THE FINE STRUCTURE OF THE NUCLEAR MATERIAL OF A BLUE-GREEN ALGA,ANABAENA CYLINDRICA LEMM

The chromatinic material of the blue-green alga Anabaena cylindrica has complex configurations in the central regions of the cells. The distribution of the chromatin within the cells varies in different filaments, probably in response to variations in the disposition of other cellular components. In electron micrographs of thin sections of organisms fixed by the method of Kellenberger, Ryter, and Séchaud (1958) the centroplasm contains fibrillar and possibly granular components which can be identified as the nuclear material by comparison with stained preparations viewed in the light microscope. The fibrils in the nuclear regions have diameters in the range of 5 to 7 mµ and are embedded in a matrix of lower density. The nuclear regions are not greatly different from the cytoplasm in their electron density. Reducing the calcium content of the fixative results in coagulation of the fibrils to form coarser structures. The significance of the observations is discussed in relation to observations on the fine structure of other classes of algae and of bacteria.     

An Electron Microscope Study of Polyoma Virus in Hamster Kidney

Electron microscope studies were made of hamster kidneys taken at daily intervals after injection of a variant of polyoma virus into newborn animals. Particular attention was paid to the period 5 to 6 days after injection at which time the necrotizing response was at its peak and virus particles were seen in greatest numbers. The most numerous particles were about 28 mµ in diameter. They were observed mainly within nuclei of stromal cells and are similar to the particles seen in large numbers in polyoma-infected mouse cells growing in vitro. They were not observed in cells of fully developed tumors. Filamentous or tubular structures closely associated with the 28 mµ particles and probably concerned in their formation are described. Considerable quantities of viral material were contained within cytoplasmic inclusions. In some of the inclusions larger particles of diameter 60 mµ were observed. The origin of these particles and their relation to the 28 mµ particles is discussed.     

STRUCTURE AND DEVELOPMENT OF VIRUSES OBSERVED IN THE ELECTRON MICROSCOPE : IV. VIRUSES OF THE RI-APC GROUP

Representative viruses of the RI-APC group were observed with the electron microscope in thin sections of infected HeLa cells. The viral particles varied in density, were approximately 60 mµ in diameter and had a center to center spacing when close packed of about 65 mµ. Many of the less dense particles exhibited an internal body averaging 24 mµ in diameter. It was suggested that within the nucleus the virus differentiated from dense granular and reticular material and formed crystals. Disintegration of the crystals and disruption of the nuclear membrane with release of virus into the cytoplasm appeared to occur at any stage. No evidence to suggest development of the virus in the cytoplasm was obtained. It was possible to deduce the structure of the viral crystal from the electron micrographs. The viral particles are packed in a cubic body—centered lattice. Correlative histochemical observations in the light microscope which are now in progress revealed that the crystals and non-crystalline aggregates of virus were strongly Feulgen-positive.     

MORPHOLOGY OF THE OMMATIDIA OF THE COMPOUND EYE OF LIMULUS

The sensory portion of the ommatidium of the compound eye of Limulus has been studied with the electron microscope. In axial longitudinal section the rhabdom appears to be made up of small polygons, and in transverse section the rhabdom appears as a banded structure of dark lines. Thus in three dimensions the rhabdom resembles a honeycomb composed of tubular units, the long axes of which lie in transverse planes and are oriented perpendicular to the retinula cell's contours. The tubular units, which are about 140 mµ in diameter in Limulus (70 mµ in diameter in the spider and Scutigera), are microvilli of the borders of the retinula cells. The walls of these microvilli are continuous with fine linear structures (membranes) in the cytoplasm of the retinula cells. In transverse sections of the ommatidium oval bodies interpreted as mitochondria are observed in an annular zone at the tips of the rhabdom's rays. These mitochondria, which are 2 to 10 µ in diameter, are crowded with irregular closed outlines about 100 mµ in diameter. Possible functions of components of the ommatidium are discussed.     

