Nippostrongylus brasiliensis: in vitro feeding activity

Nippostrongylus brasiliensis incorporated the fluorescent dye, Rhodamine B, while feeding in vitro. Uptake of dye in both sexes of helminth was time-dependent with maximal incorporation occurring after 30 min. Feeding activity was elevated as the time of removal from the host increased from 1 to 8 h. Feeding was reduced in 4- or 5-day-old helminths when compared to 7-day-old or older worms. Feeding activity increased also after temperature increases, pH decreases and solute decreases. Feeding was similar under conditions of both light and dark. Feeding by males and females of N. brasiliensis decreased as the density of worms in single- or mixed-sex groups of worms was increased. Females fed significantly more than males under crowded conditions.     

Nippostrongylus brasiliensis: feeding activity in the mouse

Nippostrongylus brasiliensis incorporated the fluorescent dye, Rhodamine B, while feeding in vivo. Uptake of dye in both sexes of helminth increased linearly from 30 to 120 min after the hosts were given dye per os. Feeding by female N. brasiliensis significantly exceeded that of the male at 4 and 5 days postinfection. Feeding declined in older helminths of both sexes. The density of helminths had no effect on their incorporation of dye in vivo. Feeding in male- and female-only groups of worms was similar to that seen in populations of mixed sexes. Feeding by helminths decreased during the first 36 h of food deprivation in the host, but increased during subsequent fasting of the host. Both sexes of N. brasiliensis resumed feeding within 15 min after the fasted hosts were fed. Growth of N. brasiliensis increased linearly from 4 to 7 days postinfection, based on dry weight. Seven-day-old and older females were significantly heavier than males.     

Nippostrongylus brasiliensis: factors influencing movement of males toward a female pheromone

Single, responding males of Nippostrongylus brasiliensis exhibited a dose-dependent movement toward a pheromone source that was derived from incubation or homogenization of female helminths in Tyrode's solution. Increases in female homogenate beyond an optimum concentration resulted in reduced male movement toward the pheromone source. Crowding of male N. brasiliensis prior to their exposure to female pheromone gradients diminished the males' response. However, crowding of females had no effect on response to the pheromone. The locomotory responses of males exposed to various mixed male and female pheromone sources reaffirmed earlier suggestions of a pheromone feedback system in which males influence the pheromone released from females.     

Survival of Nippostrongylus brasiliensis larvae after freezing over liquid nitrogen

Survival of Nippostrongylus brasiliensis larvae after freezing over liquid nitrogen. International Journal for Parasitology4: 173–176. Third stage larvae of N. brasiliensis were frozen over liquid nitrogen and after storage for 7 days were thawed rapidly and inoculated subcutaneously into rats. When ensheathed larvae were used, none survived freezing as judged by motility and infectivity trials. Separate vials of exsheathed larvae survived freezing in proportions ranging from 10 to 64 per cent. Female worms, derived from frozen exsheathed larvae, had a normal complement of eggs in the uterus and both male and female worms had a normal histological appearance. Exsheathed larvae frozen in the presence of 10 per cent dimethylsulphoxide had the same survival rate as those frozen without the addition of cryoprotectant. The addition of 10 per cent glycerol adversely effected the survival of frozen exsheathed larvae.     

Nippostrongylus brasiliensis: reversibility of reduced-energy status associated with the course of expulsion from the small intestine in rats

Gastrointestinal nematodes require energy for active establishment in the gut against intestinal flow and peristaltic motion. In this study we employed CellTiter-Glo Luminescent Cell Viability Assay to measure the ATP value of individual adult Nippostrongylus brasiliensis during the course of immune-mediated expulsion from the small intestine in rats. The ATP values of adult worms taken from the lumen of the distal small intestine were lower than worms collected from the lumen of the proximal small intestine. Moreover, values from worms in the lumen of the proximal small intestine were lower than those from worms in the mucosa, the preferred site of adult N. brasiliensis. The reduction of ATP values in worms from each region was observed not only at expulsion phase, but also at established phases of the infection suggesting that energy metabolism of the parasites is independent of host immune response. When adult worms with low ATP values on day 12 post-infection were implanted surgically into the small intestine of na?ve rats, the worms re-established in recipients and completely restored the ATP values. Short in vitro culture of adult worms under low oxygen tension resulted in low ATP value in the worms. These results suggested that adult worms were dislodged from their preferred site by intact energy metabolism activity.     

