Electron microscope studies of Fasciola hepatica. X. Egg formation

Evidence obtained would suggest that the processes involved in egg formation in Fasciola hepatica occur in the following sequence. The egg constituents, namely an ovum and some vitelline cells, pass through the proximal ootype and as they do so they are smeared by the secretions of Mehlis' gland which have accumulated there. A temporary interface is set up between the Mehlis' gland secretion and the fluid which surrounds the egg constituents. Shell globules are released by the vitelline cells and coalesce on the inner aspect of the interface. At the same time some of the Mehlis' secretion diffuses across the interface, thereby bringing about the dissociation of the interface. It is suggested that Mehlis' gland secretion brings about the fusion of the shell layer. During the initial stages of the process the developing egg is supported by the cells of the ootype epithelium. Later the egg passes into a wider, more distal part of the ootype where the process of shell deposition is continued. When complete, or almost complete, the egg passes into the uterus where changes indiciating the initial process of tanning are seen to take place. A thin, very uniform layer of Mehlis' gland secretion can still be identified on the surface of mature eggs.     

Electron microscope studies of Fasciola hepatica. 13. Fine structure of newly excysted juvenile

The gross morphology and fine structure of the newly excysted juvenile fluke are described. The tegument is organized as in the adult, with a spine-containing surface syncytium connected to a perinuclear region lying below the muscle layers. It differs from the adult, however, in having only one type of perinuclear region and one type of secretory body. The digestive system has all the morphological characteristics of a secretory epithelium, rather than an absorptive one. It has numerous dense secretory bodies, ribosomes, and GER, an irregular apical surface due to eccrine secretion, and a lumen filled with a moderately dense material derived from the dispersed secretory granules.The excretory system closely resembles that of the adult, but has, in addition, part of its ascending ducts ciliated and concretions as well as lipid droplets as visible excretory products. The muscle is identical with that of the adult and the parenchyma is also very similar to the adult, although it has more lipid droplets and is associated in a less complex way with cells of other organ systems. Groups of embryonic cells are present and are characterized by a dense, invaginated nucleus surrounded by a very thin layer of cytoplasm.     

Fasciola hepatica: ultrastructure and histochemistry of the glycocalyx of the tegument.

The morphology and histochemistry of the glycocalyx of the tegument was investigated by electron microscopy. The results showed that both the morphology and histochemistry of the glycocalyx varied depending on the environment immediately prior to fixation and also on postfixation treatments. Conventional electron microscope fixation appeared to preserve only about half the total thickness of the glycocalyx, and only histochemical tests applied en bloc gave a true morphological and histochemical picture. The glycocalyx, therefore, consists of two parts, an inner continuous layer which is tightly bound to the apical plasma membrane and is always preserved, and an outer fibrillar layer, which is not always preserved by conventional fixation. Both layers are anionic and carbohydrate-rich and therefore contain glycoproteins and sialic acids. The surface of Fasciola hepatica, therefore, has morphological and chemical features very like those proposed for the “greater membrane” by Lehninger.     

Fasciola hepatica: an electron microscope autoradiographic study of protein synthesis and secretion by gut cells in tissue slices.

A loop technique for electron microscope autoradiographic studies on slices of Fasciola hepatica is described, and used to study the synthesis of protein-containing bodies by gut cells of the fluke. Slices were pulse labeled with tritiated tyrosine, methionine, leucine, and phenylalanine, and, after appropriate chase periods in unlabeled medium, were fixed with formaldehyde and prepared for electron microscopy by a procedure involving ethylene glycol dehydration. Labeled amino acids were found to be incorporated into protein, or glyoprotein, in the gut cells of the slices by a GER-Golgi complex mediated mechanism similar to that which occurs in vertebrate tissues. The precursors appeared to enter the cells across their basal and lateral plasma membranes and mature secretory bodies were discharged at the cell apex.     

Fasciola hepatica: cytochrome c oxidoreductases and effects of oxygen tension and inhibitors

Studies were made on the mechanism of respiration in Fasciola hepatica (Trematoda). Respiration was found to be dependent on the oxygen tension. The respiratory enzyme systems, NADH-cytochrome c oxidoreductase (EC 1.6.2.1), succinate-cytochrome c oxidoreductase (EC 1.3.99.1) NADH oxidase and cytochrome c-oxygen oxidoreductase (EC 1.9.3.1) were detected in a mitochondrial preparation, the NADH oxidase activity being markedly stimulated by addition of mammalian cytochrome c. Amytal and rotenone inhibited NADH oxidase activity. Antimycin A inhibited succinoxidase activity only at relatively high concentrations. Azide was inhibitory at high concentrations. However, cyanide was found to stimulate respiration. Hydrogen peroxide was found to be an end product of respiration in F. hepatica.     