Electron Microscopy of Neurospora crassa Mycelia

The fine structure of vegetative mycelia of the filamentous Ascomycete, Neurospora crassa, has been investigated by the standard techniques of electron microscopy. Addition of uranyl nitrate to the methacrylate-embedding medium minimized disruption of the specimens—an accident often observed in the preparation of microbial material. This report describes the presence of a chitinous polysaccharide wall containing fine fibrils embedded in a homogeneous matrix. A sinuate plasma membrane lies adjacent to the inner wall surface. This membrane is often closely associated with the endoplasmic reticulum of the cytoplasm. Numerous mitochondria of the classical type, and dense particles of 10 mµ diameter occur throughout the cytoplasm. The nuclear region is surrounded by a double membrane with pore openings. Associated with the nuclear envelope is a dense area, the nucleolus. The significance of these observations and their relationship to other forms is discussed.     

THE FINE STRUCTURE AND CHEMICAL COMPOSITION OF NUCLEI DURING SPERMIOGENESIS IN THE HOUSE CRICKET : I. Initial Stages of Differentiation and the Loss of Nonhistone Protein

The early stages of nuclear differentiation in spermatids of the house cricket are described with regard to the fine structural elements and chemical components which occur. Particular attention is given to the loss of nonhistone protein from the nucleus and its relation to chromatin structure. Granular elements about 25 to 80 mµ in diameter, and fibers about 8 mµ in diameter occur in the earliest spermatid nucleus. The fibers are found in diffuse and condensed chromatin while granules are found only in diffuse material. DNA and histone parallel the chromatin fibers in distribution, while nonhistone protein and RNA parallel the granules in distribution. The granules and most of the nonhistone protein are lost, simultaneously, after the early spermatid stage. The protein loss occurs without detectable change in the structure of chromatin fibers. Chromatin fibers first show a structural change in mid spermiogenesis, when they become thicker and very contorted. Unusually thin fibers (about 5 mµ) also appear in mid spermatid nuclei; they are apparently composed of nonhistone protein and free of DNA and histone.     

Action spectra for photoreactivation of ultraviolet-irradiated Escherichia coli and Streptomyces griseus

Action spectra for photoreactivation (light-induced recovery from ultraviolet radiation injury) of Escherichia coli B/r and Streptomyces griseus ATCC 3326 were determined. The spectral region explored was 365 to 700 mµ. The action spectrum for S. griseus differed from that for E. coli, indicating that the chromophores absorbing reactivating energy in the two species were not the same. Reactivation of S. griseus occurred in the region 365 mµ (the shortest wave length studied) to about 500 mµ, with the most effective wave length lying near 436 mµ. This single sharp peak in the spectrum at 436 mµ suggested the Soret band typical of porphyrins. Reactivation of E. coli occurred in the region 365 to about 470 mµ, with the most active wave length lying near 375 mµ. The single, non-pronounced peak near 375 was probably not due to a Soret band, and the identification of the substance absorbing reactivating light in E. coli is uncertain. In neither species was the region 500 to 700 mµ active. The implications of these action spectra and their differences are discussed.     

The spectral sensitivities of the dorsal ocelli of cockroaches and honeybees; an electrophysiological study

The spectral sensitivities of the dorsal ocelli of cockroaches (Periplaneta americana, Blaberus craniifer) and worker honeybees (Apis mellifera) have been measured by electrophysiological methods. The relative numbers of quanta necessary to produce a constant size electrical response in the ocellus were measured at various wave lengths between 302 and 623 mµ. The wave form of the electrical response (ERG) of the dark-adapted roach ocellus depends on the intensity but not the wave length of the stimulating light. The roach ocellus appears to possess a single photoreceptor type, maximally sensitive about 500 mµ. The ERG's of bee ocelli are qualitatively different in the ultraviolet and visible regions of the spectrum. The bee ocellus has two types of photoreceptor, maximally sensitive at 490 mµ and at about 335 to 340 mµ. The spectral absorption of the ocellar cornea of Blaberus craniifer was measured. There is no significant absorption between 350 and 700 mµ.     

