ELECTRON MICROSCOPE OBSERVATIONS ON THE STRUCTURE AND DISCHARGE OF THE STENOTELE OF HYDRA

Sections of the stenotele type of nematocyst of Chlorohydra hadleyi have revealed that the stenotele, upon firing, completely everts its stylets and spines and the long, thin tubule, much as the eversion of the tubule of the nematocyst of the jewel anemone (Picken, 1953; Robson, 1953). Alternative mechanisms for supplying the energy necessary to forcefully discharge the stenotele contents are discussed as well as the possible significance of several regions containing highly ordered periodic structure. The origin of nematocysts as kinetosomal derivatives is discussed as a possibility suggested by the symmetry of the stenotele contents and the structure, location, and function of the cnidocil.     

THE FINE STRUCTURE OF TWO UNUSUAL STALKED BACTERIA

Two strains of bacteria that produce slender appendages (pseudostalks) from their lateral surfaces were studied using the electron microscope. The pseudostalks were shown to be extensions of the cytoplasm and peripheral membranes of the cell proper. Both strains of bacteria produce holdfasts at the poles of the cells by the means of which attachment can take place. The pseudostalks are not involved in the attachment of cells. No specialized intracytoplasmic structures are present at the point of juncture of pseudostalk and cell. A discussion of the possible functions of the pseudostalks, based on the electron microscope findings, is presented.     

AN ELECTRON MICROSCOPE STUDY OF THE FINE STRUCTURE OF FEATHER KERATIN

Thin sections of the rachis of regenerating follicles of pigmented fowl feathers and of mature non-pigmented seagull feather rachis, embedded in methacrylate and Araldite respectively, were studied in the electron microscope. The late stages of development of keratin fibrils were examined in OsO₄-fixed follicle material, and after poststaining with lead hydroxide the keratin aggregates were found to be composed of fine microfibrils approximately 30 A in diameter apparently embedded in a matrix material which had absorbed the lead stain. The centre-to-centre separation of the microfibrils was of the order of 35 A. After bulk treatment by reduction with thioglycollic acid, OsO₄ staining, and poststaining with lead hydroxide, a similar microfibrillar fine structure was observed in mature rachis. Only after lead staining could the microfibrils be delineated, and their diameter and separation were similar to that found in the keratin of the follicle. It is suggested that feather keratin resembles α-keratins in consisting of microfibrils embedded in an amorphous protein matrix. However, in comparison with α-keratins, the microfibrils are much smaller in diameter, their arrangement is less orderly, and on the basis of the reactions towards the electron staining procedures, the cystine content of the matrix appears to be not greatly different from that of the microfibrils. The significance of a microfibrillar constitution of feather keratin is discussed in relation to current structural models for this fibrous protein deduced from x-ray diffraction studies. The boundaries between the component cells of feather rachis are desmosomal in character and similar to those of related keratinous structures and a number of different types of cells; the melanin granules are dissimilar to those of mammalian epidermis in their apparent lack of melanin-protein lamellae.     

OBSERVATIONS ON THE FINE STRUCTURE OF LUTEIN CELLS

Corpora lutea from the period of delayed implantation and from early postimplantation stages of the armadillo, mink, and rat were fixed in buffered osmium tetroxide-sucrose or potassium permanganate. After rapid dehydration, the portions of the corpora lutea were embedded in either methacrylate or epoxy resin. Examination of the lutein cells by electron microscopy revealed the presence, in the better preserved material, of an extensive development of tubular agranular endoplasmic reticulum. Although the membranes of the endoplasmic reticulum are the most striking feature of the lutein cells of both stages of the three animals examined, very numerous large mitochondria with cristae that exhibit a variety of forms tending toward villiform, and protrusions and foldings of the lutein cell margins on the pericapillary space are also characteristic of these cells. Certain minor differences in the lutein cells of the species examined are also noted. No indications of conversion of mitochondria into lipid, of accumulation of lipid in the Golgi area, or of the protrusion of lutein cells into spaces between the endothelial cells, as suggested by other authors, were noted in these preparations. Some of the difficulties inherent in the visualization of the secretory activity of cells producing steroid hormones are briefly discussed.     