Molecular interplay of autophagy and endocytosis in human health and diseases

Autophagy, an evolutionarily conserved process for maintaining the physio‐metabolic equilibrium of cells, shares many common effector proteins with endocytosis. For example, tethering proteins involved in fusion like Ras‐like GTPases (Rabs), soluble N‐ethylmaleimide sensitive factor attachment protein receptors (SNAREs), lysosomal‐associated membrane protein (LAMP), and endosomal sorting complex required for transport (ESCRT) have a dual role in endocytosis and autophagy, and the trafficking routes of these processes converge at lysosomes. These common effectors indicate an association between budding and fusion of membrane‐bound vesicles that may have a substantial role in autophagic lysosome reformation, by sensing cellular stress levels. Therefore, autophagy–endocytosis crosstalk may be significant and implicates a novel endocytic regulatory pathway of autophagy. Moreover, endocytosis has a pivotal role in the intake of signalling molecules, which in turn activates cascades that can result in pathophysiological conditions. This review discusses the basic mechanisms of this crosstalk and its implications in order to identify potential novel therapeutic targets for various human diseases.     

Nippostrongylus brasiliensis: local humoral responses in the lungs and intestines of rats following vaccination with irradiated larvae

Groups of rats were infected with 2000 normal larvae of Nippostrongylus brasiliensis or larvae irradiated with 10 to 120 kR. On Day 10 after infection half the animals from each group were autopsied. The remainder were challenged with 5000 unirradiated larvae on Day 15 and killed ten days later. During the experiment enteric antibody levels were estimated by coproantibody measurement. At autopsy the worm burdens were determined and worm-specific antibodies evaluated in lung extracts and serum. It was found that the levels of coproantibody detected with adult worm metabolites were positively correlated with the number of adult nematodes recovered from the intestine after primary infection. The challenge induced a similar increase of these antibodies in all immunised rats which reflected a high immunity to reinfection of vaccinated animals. Preliminary immunochemical studies suggested that the coproantibodies had SIgA properties. In lung extracts of rats immunised with larvae irradiated at 40, 80, or 120 kR and in all animals after challenge, antibodies reacting with infective larval antigens were found. Their titres were negatively correlated with serum antibody levels. The significance of bronchial and enteric antibodies in conferring protection against challenge remains to be elucidated.     

Ultrastructural analysis of nematocyte removal in Hydra

A consecutive series of ultrathin sections through the distal one-third of a Hydra tentacle has revealed at least four categories of nematocytes: (1) normal, mounted nematocytes, in specific arrangements within the battery cells; (2) degenerating nematocytes, within the battery cells; (3) mature nematocytes, enclosed within endodermal cells; (4) a mature nematocyte, in the enteric cavity. The degenerating nematocytes within the battery cells and the nematocytes in the endoderm and enteric cavity appeared to be aging nematocytes undergoing death and removal. The results provide the first ultrastructural evidence for nematocyte degeneration within battery cells and also suggest phagocytosis of mature nematocytes by endodermal cells.     

The phospholipase B content of the intestines of rats infected with varied larvae doses of Nippostrongylus brasiliensis.