MATURATION OF RAT MAST CELLS : An Electron Microscope Study

Electron microscope study of rat mast cell maturation corroborates certain interpretations of features of mast cell differentiation based on light microscope studies. In addition, the ultrastructural variation observed in the granules of differentiating mast cells suggests that granule formation begins with the elaboration of dense granules about 70 mµ in diameter inside Golgi vacuoles. These progranules appear to aggregate inside a membrane and fuse to form dense cords 70 to 100 mµ in diameter. These dense cords are embedded in a finely granular material possibly added to the developing granule by direct continuity between perigranular membranes and cisternae of rough endoplasmic reticulum. The dense cords and finely granular material then appear to be replaced by a mass of strands about 30 mµ in diameter, thought to be a reorganization product of the two formerly separate components. A process interpreted as compaction of the strands completes the formation of the dense, homogeneous granules observed in mature rat mast cells. The similarity between mast cell granule formation and the elaboration of other granules is considered, with special reference to rabbit polymorphonuclear leukocyte azurophil granules. The relationships between the ultrastructural, histochemical, and radioautographic characteristics of mast cell granule formation are considered, and the significance of the perigranular membrane is discussed.     

ASPECTS OF CILIARY FINE STRUCTURE IN EUPLOTES PATELLA

1. The functional unity of cirri and membranelles can result structurally only from extensions of the ciliary membrane. 2. The pellicle is composed of an outer pellicular membrane and an inner cytoplasmic membrane. 3. The ciliary rootlets are composed of numerous filaments 120 A in diameter with central areas of low density. They have no periodic structure. 4. The ciliary membrane is a double-layered structure continuous with the pellicular membrane. The cilia show the typical arrangement of nine double, peripheral and two single, central fibrils. All fibrils pass into the basal region, the peripheral ones joining with the rootlet filaments, while the central fibrils from the extreme proximal position of the basal region turn back toward the pellicle and appear to unite just beneath the cytoplasmic membrane. 5. The cilia (300 mµ diameter) taper at their tips to a diameter at least as small as 50 mµ. At a diameter of about 150 mµ, the fibrils begin to show a reduction in number. 6. The central ciliary fibrils may determine the possible directions of ciliary beat. These fibrils show an intrafibrillar structure in their basal portion, which involves regularly spaced 40 A granules. 7. These observations on Euplotes, together with the other evidence cited, are consistent with the hypothesis that ciliary motion is produced by the contraction of the peripheral fibrils, while the central fibrils perhaps determine the plane in which the cilia can bend.     

AFM-based Mapping of the Elastic Properties of Cell Walls: at Tissue,Cellular, and Subcellular Resolutions

We describe a recently developed method to measure mechanical properties of the surfaces of plant tissues using atomic force microscopy (AFM) micro/nano-indentations, for a JPK AFM. Specifically, in this protocol we measure the apparent Young’s modulus of cell walls at subcellular resolutions across regions of up to 100 µm x 100 µm in floral meristems, hypocotyls, and roots. This requires careful preparation of the sample, the correct selection of micro-indenters and indentation depths. To account for cell wall properties only, measurements are performed in highly concentrated solutions of mannitol in order to plasmolyze the cells and thus remove the contribution of cell turgor pressure.In contrast to other extant techniques, by using different indenters and indentation depths, this method allows simultaneous multiscale measurements, i.e. at subcellular resolutions and across hundreds of cells comprising a tissue. This means that it is now possible to spatially-temporally characterize the changes that take place in the mechanical properties of cell walls during development, enabling these changes to be correlated with growth and differentiation. This represents a key step to understand how coordinated microscopic cellular changes bring about macroscopic morphogenetic events.However, several limitations remain: the method can only be used on fairly small samples (around 100 µm in diameter) and only on external tissues; the method is sensitive to tissue topography; it measures only certain aspects of the tissue’s complex mechanical properties. The technique is being developed rapidly and it is likely that most of these limitations will be resolved in the near future.     