Live Cell Imaging of Early Autophagy Events: Omegasomes and Beyond

Autophagy is a cellular response triggered by the lack of nutrients, especially the absence of amino acids. Autophagy is defined by the formation of double membrane structures, called autophagosomes, that sequester cytoplasm, long-lived proteins and protein aggregates, defective organelles, and even viruses or bacteria. Autophagosomes eventually fuse with lysosomes leading to bulk degradation of their content, with the produced nutrients being recycled back to the cytoplasm. Therefore, autophagy is crucial for cell homeostasis, and dysregulation of autophagy can lead to disease, most notably neurodegeneration, ageing and cancer.Autophagosome formation is a very elaborate process, for which cells have allocated a specific group of proteins, called the core autophagy machinery. The core autophagy machinery is functionally complemented by additional proteins involved in diverse cellular processes, e.g. in membrane trafficking, in mitochondrial and lysosomal biology. Coordination of these proteins for the formation and degradation of autophagosomes constitutes the highly dynamic and sophisticated response of autophagy. Live cell imaging allows one to follow the molecular contribution of each autophagy-related protein down to the level of a single autophagosome formation event and in real time, therefore this technique offers a high temporal and spatial resolution.Here we use a cell line stably expressing GFP-DFCP1, to establish a spatial and temporal context for our analysis. DFCP1 marks omegasomes, which are precursor structures leading to autophagosomes formation. A protein of interest (POI) can be marked with either a red or cyan fluorescent tag. Different organelles, like the ER, mitochondria and lysosomes, are all involved in different steps of autophagosome formation, and can be marked using a specific tracker dye. Time-lapse microscopy of autophagy in this experimental set up, allows information to be extracted about the fourth dimension, i.e. time. Hence we can follow the contribution of the POI to autophagy in space and time.     

Arylsulfatase activity in Gorgoderina attenuata and Haematoloechus medioplexus: cytochemical and biochemical observations on the digestive tracts of digenetic trematodes

Arylsulfatase (EC 3.1.6.1) activity in the gastrodermis of Gorgoderina attenuata and Haematoloechus medioplexus is operative over a pH range of 4.2–7.2. The activity is localized in the cytolysomes and microvesicles, as well as on invaginations of the basal plasmalemma and membranous remnants in the lumen.     

Cytochemical and biochemical observations on the digestive tracts of digenetic trematodes. 8. Acid phosphatase activity in Schistosoma mansoni and Schistosomatium douthitti

At the light microscope level, nonspecific acid phosphatase (AcPase) (EC 3.1.3.2) and N-acetyl glucosaminidase (NAGase) (EC 3.2.1.29) activities are in the esophageal gland cells of Schistosoma mansoni and Schistosomatium douthitti and in the gastrodermis of S. mansoni. The gastrodermis of S. douthitti is negative for these two enzymes. At the electron microscope level, AcPase activity in the esophageal gland cells of both species is observed in cytoplasmic vesicles. In S. mansoni, AcPase activity is also observed associated with the infoldings of the basal plasma membranes of the esophagus and the gastrodermis. It is hypothesized that this enzyme(s) is involved with membrane transport. AcPase activity is also associated with “droplets” and vesicles in the gastrodermis of S. mansoni. It is believed that the digestion of foodstuffs in both species occurs extracellularly.     

Dirofilaria immitis: ultrastructural aspects of oocyte development and zygote formation.

