Peculiar Epibionts in Euplotidium itoi (Ciliata, Hypotrichida)

ABSTRACT. Euplotidium itoi share with some other species of the same genus a peculiar feature: the presence of a band of particles running along the right and left borders of the cell body and forming a sort of "scarf" at the dorsal anterior end. The ultrastructural analysis, here performed, revealed that these particles (reported in the literature as extrusomes) are always external to the cell and are inserted in matching depressions on the euplotidium cortex. They are present in two different forms: type I, whose ultrastructure recalls that of bacteria, are able to reproduce by binary fission; type II are not able to divide and contain peculiar structures (a granular dome-shaped zone, a complex extrusive apparatus and a network of regularly arranged fibrils) which render them more complicated with respect to the majority of prokaryotic organisms. These observations, together with the finding that these particles contain DNA, indicate that we are dealing with epibionts, that will be referred to as "epixenosomes" (ecto-organisms), rather than extrusomes. Some ideas about the nature of "epixenosomes" and their relationship with the host cell are proposed and discussed.     

纤毛虫大核提取方法上的改进及其扫描电镜观察

以包囊游仆虫(Euplotes encysticus)为材料,对Arikawa等报道的纤毛虫大核提取方法进行了改进,提出了一种更为简便有效的方法,即：用去垢剂Triton X-100处理细胞,结合玻璃微吸管的物理破碎,得到完整的大核后,应用扫描电镜观察。     

鲂属鱼类形态度量学研究

鲂属(Megalobrama)鱼类是鲤科(Cyprinidae)鲌亚科(Cultrinae)中一群分布广的较大型经济鱼类[1],有的种已作为淡水养殖对象,如着名的武昌鱼.多年以来,由于过度捕捞,广泛的移殖和人工繁殖等诸多因素,鲂属鱼类的种质资源正受到衰退和混杂的威胁[2].以中国科学院水生生物研究所淡水鱼类标本馆收藏的标本为基础,对鲂属鱼类的广东妨、厚颌鲂、鲂、团头鲂和鳊的标本进行了测量和分析,希望对鲂属鱼类的种间差异、物种鉴定特征、物种分化过程有较全面的了解,并为鲂属鱼类资源的利用和保护提供参考.     

A preliminary optical and electron microscopic study of the β<Subscript>1</Subscript> integrin distribution pattern of human osteosarcoma-derived cells

Immunogold labelling was used to study the organisation of the ₁ integrins on osteosarcoma-derived osteoblasts (Saos-2 and MG-63). Monolayers of cells were prepared in multiwell culture plates on both uncovered and collagen-covered coverslips, and ₁ integrins were primarily labelled using mouse monoclonal antibodies to ₁ integrins. Indirect immunofluorescence labels using an anti-mouse fluorescein-conjugated goat antibody showed an even distribution of the ₁ integrins on the cell membranes of all cell types used. A concentration of 2 g/ml of the primary antibodies and a 1:100 dilution of the secondary antibodies were determined as the optimal concentration for labelling to use with indirect localisation of the primary antibodies gold conjugated to goat anti-mouse antibodies and viewed under an electron microscope. Ten nanometre gold particles were used for transmission electron microscopy (TEM) and 40 nm gold particles for scanning electron microscopy. TEM showed that ₁ integrins were mainly clustered on the cell membrane processes with less labelling on the cell membranes themselves. The distribution of ₁ integrins on osteosarcoma cells supports the concept that integrins may function by forming focal adhesions at the site of the cytoplasmic membrane processes.     

Aggregation-induced alterations in fibroblast morphology

Summary The different stages during aggregation of diploid human skin fibroblasts have been examined by transmission and scanning electron microscopy. As a result of aggregation, fibroblasts form a complex tissue configuration. Numerous intercellular junctions can be observed, while the cells remain polygonal and do not develop an organised intracellular cytoskeleton. Cell division occurs only rarely. After aggregation, signs of progressive auto-digestion develop.Adhesion to a substrate results in outgrowth of the cells and monolayer formation, even when extensive cell damage had occurred. The morphology of fibroblasts in aggregates and in the monolayers, from which they were derived, is compared and the contribution of the aggregate system to the study of fibroblast behavior is discussed.J.J. Cassiman is Aangesteld Navorser van The National Foundation for Scientific Research, Belgium     

Adhesion forces in the self-recognition of oligosaccharide epitopes of the proteoglycan aggregation factor of the marine sponge <Emphasis Type="Italic">Microciona prolifera</Emphasis>

