NEURONAL-GLIAL MEMBRANE CONTACTS DURING PESSIMAL ELECTRICAL STIMULATION

After the creation of a method for obtaining inter-neuronal gap junctions in a nervous system devoid of glia, it is expedient to reproduce gap neuronal-glial contacts on a model that also contains hybrid neuronal-glial gap junctions, which, as you know, are functionally fundamentally different from inter-neuronal contacts. The experiments were carried out on the truncus sympathicus ganglia of laboratory rats using pessimal electrical stimulation and transmission electron microscopy. Electrical activation of ganglia with a frequency of up to 100 Hz revealed local and widespread variants of various neuronal-glial connections (contacts, bridges), fringed with peri-membrane filamentous proteins. They had a blurred veil that masked two-layer neuro-membranes. Some of the contacts resembled slit or dense 5-layer structures without a visible inter-neuronal slit, but with an extreme decrease in the thickness of the contact slit. The main result of the experiments was the formation, in addition to slotted, multiple septate (ladder) contacts. Relatively independent aggregates of the electron-dense substance of the septa were located inside the intercellular gaps, crossing both adjacent membranes, and, possibly, permeate of them. Near-membrane, poorly outlined pyramid-like protein cones associated with both cell membranes were also formed. Such membranes appeared to be dotted-dashed, that is, not continuous. A significant number of septic contact membranes had endocytic invaginations (invaginations) facing neuroplasm with pyramid-like marginal projections. All reactive altered structures that have arisen de novo are considered by the authors as developed under the influence of frequency electrical stimulation of denaturation and aggregation of intrinsic and perimembrane proteins.

Sperm ultrastructure of Pochazia shantungensis (Chou & Lu) and Ricania speculum (Walker) (Hemiptera, Ricaniidae) with phylogenetic implications

The sperm ultrastructure of two ricaniid species, Pochazia shantungensis (Chou & Lu) and Ricania speculum (Walker), was investigated using light and transmission electron microscopy. Both species have monoflagellate sperm, the shape and ultrastructure of the mature spermatozoon of these two species are similar in morphology, and 128 spermatozoa are organized into sperm bundles with their heads embedded in a homogenous matrix forming the spermatodesmata. The individual sperm is filiform and includes the head, neck and flagellum. The head is needle-like, with a bilayer acrosome and an inferior elongated nucleus which is formed of homogeneously compact and electron-dense chromatin. The neck region is indistinct and is comprised of the centriole and centriole adjunct with a homogeneous dense substance. The long flagellum has the typical 9 + 9 + 2 axoneme microtubule pattern and two symmetrical mitochondrial derivatives with an orderly array of cristae flanking both sides, and a pair of well-developed fishhook-shaped accessory bodies. Current evidence shows that ricaniid species have D-shaped mitochondrial derivatives in cross-section and a serrated electron-dense region. The phylogenetic relationship of Fulgoroidea with other superfamilies in Auchenorrhyncha is briefly discussed.

Effect of kinetin and chloramphenicol on chlorophyll synthesis and chloroplast development in detached lupin cotyledons under low light intensity

Chlorophyll synthesis in detached lupin cotyledons under low light intensity was stimulated by kinetin at 20 mg/l and inhibited by chloramphenicol at 50 mg/1. Kinetin not only conteracted the inhibitory effect of chloramphenicol, but stimulated1 the chlorophyll syntesis to a greater level than in the control material. Kinetin accelerated the starch degradation and the development of chloroplast; its prolonged, action, however, produced some abnormalities, such as an excessive growth of plastids resulting in some cases in bursting of their envelopes, the formation and release f r om plastids of numerous membrane - bound bodies and the accumulation in released and swollen thylakoids of electron - dense substance. In the presence of chloramphenicol, some disturbances in structure of the stroma thylakoids and the appearance of vesicular structures in the stroma and the enlargement of grana and swelling of their thylakoids were observed. Kinetin prevented some of these abnormalities.