Ultrastructural observations of the ovary and uterus of Dirofilaria immitis reveal some characteristics of oogonia, oocytes, and uterine sperm. Oogonia are confined to the distal portion of the ovary including a blind tip, where a morphologically distinct terminal cap cell was not observed. These cells contain a nucleus with a nucleolus, numerous dense bodies, scanty ribosomes, lipid droplets, and an occasional mitochondrion. Endoplasmic reticulum is lacking and Golgi complexes were observed only in fully grown oogonia. Primary oocytes located in the middle portion of the ovary are large, elongate, and have a complete set of organelles including many small mitochondria, fragmentary endoplasmic reticulum, ribosomes, Golgi complexes, and very few dense bodies. These cells are arranged into many rosettes about central cytoplasmic masses, the rachises, to which they maintain cytoplasmic continuity by pseudopodlike processes. The rachises contain no organelle except a few dense granules and are bound by winding membranes. Oocytes from the proximal portion differ from those of the middle portion of the ovary in their larger size, round shape, absence of many organelles, presence of small dense granules, and lacking a rachis. Dense bodies are specific to the oogonia and exhibit DNase susceptibility and a positive reaction for a mitochondrial enzyme. These findings together with their decreased number and a concomitant increase of mitochondria in the oocytes suggest a relationship between these bodies and mitochondria.Uterine sperm of D. immitis are of the amoeboid type and contain several chromatin masses without a nuclear envelope, many mitochondria, and specialized membranous organelles referred to as mesosomelike vesicles. The vesicles are probably originated from the sperm plasma membrane. Upon fertilization, the entire spermatozoon penetrates the oocyte and its contents are gradually dissolved in the ooplasm with a simultaneous appearance of large numbers of ribosomes at the site of dissolution. Ribosomes were later found in the nucleus. A pronucleus was not observed. These findings are basically in agreement with those described for Ascaris but differ in the morphologic features and number of rachises, presence of dense bodies, absence of refringent granules in the oocytes and the absence of a refringent body and presence of several chromatin masses in the sperm.     

Interaction studies of novel cell selective antimicrobial peptides with model membranes and E. coli ATCC 11775

Cationic antimicrobial peptides (CAMPs) are novel candidates for drug development. Here we describe design of six short and potent CAMPs (SA-1 to SA-6) based on a minimalist template of 12 residues H+HHG+HH+HH+NH2 (where H: hydrophobic amino acid and +: charged hydrophilic amino acid). Designed peptides exhibit good antibacterial activity in micro molar concentration range (1-32 μg/ml) and rapid clearance of Gram-positive and Gram-negative bacterial strains at concentrations higher than MIC. For elucidating mode of action of designed peptides various biophysical studies including CD and Trp fluorescence were performed using model membranes. Further based on activity, selectivity and membrane bound structure; modes of action of Trp rich peptide SA-3 and template based peptide SA-4 were compared. Calcein dye leakage and transmission electron microscopic studies with model membranes exhibited selective membrane active mode of action for peptide SA-3 and SA-4. Extending our work from model membranes to intact E. coli ATCC 11775 in scanning electron micrographs we could visualize different patterns of surface perturbation caused by peptide SA-3 and SA-4. Further at low concentration rapid translocation of FITC-tagged peptide SA-3 into the cytoplasm of E. coli cells without concomitant membrane perturbation indicates involvement of intracellular targeting mechanism as an alternate mode of action as was also evidenced in DNA retardation assay. For peptide SA-4 concentration dependent translocation into the bacterial cytoplasm along with membrane perturbation was observed. Establishment of a non specific membrane lytic mode of action of these peptides makes them suitable candidates for drug development.     

Characterization of growth and Oryctes rhinoceros nudivirus production in attached cultures of the DSIR-HA-1179 coleopteran insect cell line

The DSIR-HA-1179 coleopteran cell line is a susceptible and permissive host to the Oryctesrhinoceros nudivirus (OrNV), which has been used as a biocontrol agent against the coconut rhinoceros beetle (Oryctes rhinoceros); a pest of palms in the Asia-Pacific region. However, little is known about growth and metabolism of this cell line, knowledge of which is necessary to develop an in vitro large-scale OrNV production process. The strong anchorage-dependent characteristics of the cell line, its particular fragility and its tendency to form dense clumps when manipulated, are the most likely reasons that have precluded further development of the cell line. In order to characterize DSIR-HA-1179 cells, there was first a need for a reliable technique to count the cells. A homogenous cell suspension suitable for enumeration could be produced by treatment with TrypLE Express™ with optimum mean time for cell release calculated as 30 min. The cell line was adapted to grow in four serum-supplemented culture media namely TC-100, IPL-41, Sf-900 II and Sf-900 III and cell growth, glucose consumption, lactate and ammonia production were assessed from static-batch cultures. The maximum viable cell density was reached in Sf-900 II (17.9 × 10⁵ cells/ml), with the maximum specific growth rate observed in this culture medium as well (0.0074 h⁻¹). Higher production of OrNV was observed in IPL-41 and TC-100 (4.1 × 10⁷ TCID₅₀/ml) than in cultures infected in Sf-900 III (2.0 × 10⁷ TCID₅₀/ml) and Sf-900 II (1.4 × 10⁷ TCID₅₀/ml). At the end of the growth period, glucose was completely consumed in cultures grown in TC-100, while remained in excess in the other three culture media. The cell line produced lactate and ammonia to very low levels in the TC-100 culture medium which is a promising aspect for its cultivation at large-scale.