Phospholipase B activity, bone marrow eosinophilia and worm burden were measured in separate groups of rats that received initial infections of 100, 400, 800, 1200, 2400, and 4800 Nippostrongylus brasiliensis larvae. The results indicated that as the numbers of larvae in the inocula were increased, the resulting higher worm burdens caused the development of earlier and more intense bone marrow eosinophilias and earlier and greater phospholipase B activities. The expulsion of the adult worms from the host was followed by equally rapid decreases in bone marrow eosinophilia and enzyme activity. It was found that as the number of intestinal adult worms was increased from low to large numbers, the degree of the inflammatory response changed from mild to severe. This suggests that the inflammatory response was responsible for the elevated phospholipase B levels.     

Ultrastructural localization of calcium, by electron-probe X-ray microanalysis, in the small intestine of suckling rats

The subcellular distribution of calcium has been investigated in samples, from the intestinal mucosa of 10-day rats, prepared for X-ray microanalysis by various techniques designed to minimize the loss of this element. Calcium retention and its threshold of detection was most satisfactory in freeze-dried frozen thin sections. In resin-embedded samples the best retention of calcium was found in specimens fixed in absolute ethanol, embedded without osmication, and sectioned onto glycerol. The results of this investigation indicate the presence of calcium in the supranuclear vacuole of enterocytes in the distal intestine of the neonatal rat. This calcium is probably taken up during the endocytosis of material from the intestinal lumen. The same mechanism may also be important in the uptake of other metals by suckling animals.     

The phospholipase B content of the intestines of sensitized rats challenged with varied larvae doses of Nippostrongylus brasiliensis.

The effects of challenging previously infected (sensitized) groups of rats with 25, 100 and 400 Nippostrongylus brasiliensis larvae on the relation between phospholipase B activity, bone marrow eosinophilia and worm burden were studied. The results indicated that the immune expulsion of worms from sensitized rats was temporally related to an increased phospholipase B activity and bone marrow eosinophilia. All three parameters demonstrated an anamnestic-type of response in the previously infected rats when compared to the infected control animals. The results also demonstrated that the size of the worm burden influenced the induction and concentration of phospholipase B and the numbers of bone marrow eosinophilis.     

The first stage larva of brugia pahangi in Aedes togoi: An ultrastructural study

Lehane M. J. 1978. The first stage larva of Brugi pahangi in Aedes togoi: an ultrastructural study. International Journal for Parasitology8: 207–218. The ultrastructure of the first stage larva in the mosquito is described up to the onset of the first cuticular moult. The following structural changes from the microfilarial stage have been noted at this time; the numbers of muscle cells have increased, usually to four with a maximum of six in each intercordai quadrant ; part of the pharyngeal thread has formed into a knot ; the intestinal lumen has developed and is surrounded in any given transverse section by up to four intestinal cells and the inner body has largely been lost; the anal apparatus has enlarged and developed; the excretory apparatus has largely degenerated. The functional morphology of the various organ systems described is discussed.     

Ultrastructural characterization of the delimiting membranes of isolated autophagosomes and amphisomes by freeze-fracture electron microscopy

The delimiting membranes of isolated autophagosomes from rat liver had extremely few transmembrane proteins, as indicated by the paucity of intramembrane particles in freeze-fracture images (about 20 particles/microm2, whereas isolated lysosomes had about 2000 particles/microm2). The autophagosomes also appeared to lack peripheral surface membrane proteins, since attempts to surface-biotinylate intact autophagosomes only yielded biotinylation of proteins from contaminating damaged mitochondria. All the membrane layers of multilamellar autophagosomes were equally particle-poor; the same was true of the autophagosome-forming, sequestering membrane complexes (phagophores). Isolated amphisomes (vacuoles formed by fusion between autophagosomes and endosomes) had more intramembrane particles than the autophagosomes (about 90 particles/microm2), and freeze-fracture images of these organelles frequently showed particle-rich endosomes fusing with particle-poor or particle-free autophagosomes. The appearence of multiple particle clusters suggested that a single autophagic vacuole could undergo multiple fusions with endosomes. Only the outermost membrane of bi- or multilamellar autophagic vacuoles appeared to engage in such fusions.     