Photoreversal of nuclear and cytoplasmic effects of short ultraviolet radiation on Paramecium caudatum

1. Irradiation with three short ultraviolet (UV) wave lengths, 226, 233, and 239 mµ rapidly immobilizes Paramecium caudatum, the dosage required being smaller the shorter the wave length. 85 per cent of paramecia immobilized with wave length 226 mµ recover completely. Recovery from immobilizing doses is less the longer the wave length. 2. Irradiation continued after immobilization kills the paramecia in a manner which is markedly different for very short (226, 233, and 239 mµ) and longer (267 mµ) wave lengths. 3. An action spectrum for immobilization in P. caudatum was determined for the wave lengths 226, 233, 239, 248, and 267 mµ, and found to resemble the absorption of protein and lipide in the wave length region below 248 mµ. Addition of these data to those of Giese (1945 b) gives an action spectrum resembling the absorption by albumin-like protein. 4. Division of P. caudatum is delayed by doses of wave lengths 226, 233, and 239 mµ which cause immobilization, the longest wave length being most effective. 5. Immobilization at any of the wave lengths tested (226, 233, 239, 248, 267 mµ) is not photoreversible when UV-treated paramecia are concurrently illuminated. 6. Division delay resulting from immobilizing doses of 226, 233, and 239 mµ is photoreversible by exposure to visible light concurrently with the UV. 7. Division delay induced by exposure to wave length 267 mµ is reduced by exposure to visible light applied concurrently with UV or immediately afterwards. 8. The data suggest that the shortest UV wave length tested (226 mµ) affects the cytoplasm selectively, because it is absorbed superficially as indicated by unilateral fluorescence in UV. Consequently it immobilizes paramecia rapidly but has little effect on the division rate because little radiation reaches the nucleus. 9. The data support the view that nuclear effects of UV are readily photoreversed but cytoplasmic effects are not.     

Structure of the Red Fluorescence Band in Chloroplasts

Using Weber's method of "matrix analysis" for the estimation of the number of fluorescent species contributing to the emission of a sample, it is shown that the fluorescence¹ band in spinach chloroplast fragments at room temperature originates in two species of chlorophyll a. Emission spectra obtained upon excitation with different wavelengths of light (preferentially absorbed in chlorophyll a or b) are presented. Upon cooling to - 196°C, the fluorescence efficiency increases about twentyfold. Two additional bands, that now appear at 696 and 735 mµ, suggest the participation of four molecular species. Emission spectra observed at different concentrations of chloroplast fragments with excitation in chlorophyll a and b and excitation spectra for different concentrations of chloroplast fragments and measurements at 685 and 760 mµ are presented. Two of the four emission bands may belong to pigment system I and two to system II. The 685, 696, and 738 mµ bands respond differently to temperature changes. In the -196°C to -150°C range, the intensity of the 685 mµ band remains constant, and that of the 696 mµ band decreases twice as fast as that of the 738 mµ band.     

MICROTUBULATION OF THE INNER MEMBRANE OF THE NUCLEAR ENVELOPE

In the course of a light and electron microscopy study of spermatogenesis in the European crayfish, Astacus fluviatilis, spermatocytes of abnormal appearance were observed in two instances in individuals that had passed the mating period. The electron microscope showed that the inner membrane of the nuclear envelope of these cells was erupting into a mass of microtubules, 15 to 18 mµ in diameter and 0.5 µ or more in length, while the outer membrane transformed into cytoplasmic vesicles. Stages in the formation of these novel processes were followed. The plasma membrane of the affected cells was seen in some cases to erupt into similar although shorter microtubules. It is concluded that the phenomenon is part of a degenerative process in which the spermatocytes are being absorbed by sustentacular cells. It is suggested that the observations provide further evidence for a fundamental functional as well as a morphological similarity between the membranes bounding the nucleus and the plasma membrane.     