OBSERVATIONS ON THE FINE STRUCTURE OF CALLIXYLON WOOD

Secondary wood of three species of Callixylon of Lower Mississippian age, preserved by three different modes (fusinization, silicification, and phosphatization), have been studied and characterized in detail. Problems of interpretation at the SEM level of permineralized woods, both containing (silicified wood of Callixylon erianum) and essentially lacking (phosphatized wood of C. arnoldii) original organic cell wall material, are analyzed and discussed. In particular, it is concluded that the flat to curved surfaces showing no evidence of apertures, observed in bordered pit pairs, commonly represent pit membrane surfaces. It is accepted, however, that some concave surfaces might be the mineral accretion surfaces of incomplete pit cavity casts as proposed by Leo and Barghoorn (1976). Regions between groups of pits, previously interpreted as crassulae, may be artifacts of preservation. The fusinized wood has the general appearance of charcoal, but unlike commercially produced charcoal provides evidence of its original microfibrillar structure. The origin of fusain in the fossil record is discussed, and it is concluded that it probably had several origins, including forest fire. Since charcoal can be produced naturally in the absence of O₂ (Brown and Davis, 1973), the suggestion that fusain (charcoal) in the geologic column provides a basis for “assessing oxygen levels in paleoatmospheres” (Cope and Chaloner, 1980) is not supported. Natural sites of fusain production in the absence of O₂ are regions of vulcanism and organic sediments inhabited by anaerobic microorganisms. A circular pattern of crystal orientation in the pit borders of C. arnoldii is interpreted to represent the original microfibrillar pattern. Pit apertures in C. arnoldii are shown to be circular to slightly elliptical. Interpretive evidence of heterogeneous pit membranes in C. arnoldii suggests but does not prove the presence of a torus. The distinctive central region in some pit membranes of the fusinized wood of Callixylon sp. might represent accumulations of waste metabolites. It is argued that a torus would be highly adaptive in large pits with circular apertures.     

白刺珠心组织及其营养功能的超微结构观察

白刺具厚珠心,珠心组织发达。原胚期,根据超微结构特征,珠心组织大体可区分为三层:外围不活跃细胞层、特化细胞层和胚囊外围解体细胞层。珠心细胞解体时,细胞壁首先以微纤丝状态分散,原生质不同程度破坏,并累积脂滴。近胚囊壁降解后,珠心细胞即成为开放细胞,原生质团游离到胚囊周围。胚囊壁在珠孔区和合点区间断,因之,珠心细胞的游离原生质团可直接迁入胚囊内。在胚囊壁和中央细胞质膜之间存在明显间隙区域,其中有大量壁旁体存在,是珠心细胞降解物质进入胚囊的另一重要途径。     

ELECTRON MICROSCOPE STUDIES OF EPIDERMAL MELANOCYTES, AND THE FINE STRUCTURE OF MELANIN GRANULES

Melanocytes and melanin granules have been studied by electron microscopy in normal human and cat skin, and in hyperplastic human skin lesions. The melanocytes have always been found as free cells within the epidermis,i.e., on the epidermal side of the dermal membrane. Melanocytes frequently rest on the dermal membrane or bulge towards the dermis. In such cases the uninterrupted dermal membrane is uniformly thin and smooth in appearance, in contrast with the regions alongside Malpighian cells, where it appears appreciably thicker and seemingly anchored to the basal cell layer. Two types of melanin granules have been distinguished according to their location in the melanocytes and to morphological characteristics which may only express different stages in the maturation of the granules: (a) light melanin granules in which a structure resembling a fine network is apparent; (b) dense melanin granules which, in osmium-fixed preparations, appear as uniformly dense masses surrounded by a coarsely granular, intensely osmiophilic shell. Treatment of sections of osmium-fixed tissues with potassium permanganate has revealed within the dense granules the existence of an organized framework in the form of a regular, crystalline-like lattice. It is suggested that this basic structure is protein in nature and may include the enzymatic system capable of producing melanin. The existence is reported of fine filaments located in the cytoplasm of melanocytes and morphologically distinct from the tonofilaments found in Malpighian cells.     