Cell aggregation in the marine sponge Microciona prolifera is mediated by a multimillion molecular-mass aggregation factor, termed MAF. Earlier investigations revealed that the cell aggregation activity of MAF depends on two functional domains: (i) a Ca²⁺-independent cell-binding domain and (ii) a Ca²⁺-dependent proteoglycan self-interaction domain. Structural analysis of involved carbohydrate fragments of the proteoglycan in the self-association established a sulfated disaccharide β-d-GlcpNAc3S-(1→3)-α-l-Fucp and a pyruvated trisaccharide β-d-Galp4,6(R)Pyr-(1→4)-β-d-GlcpNAc-(1→3)-α-l-Fucp. Recent UV, SPR, and TEM studies, using BSA conjugates and gold nanoparticles of the synthetic sulfated disaccharide, clearly demonstrated self-recognition on the disaccharide level in the presence of Ca²⁺-ions. To determine binding forces of the carbohydrate–carbohydrate interactions for both synthetic MAF oligosaccharides, atomic force microscopy (AFM) studies were carried out. It turned out that, in the presence of Ca²⁺-ions, the force required to separate the tip and sample coated with a self-assembling monolayer of thiol-spacer-containing β-d-GlcpNAc-(1→3)-α-l-Fucp-(1→O)(CH₂)₃S(CH₂)₆S- was found to be quantized in integer multiples of 30 ± 6 pN. No binding was observed between the two monolayers in the absence of Ca²⁺-ions. Cd²⁺-ions could partially induce the self-interaction. In contrast, similar AFM experiments with thiol-spacer-containing β-d-Galp4,6(R)Pyr-(1→4)-β-d-GlcpNAc-(1→3)-α-l-Fucp-(1→O)(CH₂)₃S(CH₂)₆S- did not show a binding in the presence of Ca²⁺-ions. Also TEM experiments of gold nanoparticles coated with the pyruvated trisaccharide could not make visible aggregation in the presence of Ca²⁺-ions. It is suggested that the self-interaction between the sulfated disaccharide fragments is stronger than that between the pyruvated trisaccharide.     

Structural characterization of the hydrocarbon degrading bacteria–oil interface: implications for bioremediation

Hydrocarbon degrading bacteria, enriched from an in situ bioremediation site in Long Valley, AZ emulsified and colonized the surface of waste engine oil. The application of a partial dehydration conventional embedding protocol for ultrathin-section transmission electron microscopy preserved the hydrocarbon degrading bacteria–surfactant–oil interface. Bacterial adsorption to oil occurred in association with a highly charged, amphipathic bacterial surfactant interface (25–50 nm thick). This biosurfactant completely encapsulated the emulsified oil droplets demonstrating that less than 1% surfactant (by volume) is required to emulsify waste hydrocarbon during or to promote biodegradation. Growth on oil appeared to occur by the uptake of tens of nm-sized droplets of emulsified oil.     

A RAPID, HIGHLY EFFICIENT METHOD FOR COLLECTING, FIXING, AND EMBEDDING PLANKTONIC AND OTHER SMALL CELLS FOR ELECTRON MICROSCOPY

A rapid, highly efficient method is described for fixation, dehydration, and embedding of small (e.g. planktonic) cells dispersed in large volumes of culture medium. The entire protocol, based on continuous filtration, can be completed within about an hour, and the yield of cells is very high. Fixation quality has been excellent with several different types of samples.     

Observations on the marginal ruffles of an established fibroblast-like cell line

Summary LW13K2 cells, a clone of a spontaneously in vitro transformed derivative of embryonic Lewis rat fibroblastic cells, were studied by phase contrast cine-light microscopy, scanning electron microscopy (SEM) and transmission electron microscopy (TEM). The ruffles found at the advancing edge of cells grown on glass substrates in vitro form and recede in a period of less than one min if they do not make an attachment of the substrate. If they fail to make an attachment they may form pinocytotic channels near the leading edge as described by Price (1972) and/or collapse, generally backwards, towards the cell body. The spines which appear to reinforce the membranous ruffles are the last structures to disappear, and accumulate in an irregular array behind the ruffling edge; this area is behind that in which pinocytosis occurs. In comparison with the sparse numbers of ribosomes found in the trailing edge, they are present in notable concentrations near the leading, ruffling edge of the cell. No membrane vesicles have been found in or near the ruffling edges at the ruffle-spine concentration zone.     

A Propos D'observations Sur L'ultrastructure Du Cilié Cyrtolophosis Mucicola Stokes, 1885

RESUME L'étude détaillée du cortex et des organelles buccaux de Cyrtolophosis mucicola montre que ce Cilié possède une organisation structural corticale comparable à celle de Woodruffia, Platyophrya, Kuklikophrya et des Colpoda . Tout en restant conforme au plan général d'organisation de ces derniers, il a des variations spécifiques décelées au niveau des organelles buccaux qui confirment la position de C. mucicola dans la famille des Cyrtolophosididae , incluse dans le sous-ordre des PLATYOPHRYINA.     

