 6.0 near 20 degrees . Aging of mitochondria at 0 degrees causes loss of thyroxine sensitivity which correlates closely with the loss of bound DPN from the mitochondria, but not with loss of activity of the respiratory chain or with the efficiency of oxidative phosphorylation. Tests with various respiratory chain inhibitors showed that the oxidation state of bound DPN may be a major determinant of thyroxine sensitivity; the oxidation state of the other respiratory carriers does not appear to influence sensitivity to thyroxine. These facts and other considerations suggest that a bound form of mitochondrial DPN is the "target" of the action of thyroxine. The thyroxine-induced swelling is not reversed by increasing the osmolar concentration of external sucrose, but can be "passively" or osmotically reversed by adding the high-particle weight solute polyvinylpyrrolidone. The mitochondrial membrane becomes more permeable to sucrose during the swelling reaction. On the other hand, thyroxine-induced swelling can be "actively" reversed by ATP in a medium of 0.15 M KCl or NaCl but not in a 0.30 M sucrose medium. The action of ATP is specific; ADP, Mn(++), and ethylenediaminetetraacetate are not active. It is concluded that sucrose is an inhibitor of the enzymatic relationship between oxidative phosphorylation and the contractility and permeability properties of the mitochondrial membrane. Occurrence of different types of mitochondrial swelling, the intracellular factors affecting the swelling and shrinking of mitochondria, as well as the physiological significance of thyroxine-induced swelling are discussed.     

Critical helix content in gelatin gels

Aqueous gelatin solutions of different concentrations have been investigated at various quench temperatures by viscosity measurements to determine the gel times and by optical rotation measurements to derive the evolution of the helix content by reference to native collagen. As a result, it appears that the gelation of the different aqueous gelatin solutions tested takes place at a common helix concentration independent of the initial gelatin concentration and quench temperature. Further, for each concentration, the dependence of gel time as a function of quench temperature has revealed the existence of two domains: a higher temperature domain where gel times increase strongly with quench temperature and a lower temperature domain where gel times are short and only slightly dependent on quench temperature.