ULTRASTRUCTURAL FEATURES OF THE BASAL DINOFLAGELLATE Ellobiopsis chattoni (ELLOBIOPSIDAE, ALVEOLATA), PARASITE OF COPEPODS

Ellobiopsis chattoni is the type species of the ellobiopsids, an enigmatic lineage of parasitic alveolates that branched between the syndinean dinoflagellates and the perkinsids. We have investigated the ultrastructure of four trophonts from three calanoid copepod hosts collected from the port of Valencia, northwestern Mediterranean Sea. The cell wall showed a thick and homogenous layer and flask-shaped mucocysts that excreted an electron-dense substance that forms the outer layer. The cell wall in the attachment peduncle of Ellobiopsis was thicker and with numerous invaginations. The inner section showed numerous longitudinal channels here interpreted as conduits for the transport of host fluids. Trophomere and gonomere were separated by a thin septum with a central pore. Before the mature gonomere detached from the trophomere, the area of junction became undulated. Deficiencies in the fixation of the membrane organelle preclude discussing on other ultrastructural features. To date the ultrastructure of three ellobiopsid genera have been examined. The trophonts of Ellobiopsis and Thalassomyces showed a high similarity in the cell wall, with characteristic flaskshaped mucocysts. The lack of flask-shaped mucocysts in Ellobiocystis and other morphological and ecological differences argue against the monophyly of the ellobiopsids. Caracteres ultrastucturales del dinoflagelado basal Ellobiopsis chattoni (Ellobiopsidae, Alveolata), un parásito de copépodos Ellobiopsis chattoni es la especie tipo de los ellobiópsidos, un enigmático linaje de alveolados parásitos que se sitúa entre los dinoflagelados Syndiniales y los perkinsoides. Hemos examinado la ultraestructura de cuatro trofontes que parasitaban tres copépodos calanoides procedentes del puerto de Valencia, Mediterráneo noroccidental. La pared celular presenta una capa gruesa y homogénea con mucocistos con forma de matraz que excretan una substancia electro-densa que forma la capa externa. El pedúnculo de adhesión de Ellobiopsis presenta una pared celular más ancha y con numerosas invaginaciones. El pedúnculo en su sección interna muestra numerosos canales longitudinales cuya función se ha interpretado como conductos para el transporte de los fluidos del hospedador. El trofonte y el gonómero están separados por un fino septo con un poro central. Esa región de unión es undulada cuando el gonomero maduro se separe del trofonte. Otros caracteres ultrastructurales no pueden ser descritos debido a deficiencias en la fijación de las membranas de los orgánulos. Hasta ahora se ha examinado la ultrastructura de tres géneros de ellobiopsidos. Los trofontes de Ellobiopsis y Thalassomyces muestran una gran similitud en su pared celular que presenta el mismo tipo de mucocistos. En contraste, la falta de mucocistos con forma de matraz en Ellobiocystis, además de otras diferencias morfológicas y ecológicas, pone en duda el supuesto origen monofilético de los ellobiópsidos.

Herpes Simplex Virus 1 Envelopment Follows Two Diverse Pathways

ABSTRACT Herpesvirus envelopment is assumed to follow an uneconomical pathway including primary envelopment at the inner nuclear membrane, de-envelopment at the outer nuclear membrane, and reenvelopment at the trans-Golgi network. In contrast to the hypothesis of de-envelopment by fusion of the primary envelope with the outer nuclear membrane, virions were demonstrated to be transported from the perinuclear space to rough endoplasmic reticulum (RER) cisternae. Here we show by high-resolution microscopy that herpes simplex virus 1 envelopment follows two diverse pathways. First, nuclear envelopment includes budding of capsids at the inner nuclear membrane into the perinuclear space whereby tegument and a thick electron dense envelope are acquired. The substance responsible for the dense envelope is speculated to enable intraluminal transportation of virions via RER into Golgi cisternae. Within Golgi cisternae, virions are packaged into transport vacuoles containing one or several virions. Second, for cytoplasmic envelopment, capsids gain direct access from the nucleus to the cytoplasm via impaired nuclear pores. Cytoplasmic capsids could bud at the outer nuclear membrane, at membranes of RER, Golgi cisternae, and large vacuoles, and at banana-shaped membranous entities that were found to continue into Golgi membranes. Envelopes originating by budding at the outer nuclear membrane and RER membrane also acquire a dense substance. Budding at Golgi stacks, designated wrapping, results in single virions within small vacuoles that contain electron-dense substances between envelope and vacuolar membranes.

Localization of phycoerythrin at the lumenal surface of the thylakoid membrane in Rhodomonas lens.