THE FINE STRUCTURE OF GREEN BACTERIA

The fine structure of several strains of green bacteria belonging to the genus Chlorobium has been studied in thin sections with the electron microscope. In addition to having general cytological features typical of Gram-negative bacteria, the cells of these organisms always contain membranous mesosomal elements, connected with the cytoplasmic membrane, and an elaborate system of isolated cortical vesicles, some 300 to 400 A wide and 1000 to 1500 A long. The latter structures, chlorobium vesicles, have been isolated in a partly purified state by differential centrifugation of cell-free extracts. They are associated with a centrifugal fraction that has a very high specific chlorophyll content. In all probability, therefore, the chlorobium vesicles are the site of the photosynthetic apparatus of green bacteria.     

OBSERVATIONS ON THE FINE STRUCTURE OF THE MACRONUCLEUS OF TOKOPHRYA INFUSIONUM

The macronucleus in Tokophrya infusionum is composed of numerous Feulgen-positive chromatin bodies (about 0.5 µ in diameter) which appear in thin sections as a dense spongework, homogeneous throughout. The same appearance characterizes metaphase chromosomes of higher forms. Some chromatin bodies of the macronucleus were found to possess a highly organized structure in certain old organisms. This structure appears in cross-sections as a honeycomb and in longitudinal sections as parallel lines about 120 A in diameter evenly spaced (about 230 A). As far as is known this is the first time a regular structure has been found in bodies of chromosomal character at the dimensional level presently explored by electron microscopy. The demonstration that OsO₄ can preserve order in chromatin material is another significant aspect of these findings.     

OBSERVATIONS ON THE FINE STRUCTURE OF SPHEROPLASTS OF RHODOSPIRILLUM RUBRUM

Spheroplasts of the photosynthetic bacterium Rhodospirillum rubrum were prepared from cultures grown in either the presence or absence of light. Cells were converted into spheroplasts by using lysozyme and Versene and fixed in a sucrose-veronal-acetate buffer mixture containing osmium tetroxide. Some preparations were shadow-cast and examined whole; others were embedded in Epon 812 and sectioned. The action of lysozyme and Versene appears to result in removal of the cell wall in strips. The relationship of the chromatophores to the cytoplasmic membrane is readily visualized in sections of broken spheroplasts, and in areas the chromatophores are seen to be continuous with the membrane. In all preparations examined, no definite connections between individual chromatophores were observed. In some cells large spherical granules were evident which either possessed or lacked a clearly visible limiting membrane. On serial sectioning, all granules appeared bounded by a single membrane 40 A wide. The granule membrane was well defined only if the section came from the center of the granule. Sections at other levels showed either a diffuse membrane or no membrane at all. The reasons for this are discussed.     

ELECTRON MICROSCOPE OBSERVATIONS ON THE FUSION OF CHICK MYOBLASTS IN VITRO

The process of myoblast fusion was observed in embryonic chick skeletal muscle cells grown in monolayer cultures at the fine structural level. At the first step, the sarcolemmas of cells destined to fuse are closely applied to each other. They are linked in some places by fasciae adherentes; in other places, engulfment of small processes of one cell by another is seen. At a somewhat more advanced stage of myogenesis, vesicles and tubules are formed between the adjacent cytoplasms; presumably, the apposed membranes have opened at several points and their edges have fused to each other. Finally, remnants of cell membranes (vesicles and tubules) disappear completely, and the confluent cytoplasm is formed. The cytoplasmic contents of the multinucleated cells are often poorly admixed, giving the cytoplasm a mosaic appearance in which different zones can be designated as arising from separate cells. This observation suggests, however, that there is slow diffusion of myoblast contents (ribosomes and, possibly, other materials) into the myotube. In agreement with the previous works at the light microscopic level, the present study suggests the occurence of fusion between mononucleated cells, between mononucleated cells and multinucleated myotubes, and between nascent multinucleated myotubes.     