Determination of bacterial cell number and cell volume by means of flow cytometry, transmission electron microscopy, and epifluorescence microscopy

Comparative measurements of bacterial total counts and volumes of flow cytometry (FCM), transmission electron (TEM), and epifluorescence microscopy (EFM), were undertaken during a four week mesocosm experiment. Total counts of bacteria measured by TEM, EFM, and FCM were in the range of 1 · 10⁶−6 cells ml⁻¹, 1 · 10⁶−3 · 10¹⁶ cells ml⁻¹, and 5 · 10⁵ cells ml⁻¹ respectively. The mean volume of the bacterial community, measured by means of EFM and TEM, increased from 0.12–0.15 μm³ at the start of the experiment to 0.39–0.53 μm³ at the end. Generally, there was good agreement between the two methods and regression analyses gave r = 0.87 (p < < 0.01) for cell volume and r = 0.97 (p < < 0.01) for cell number. DAPI stained bacteria with volumes less than 0.2 μm³ were not detected by flow cytometry and these were generally an order of magnitude lower than counts made by TEM and EFM. For samples where the mean bacterial cell volume was longer than 0.3 μm³, all three methods were in agreement both with respect to counts and volume estimates.     

Lateral connections between heart muscle cells as revealed by conventional and high voltage transmission electron microscopy

Summary The lateral surfaces of heart muscle cells are interconnected by a varied and extensive network of structures that exist in addition to intercalated discs. Ultrastructural images of this network are vastly improved over those from epoxy-embedded material, particularly for low density components, through the application of a method for removing the embedding matrix from thin or thick sections that are then stereoscopically analyzed with standard or high voltage transmission electron microscopy. The connections include cables, 3–20 nm in diameter, multi-strand cables, 10–40 nm-granules, meshlike mats, and sheets, all extensively interwoven. It is suggested that intercellular connections of varying strength and distribution aid in the integration of mechanical performance of the large population of myocytes during the contractile cycle of the heart.This study was supported by a grant from NIH Biotechnology Resources through the University of Colorado High Voltage E.M. Laboratory, NIH Research Grant HL 24336, a N.Y. Heart Association Grant-in-Aid, and NIH Research Career Development Award HL 00568I thank Dr. E.H. Sonnenblick for continual aid and encouragement and Dr. R. Terry, Ms. Y. Kress, and Ms. J. Fant for use of facilities. I also thank Dr. K.R. Porter for guidance in the use of the HVEM technique, Dr. J.J. Wolosewick and Dr. M. Fotino for valuable suggestions, and Ms. J. Fleming, Mr. G. Wray, and Mr. G. Charlie of HVEM staff at Boulder. I acknowledge Dr. F. Pepe for use of facilities, Dr. R. Bloodgood for comments, and Mrs. L. Cohen-Gould, Ms. T. Downey, Mr. F. Reingold, Mrs. T. Maio, and Mrs. R. Shamoon for excellent assistance     

Ultrastructural alterations in ciliary cells exposed to ionizing radiation

Summary Early effects of ionizing radiation were investigated in an experimental in vitro system using the ciliary cells of the tracheal mucous membrane of the rabbit, irradiated at 30° C and at more than 90% humidity. The changes in physiological activities of the ciliary cells caused by irradiation were continuously registered during the irradiation. The specimens were examined immediately after irradiation electron microscopically. The morphological changes in irradiated material after 10–70 Gy are compared with normal material. After 40–70 Gy, scanning electron microscopy revealed the formation of vesicles on cilia, and club-like protrusions and adhesion of their tips. After 30–70 Gy, a swelling of mitochondrial membranes and cristae was apparent transmission electron microscopically. The membrane alterations caused by irradiation are assumed to disturb the permeability and flow of ATP from the mitochondria, which in turn leads to the recorded changes in the activity of the ciliated cells.This investigation was supported by grants from Konung Gustaf V:s Jubileumsfond, John and Augusta Perssons Stiftelse, B. Kamprads Fond, the Faculty of Medicine, University of Lund, Sweden and the Swedish Medical Research Council (No. B77-17X-03897-05)The authors are greatly indebted to Miss Inger Norling, Miss Marianne Palmegren and Miss Birgitta Sandström for their excellent technical assistance     

The demonstration of close nerve-Purkinje fibre contacts in false tendons of sheep heart