The thylakoids of cryptomonads are unique in that their lumens are filled with an electron-dense substance postulated to be phycobiliprotein. In this study, we used an antiserum against phycoerythrin (PE) 545 of Rhodomonas lens (gift of R. MacColl, New York State Department of Health, Albany, NY) and protein A-gold immunoelectron microscopy to localize this light-harvesting protein in cryptomonad cells. In sections of whole cells of R. lens labeled with anti-PE 545, the gold particles were not uniformly distributed over the dense thylakoid lumens as expected, but instead were preferentially localized either over or adjacent to the thylakoid membranes. A similar pattern of labeling was observed in cell sections labeled with two different antisera against PE 566 from Cryptomonas ovata. To determine whether PE is localized on the outer or inner side of the membrane, chloroplast fragments were isolated from cells fixed in dilute glutaraldehyde and labeled in vitro with anti-PE 545 followed by protein A-small gold. These thylakoid preparations were then fixed in glutaraldehyde followed by osmium tetroxide, embedded in Spurr, and sections were labeled with anti-PE 545 followed by protein A-large gold. Small gold particles were found only at the broken edges of the thylakoids, associated with the dense material on the lumenal surface of the membrane, whereas large gold particles were distributed along the entire length of the thylakoid membrane. We conclude that PE is located inside the thylakoids of R. lens in close association with the lumenal surface of the thylakoid membrane.

Further studies of the secretory pathway in thrombin-stimulated human platelets

The pathway followed by secretory products stored in platelet alpha granules during the release reaction remains controversial. Tannic acid has been used in the present study as an electron-dense stain to follow the secretory process in thrombin-stimulated platelets. Preliminary experiments demonstrated that tannic acid precipitates fibrinogen, and binds osmium tetroxide to fibrinogen and fibrin strands. Examination of platelets fixed at short intervals after exposure to thrombin and incubated in solutions containing tannic acid revealed electron-dense deposits of osmium not apparent in resting platelets. Granules and lumina of channels making up the open canalicular system (OCS) were unstained in discoid cells. However, exposure to thrombin at concentrations of 1 to 5 U/mL for thirty seconds or more resulted in intense staining of alpha granules by osmium. Some granules communicated directly with dilated channels of the OCS, and several were frequently connected to the same canaliculus. The electron-dense substance in swollen granules and channels appeared to be in the process of extrusion through narrow or dilated openings of the OCS onto the platelet surface. Granule-to-granule fusion and formation of sealed vacuoles of fused granule products unstained by tannic acid-osmium were not observed. The findings support the concept that secretion by stimulated human platelets results from development of direct communications between granules and channels of the OCS and subsequent extrusion of products through channel pores to the surrounding medium.

Further studies of the secretory pathway in thrombin-stimulated human platelets

The pathway followed by secretory products stored in platelet alpha granules during the release reaction remains controversial. Tannic acid has been used in the present study as an electron-dense stain to follow the secretory process in thrombin-stimulated platelets. Preliminary experiments demonstrated that tannic acid precipitates fibrinogen, and binds osmium tetroxide to fibrinogen and fibrin strands. Examination of platelets fixed at short intervals after exposure to thrombin and incubated in solutions containing tannic acid revealed electron-dense deposits of osmium not apparent in resting platelets. Granules and lumina of channels making up the open canalicular system (OCS) were unstained in discoid cells. However, exposure to thrombin at concentrations of 1 to 5 U/mL for thirty seconds or more resulted in intense staining of alpha granules by osmium. Some granules communicated directly with dilated channels of the OCS, and several were frequently connected to the same canaliculus. The electron-dense substance in swollen granules and channels appeared to be in the process of extrusion through narrow or dilated openings of the OCS onto the platelet surface. Granule-to-granule fusion and formation of sealed vacuoles of fused granule products unstained by tannic acid-osmium were not observed. The findings support the concept that secretion by stimulated human platelets results from development of direct communications between granules and channels of the OCS and subsequent extrusion of products through channel pores to the surrounding medium.

Intracellular infection of aposymbiotic Hydra viridis by a foreign free-living Chlorella sp.: initiation of a stable symbiosis

An aposymbiotic strain of Hydra viridis became infected with free-living Chlorella sp. A stable symbiosis formed that differed in its characteristics from other known Chlorella/Hydra symbioses. The algae reproduced and formed clusters in host endodermal cells, inside large vacuoles filled with an electron-dense substance. A few algae were found to be digested by the hydra, but the apparently uncontrolled reproduction rate of the algae more than compensated for this loss. Surplus algae were expelled into the coelenteron and eventually into the surrounding medium. The expelled algae were repeatedly re-engulfed by the hydra during its feeding, forming a process of continuous reinfection. We suggest that such repeated reinfection of the hydra by the expelled algae provides the host with an endless number of Chlorella from which it might in time select a suitable adapted, controllable symbiont. The present newly formed symbiosis might serve as a model for the study of evolution of algal endosymbioses.