Single and Multiple Visual Systems in Arthropods

Extraction of two visual pigments from crayfish eyes prompted an electrophysiological examination of the role of visual pigments in the compound eyes of six arthropods. The intact animals were used; in crayfishes isolated eyestalks also. Thresholds were measured in terms of the absolute or relative numbers of photons per flash at various wavelengths needed to evoke a constant amplitude of electroretinogram, usually 50 µv. Two species of crayfish, as well as the green crab, possess blue- and red-sensitive receptors apparently arranged for color discrimination. In the northern crayfish, Orconectes virilis, the spectral sensitivity of the dark-adapted eye is maximal at about 550 mµ, and on adaptation to bright red or blue lights breaks into two functions with λ_max respectively at about 435 and 565 mµ, apparently emanating from different receptors. The swamp crayfish, Procambarus clarkii, displays a maximum sensitivity when dark-adapted at about 570 mµ, that breaks on color adaptation into blue- and red-sensitive functions with λ_max about 450 and 575 mµ, again involving different receptors. Similarly the green crab, Carcinides maenas, presents a dark-adapted sensitivity maximal at about 510 mµ that divides on color adaptation into sensitivity curves maximal near 425 and 565 mµ. Each of these organisms thus possesses an apparatus adequate for at least two-color vision, resembling that of human green-blinds (deuteranopes). The visual pigments of the red-sensitive systems have been extracted from the crayfish eyes. The horse-shoe crab, Limulus, and the lobster each possesses a single visual system, with λ_max respectively at 520 and 525 mµ. Each of these is invariant with color adaptation. In each case the visual pigment had already been identified in extracts. The spider crab, Libinia emarginata, presents another variation. It possesses two visual systems apparently differentiated, not for color discrimination but for use in dim and bright light, like vertebrate rods and cones. The spectral sensitivity of the dark-adapted eye is maximal at about 490 mµ and on light adaptation, whether to blue, red, or white light, is displaced toward shorter wavelengths in what is essentially a reverse Purkinje shift. In all these animals dark adaptation appears to involve two phases: a rapid, hyperbolic fall of log threshold associated probably with visual pigment regeneration, followed by a slow, almost linear fall of log threshold that may be associated with pigment migration.     

AN ELECTRON MICROSCOPE STUDY OF THE CYTOLOGY OF THE PROTOZOAN EUPLOTES PATELLA

1. Structurally the "sensory bristles" in Euplotes patella are typical cilia, but no ciliary rootlets connect their bases. 2. The "neuromotor fibrils" are composed of filaments 21 mµ in diameter. At the point of junction of the filaments with the peripheral ciliary fibrils a granular structure 65 to 90 mµ in diameter is seen which has dense central and peripheral zones separated by a less dense layer. Information on the interconnection of organelles is expanded. 3. A system of subpellicular fibrils is described. The external fibrillar system described by others could not be found. 4. The motorium is shown to be a mass of intertwining rootlet filaments. 5. The micronucleus is shown to have a spongy, dense material in a less dense material, all of which is surrounded by a double-layered membrane. 6. The double-layered macronuclear membrane contains annuli whose outside diameter is 70 mµ; the macronuclear bodies are sometimes closely applied to the membrane. In the macronuclear reorganization bands, the solution plane is a fine network, while the reconstruction plane is devoid of structure at the level of resolution observed. 7. The mitochondria are composed of tubules, only occasionally oriented, usually embedded in a surrounding material of lower density. 8. Microbodies whose diameters are 250 to 350 mµ are frequently observed in close association with mitochondrial surfaces. 9. The food vacuoles, contractile vacuoles, and ciliary vacuoles are bounded by single-layered membranes. In the food vacuoles, the bacteria are surrounded by membranes individually or in small groups. 10. Cytoplasmic rods localized in the oral region, and cytoplasmic granules dispersed at random, are described. No typical ergastoplasm, endoplasmic reticulum, or Golgi material was observed.     