ELECTRON MICROSCOPE OBSERVATIONS ON FROZEN-DRIED CELLS

To explore the problem of artefacts which may be produced during usual fixation, dehydration, and embedding, the authors have examined pancreas, liver, and bone marrow frozen at about -180°C., dried, at -55 to -60°C., embedded in methacrylate, sectioned, and floated on a formol-alcohol mixture. By these treatments the labile structure of living cells can be fixed promptly and embedded in methacrylate avoiding possible artefacts caused by direct exposure to chemical fixatives. Cell structures are ultimately exposed to a fixative when the sections are floated on formol-alcohol, but at this stage artefacts due to chemical fixation are expected to be minimized, as the fixatives act on structures tightly packed in methacrylate polymer. In the central zone of tissue blocks so treated, the cells are severely damaged by ice crystallization but at the periphery of the blocks the cell structure is well preserved. In such peripherally located cells, elements of the endoplasmic reticulum (ER), Palade's granules, homogeneously dense mitochondria, and nuclear envelopes and pores, can be demonstrated without poststaining with OsO₄. The structural organization in the nucleus is distorted by vacuolization. The mitochondrial membranes and cristae, cellular membrane, and the Golgi apparatus, however, are detected only with difficulty. The Golgi region generally appears as a light zone, in which some ambiguous structures are encountered. After staining the sections with OsO₄ or Giemsa solution, an inner mitochondrial structure which resembles the cristae seen in conventional OsO₄-fixed specimens appears, but the limiting membrane is absent. Treatment with OsO₄ or Giemsa solution also renders more distinct the membrane of the ER and Palade's granules but not the Golgi apparatus and cellular membrane. Treatment with ribonuclease results in the disappearance of Palade's granules. On the strength of these observations the authors conclude that OsO₄ fixation gives a satisfactory preservation of such cell structures as the nuclear envelope, endoplasmic reticulum, and Palade's granules, though it may induce slight swelling of these cell components.     

ELECTRON MICROSCOPE OBSERVATIONS ON THE SURFACE ADENOSINE TRIPHOSPHATASE-LIKE ENZYMES OF HELA CELLS INFECTED WITH HERPES VIRUS

HeLa cells infected with herpes simplex virus have been examined in thin sections by electron microscopy after cytochemical staining for the presence of surface enzymes splitting adenosine triphosphate. As with uninfected HeLa cultures (18), the opaque enzyme reaction product was localized at the plasma membranes of about half the cells, tending to be present where there were microvilli and absent on smooth surfaces. Where mature extracellular herpes particles were found in association with cell membranes showing the enzyme activity, they were invariably likewise stained, and conversely, those mature particles which lay close against cells without reaction product at the surface were themselves free of it. Particles found budding into cytoplasmic vacuoles were also always without opaque deposit since this was never seen at vacuolar membranes, even in cells having the activity at the surface. The enzyme reaction product thus provided a marker indicating the manner in which the particles escape from cells and mature by budding out through cellular membranes, carrying, in the process, a portion of the latter on to themselves to form the outer viral limiting membrane. In some instances, virus particles were observed with more opaque material covering them than was present at the cell membrane with which they were associated. This finding has been taken as evidence for a physiological waxing and waning of surface enzyme activity of adenosine triphosphatase type. The fine structure of the mature extracellular virus as prepared here, using glutaraldehyde fixation, is also recorded. The observations and interpretations are discussed in full.     