Postinsemination changes in the amoeboid sperm of a nematode,nippostrongylus brasiliensis

Nematode sperm undergo a capacitationlike change called activation, after insemination into the female reproductive tract. In this paper we examine the morphological and functional consequences of activation of the sperm of Nippostrongylus brasiliensis by comparing the ultrastructure of sperm in the reproductive tracts of males and females and by analysing locomotion using time-lapse cinematography. Sperm in males consist of a long tail, composed of highly condensed chromatin, and a cytoplasmic region containing numbrane-bound secretory products of the Golgi called membranous organelles (MOs) as well as mitochondria. Following activation, the MOs fuse with the plasma membrane, release their electron-dense contents, and remain connected to the outside by a narrow channel. A small region at the anterior of sperm from males is amoeboid and can produce small pseudopodia. In sperm from the uterus this region enlarges considerably, the groundplasm changes from a granular to a filamentous from and the sperm become fully motile. During locomotion a cycle of events occurs at the leading edge beginning with the extension of a small pseudopodium. A constriction ring forms across its base and as the sperm progresses the constriction ring moves back along the cell, but remains stationary relative to the substrate. The location of new pseudopodia at the leading edge dictates the direction of movement of the spermatozoon. Since Cytochalasin B had no effect on the translocation of sperm, the polymerization of actin appears not to play a part in locomotion.     

Increased permeability of gut mucosa in rats infected with Nippostrongylus brasiliensis.

The permeability of the gut mucosa to macromolecules has been examined in 4 different strains of rats infected with Nippostrongylus brasiliensis (N. brasiliensis). There is a marked increase in mucosal permeability during the infection. In addition, a considerable strain difference is observed in both worm burden kinetics and the kinetics of intestinal permeability. The dose-response and drug treatment experiments in outbred Wistar rats suggest that increased mueosal permeability is a function of the worm burden. However, the increased permeability is related neither to the rapid phase of worm expulsion nor to the rise in intestinal mast cell numbers.     

Maturation of Autophagic Vacuoles in Mammalian Cells

The autophagic process was first described in mammalian cells several decades ago. After their formation as double-membraned vacuoles containing cytoplasmic material, autophagic vacuoles or autophagosomes undergo a stepwise maturation including fusion with both endosomal and lysosomal vesicles. However, the molecular mechanisms regulating these fusion steps have begun to emerge only recently. The list of newly discovered molecules that regulate the maturation of autophagosomes to degradative autolysosomes includes the AAA ATPase SKD1, the small GTP binding protein Rab7, and possibly also the Alzheimer-linked presenilin 1. This review combines previous data on the endo/lysosomal fusion steps during autophagic vacuole maturation with recent findings on the molecules regulating these fusion steps. Interestingly, autophagic vacuole maturation appears to be blocked in certain human diseases including neuronal ceroid lipofuscinosis and Danon disease. This suggests that autophagy has important housekeeping or protective functions, because a block in autophagic maturation causes a disease.     

Comparative conformational analysis of cholesterol and ergosterol by molecular mechanics

A comparative conformational analysis of cholesterol and ergosterol has been carried out using molecular mechanics methods. These studies are aimed at giving a better understanding of the molecular nature of the interaction of these sterols with polyene macrolide antibiotics. Structures of cholesterol and ergosterol determined by X-ray methods have been used as initial geometries of these molecules for force field calculations. The calculation of steric energy has also been made for conformations which do not appear in the crystal. The latter conformers have different conformations of the side chain as well as different conformations of rings A and D. The rotational barriers around bonds C17–C20 and C20–C22 have also been calculated. The results obtained on differences and similarities in the conformations of cholesterol and ergosterol allow us to postulate a mechanism for differential interaction with the antibiotics. The relatively rigid side chain of ergosterol (stretched molecule) in comparison with the flexible side chain of cholesterol (bent molecule), allows better intermolecular contact of the first sterol molecule with a polyene macrolide and in consequence facilitates complex formation involving Van der Waal's forces.