The Components of the Visual System of a Dragonfly

In this study of the electroretinograms of dragonflies (adults and nymphs) the objectives were to determine the number of classes of photoreceptors present in the visual system and to allocate these to particular morphological regions. There are probably five classes of photoreceptors present with peak sensitivities near 550, 530, 518, 420, and < 380 mµ. The dorsal ocelli contain two classes (518 mµ and < 380 mµ). The ventral (anterior) ommatidia of the adult compound eye contain at least two classes (near 518 mµ and < 380 mµ) and probably a third class (near 550 mµ). The dorsal ommatidia of the adult compound eye contain one class (420 mµ) and possibly another class (< 380 mµ). The compound eye of the nymph contains one class (530 mµ) and possibly another class (420 mµ).     

The photosensitive retinal pigments of fishes from relatively turbid coastal waters

Digitonin extracts have been prepared from the retinae of a dozen species of marine and euryhaline teleost fishes from turbid water habitats. Spectrophotometric analysis of the extracts shows that the photosensitive retinal pigments of these species have maximum absorption above 500 mµ. In nine species there are retinene₁ pigments with λ_max between 504 and 512 mµ. In the marine but euryhaline mullet, Mugil cephalus, there is a porphyropsin with λ_max 520 mµ. A mixture of rhodopsin and porphyropsin in an extract of a marine puffer, Sphoeroides annulatus, was disclosed by partial bleaching with colored light. In addition, one other species has a 508 mµ pigment, of which the nature of the chromophore was not determined. The habitats in which these fishes live are relatively turbid, with the water greenish or yellowish in color. The spectral transmission of such waters is probably maximal between 520 and 570 mµ. It is suggested that the fishes have become adapted to these conditions by small but significant shifts in spectral absorption of their retinal pigments. These pigments are decidedly more effective than rhodopsin in absorption of wavelengths above 500 mµ. This offers a possible interpretation of the confusing array of retinal pigments described from marine and euryhaline fishes.     

ANALYTICAL DIFFUSION OF INFLUENZA VIRUS AND MOUSE ENCEPHALOMYELITIS VIRUS

Analytical diffusion has been applied to a study of influenza A virus in mouse lung, influenza A virus in the extra-embryonic fluids of the chick, and mouse encephalomyelitis virus in mouse brain. The results from influenza in mouse lung suggested that about 99 per cent of the infectivity was present in particles 200 mµ in diameter, and 1 per cent in particles 6 mµ in diameter. The results from influenza in extra-embryonic fluids indicated that the preparation was inhomogeneous and that the smallest virus units were about 6 mµ in diameter. The results from mouse encephalomyelitis virus indicated that the preparation was also inhomogeneous, with 10 per cent of the infectivity in particles about 15 mµ in diameter. It has been suggested that in virus preparations normal colloidal particles can act as carriers of much smaller virus units.     

ELECTRON MICROSCOPIC STUDY OF THE CYTOPATHOLOGY OF ECHO VIRUS INFECTION IN CULTIVATED CELLS

The cultivated monkey kidney cell is subject to changes when infected with ECHO viruses 6, 9, and 19. The electron microscope reveals three stages of infection: (a) initial stage. The nucleus appears granular with chromatin condensation on the nuclear envelope. The cytoplasm contains electron transparent vesicles and vacuoles forming nests. (b) Intermediate stage. The nucleus seems to diminish, appearing more pycnotic and displaced toward the periphery. The cytoplasm is filled with electron transparent vacuoles and vesicles, and dense masses as well as some spiral bodies are seen. The mitochondria retain their shape. Dense particles are seen, which are possibly of viral nature. (c) Final stage. The nucleus is contracted to a narrow strip close to the cellular membrane or is completely destroyed. The cytoplasm shows no apparent changes. Crystals are frequently observed in cells infected with ECHO viruses 6 and 19, consisting of dense particles with an average diameter of 14.4 mµ ranging from approximately 13.2 to 15.6 mµ for ECHO virus 6, and 14.5 mµ ranging from approximately 12.5 to 16.5 mµ for ECHO virus 19. These particles are clustered in hexagonal packages forming angles of 75° and 105°. The particles in most crystals are arranged in rows separated by a constant distance, the latter varying from one crystal to another and being approximately 1.5 and 2.5 times the distance between particles. Other particles were observed which, however, are not considered to be of viral nature.