SOME OBSERVATIONS ON THE FINE STRUCTURE OF THE SYMPATHETIC GANGLION OF BULLFROG

Electron microscope observations of abdominal sympathetic ganglia of American bullfrogs, Rana catesbiana, have demonstrated the presence of specific areas of cytoplasm in the superficial zone of the perikaryon which are devoid of granulated endoplasmic reticulum. These areas are occupied almost exclusively by granules 200 to 400 A in diameter which can be stained intensely with lead hydroxide but faintly with uranyl acetate. Each granule shows subgranular internal structure after the lead staining. Granules of similar properties are found in synapses also, and may be glycogen. From the satellite cell there extends a number of leaf- or finger-like cytoplasmic projections around the root portion of the nerve process. Some of these projections directly cover the surface of the nerve process. Many others, however, are separated from the neuron by a fairly wide interspace. Multivesicular bodies of the neuron are occasionally observed in a configuration which suggests that they are being extruded from the root of the nerve process into the interspace. Filaments about 100 A in thickness are found in the satellite cell cytoplasm. They are arranged more or less parallel to each other and are especially well developed around synapses and nerve fibers.     

FURTHER OBSERVATIONS ON THE FINE STRUCTURE OF THE PARIETAL EYE OF LIZARDS

An electron microscopical study of the third eye of the Western Fence Lizard, Sceloporus occidentalis, fixed with 1 per cent osmium tetroxide, pH 7.4–7.6, for 16 to 20 hours at 0°C., revealed the following new facts. The fibrillar system of the retinal photoreceptor consists of nine double fibrils enclosed in a sheath. Pigment cells and lens cells possess similar systems. Two short cylindrical centrioles are associated with the fibrillar apparatus: one, from which striated rootlets extend inward, lies at the base of the fibrils, with the other at an oblique angle to the axis of the system. A Golgi complex, whorls of endoplasmic reticulum, lipid (?) droplets, and other organelles and inclusions in the photoreceptors are described. An axon leads from the base of the photoreceptor into the nervous layer of the retina which consists of many nerve fibers and large ganglion cells. Although the pattern of neural connections has not yet been determined, some synapses were found. The parietal nerve consists of about 250 non-medullated fibers. The capsule of the eye usually has a layer of iridocytes, which contain rows of guanine (?) rods. A few parietal eyes of the Granite Night Lizard, Xantusia henshawi, were also examined. Large lipid (?) droplets occur in the bases of their receptoral processes.     

FURTHER OBSERVATIONS ON THE FINE STRUCTURE OF THE MACRONUCLEUS IN TOKOPHRYA INFUSIONUM

In a previous paper (8) an organized structure was described in the macronuclei of certain old organisms of Tokophrya infusionum. It was found that the same honeycomb structure appears in great abundance in the macronuclei of overfed organisms. This permitted a better three-dimensional reconstruction of the described structure. Since the defined structure may be experimentally induced, it offers an opportunity for further more detailed studies as to its nature and meaning.     

OBSERVATIONS ON THE FINE STRUCTURE OF THE DEVELOPING SPERMATID IN THE DOMESTIC CHICKEN

The kinetic apparatus, the acrosome and associated structures, and the manchette of the spermatid of the domestic chicken have been studied with the electron microscope. The basic structural features of the two centrioles do not change during spermiogenesis, but there is a change in orientation and length. The proximal centriole is situated in a groove at the edge of the nucleus and oriented normal to the long axis of the nucleus and at right angles to the elongate distal centriole. The tail filaments appear to originate from the distal centriole. The plasma membrane is invaginated along the tail filaments. A dense structure which appears at the deep reflection of the plasma membrane is identified as the ring. The fine structure of the ring has no resemblance to that of a centriole and there is no evidence that it is derived from or related to the centrioles. The tail of the spermatid contains nine peripheral pairs and one central pair of tubular filaments. The two members of each pair of peripheral filaments differ in density and in shape: one is dense and circular, and the other is light and semilunar in cross-section. The dense filaments have processes. A manchette consisting of fine tubules appears in the cytoplasm of the older spermatid along the nucleus, neck region, and proximal segment of the tail. The acrosome is spherical in young spermatids and becomes crescentic and, finally, U-shaped as spermiogenesis proceeds. A dense granule is observed in the cytoplasm between acrosome and nucleus. This granule later becomes a dense rod which is interpreted as the perforatorium.