Summary An observation of intimate nerve-Purkinje fibre associations in false tendons of sheep heart is reported. Nerve bundles were observed in deep clefts of Purkinje fibres, in channels running between coupled Purkinje cells and embedded within Purkinje cells, as well as in the outer connective tissue sheath. Most nerve terminals in these areas were filled with small clear vesicles and a few large dense-cored vesicles. Only a few axons with many small dense-cored vesicles were observed.Intimate associations (separation, 60 to 90 nm) between the Purkinje cell and nerve varicosity were observed in the deep clefts. Similar close appositions were also present where nerves were embedded in Purkinje cells. In these cases the Purkinje cell enclosing the nerve bundle formed intercellular junctions with its own sarcolemma.Elaborate sarcolemmal folds with multi-vesicular bodies were also frequently observed near nerve bundles and varicosities. The identity of the transmitter is unknown although the nerves forming intimate associations with Purkinje cells have a morphology typical of cholinergic nerves.     

The oligodendrocytic junctional complex

Summary The junctional complex of oligodendrocytes was studied by means of different electron microscopical techniques. This complex is composed of the following junctional membrane formations: 1) tight Junctional domains in the oligodendrocytic membrane near the soma of the cells, 2) fasciae occludentes or focal tight junctions on the outer oligodendrocytic loop of myelin and on the outermost myelin membrane, 3) gap junctions of considerable size variations, either on membranes near the soma or on peripheral oligodendrocytic processes, and 4) non-paranodal transverse bands. The different types of oligodendrocytic junctions are discussed in terms of their functional implications.Supported by a grant from the Deutsche Forschungsgemeinschaft (SFB 114 Bionach) to R. DermietzelThe authors are indebted to Mrs. P. Kalweit and Mr. R. Eichner for technical assistance and Mr. A. Stapper for preparing the drawing. We also thank Mr. U. Malewski and Mr. Khing for their help with the English translation     

Cytomorphological study on human submandibular gland following treatment with secretagogue drugs

Using specimens of human submandibular glands, we have investigated in vitro the morphological modifications induced by clozapine, a dibenzodiazepine derivative that is used in psychotic patients and that provokes hypersalivation, a side-effect of therapy. The effects of the drug, used alone or in combination with carbachol, have been compared with those observed after treatment with drugs acting on specific receptors. To quantify the response to stimulation, we have calculated (with statistical methods) the number of microvilli and microbuds (corresponding to pits seen in images obtained by transmission electron microscopy) per square micrometre of the cytoplasmic surface of the intercellular canaliculi luminal membrane in images obtained by high-resolution scanning electron microscopy. Clozapine, when directly acting on human submandibular specimens, induces a small secretory response in serous cells; this is partially decreased by muscarinic and adrenergic antagonists and by combined incubation with carbachol, thus confirming its behaviour as a partial agonist to muscarinic receptors. We also suggests that the drug acts on the nerve terminals contained within the glandular specimens.This work was funded by MIUR (Italian Ministry for University and Research) and COFIN.     

Studies on the Tintinnida of Enewetak Atoll*

SYNOPSIS. Twenty-six species of Tintinnida were identified in the plankton at Enewetak Atoll. The majority of species in this habitat had hyaline loricae. The agglutinated forms had a high degree of specificity for the types of calcium-containing particles that they incorporated into the loricae. Scanning electron micrographs of loricae are presented for 10 species.     

Cytoskeleton structure,pattern of mitochondrial activity and ultrastructure of frozen or vitrified sheep embryos

Even though sheep embryo cryopreservation is a commonly used procedure the survival and pregnancy outcomes can vary greatly. This study investigated whether cryopreservation was causing subtle changes in ultrastructure, mitochondrial activity or cytoskeletal integrity. Sheep embryos were either slow cooled in 1.5 M EG (n = 22), or vitrified in 20% EG + 20% DMSO with 0.5 M sucrose in Open Pulled Straws (OPS) (n = 24). One hour after warming the cryopreserved embryos differed from control embryos in that they had no mitochondrial activity combined with cytoskeletal disorganization and large vesicles. Vitrified embryos also showed many points of cytoskeleton disruption. Ultrastructural alterations resulting from actin filaments disorganization were observed in both cryopreserved groups. This includes areas presenting no cytoplasmic organelles, Golgi complex located far from the nucleus and a decrease of specialized intercellular junctions. Additionally, large vesicles were observed in vitrified morulae and early blastocysts. The alterations after cryopreservation were proportional to embryo quality as assessed using the stereomicroscope. Even in the absence of mitochondrial activity, grade I and II cryopreserved embryos contained mitochondria with normal ultrastructure. Embryos classified as grade I or II in the stereomicroscope revealed mild ultrastructural alterations, meaning that this tool is efficient to evaluate embryos after cryopreservation.