Fasciola hepatica and Schistosoma mansoni: immunofluorescent antigen localization and cross-reactivity

In an attempt to identify the tissue sources of biochemically purified antigenic fractions of Fasciola hepatica and Schistosoma mansoni, antisera were tested against plastic-embedded sections of worms of various ages by an indirect fluorescent-antibody-labeling technique. Antibodies prepared against antigens purified by chromatography of F. hepatica whole worm extract through concanavalin A-Sepharose 4B labeled the parenchyma and tegument of adult F. hepatica strongly while antibodies developed against antigens purified by antibody-affinity chromatography against antibodies of S. mansoni labeled only the parenchyma. Antigens common to these two groups clearly originated from F. hepatica parenchyma. Certain of these common antigens are known to provide significant protection in mice to challenge with S. mansoni cercariae, and in the present study antisera against F. hepatica extracts cross-labeled S. mansoni adult male parenchyma. Reciprocal cross-reactions between antisera against S. mansoni and the parenchyma of adult F. hepatica were also noted. FhFIIb, an extract of F. hepatica which Tailliez described as not cross-reacting with S. mansoni, was found to contain no F. hepatica parenchymal antigens. Antigenic fractions of F. hepatica and S. mansoni collected from the surface of worms after incubation in nonionic detergent were unexpectedly found to contain much parenchymal antigen, suggesting leakage of internal components into the supernatant during preparation. Antisera to F. hepatica developed during a natural infection in rabbits labeled tegumental components and gut strongly but did not react with parenchymal tissue. Antisera against extracts of adult schistosomes labeled the parenchyma of male worms and the glycocalyx of the cercarial tegument, indicating the presence of common antigens in the adult and the cercarial stage. Reciprocal reactions between anticercarial sera and adult sections provided further evidence of shared antigenicity. Antisera against S. mansoni egg antigens strongly labeled sections of eggs in liver tissue and cross-reacted with cercarial glycocalyx, indicating the existence of common antigens between these two stages. The antisera also cross-reacted with what appeared to be non-membrane-bound protein in the tegument of F. hepatica. The soluble egg antigen extract shared antigenicity with the parenchyma of both S. mansoni and F. hepatica but circumoval precipitin had no cross-reactivity with this tissue. Thus S. mansoni eggs contain nondiffusable components sharing antigenic specificity with adult parenchymal tissue.(ABSTRACT TRUNCATED AT 400 WORDS)     

Golgi-mediated vicilin accumulation in pea cotyledon cells is re-directed by monensin and nigericin

Summary A range of drugs was applied to developing pea seed cotyledons in an attempt to perturb the intracellular transport of newly synthesized vicilin through the endoplasmic reticulum and Golgi vesicles to its site of storage, the vacuole. The most pronounced effects, produced by the ionophores monensin and nigericin, were on Golgi-mediated transport. Unlike the situation in most other tissues that have been studied the number of Golgi vesicles did not increase, suggesting that their movement is not slowed or stopped. However, the Golgi-mediated transport of vicilin was redirected from the vacuole tonoplast to the plasmalemma and the newly synthesized vicilin was released from the cotyledon cells to accumulate between the plasmalemma and the cell wall.     

Preparation and discharge of secretion in the subcommissural organ of the rat

Summary The secretion of the subcommissural organ (SCO) of the rat was studied by means of immunocytochemistry at the electron-microscopic level with the use of (1) the polar embedding medium Lowicryl K4M at -30° C, (2) the protein A-gold technique, and (3) a rabbit antiserum against bovine Reissner's fiber (see Sterba et al. 1981).Two different substructures of the ependymal and the hypendymal SCO-cells display a positive immunocytochemical reaction: (1) sacs containing flocculent secretion, which originate from the granular endoplasmic reticulum, and (2) vacuoles filled with fine granular secretion, which are pinched off from the Golgi apparatus. The secretory material of the sacs and the vacuoles is discharged both (i) apically into the cerebrospinal fluid and (ii) basally into intercellular spaces of the SCO-hypendyma. The apically released secretion is condensed to a lamina-like formation, which more caudally assumes the form of Reissner's fiber. The route of the basally released secretion remains, however, vague. The periodically striated bodies, which were thought to be morphological mediators of the discharge of the secretion into the capillaries, are never labeled by gold particles.Supported by grants from the Ministry for Science and Technology of the German Democratic RepublicThe expert technical assistance of Mrs. B. Wolff, Mrs. S. Mehnert, Mrs. E. Siebert, Mrs. Ch. Schneider, and Mrs. I. Seifert is gratefully acknowledged     

Distribution of myosin and the glial fibrillary acidic protein (GFA Protein) in rat spinal cord and in the human frontal cortex as revealed by immunofluorescence microscopy

Summary The glial fibrillary acidic (GFA) protein and myosin were localized in rat spinal cord and human frontal cortex using specific antibodies against GFA protein from human spinal cord and highly purified smooth myosin from chicken gizzard by means of an indirect immunofluorescence microscopical approach. A strong GFA protein and myosin immunoreactivity was found in astrocytes of the white and grey matter and in the external glial limitans membrane. The very fine branches of astrocytic processes stained with antiGFA protein, but not with anti-myosin. Similar results were obtained with the human frontal cortex, where myosin antibodies failed to reveal the very fine branches of protoplasmic astrocytes.As a whole, staining with the GFA protein antiserum was more crisp than with the myosin antibody.Thanks are due to Professor J.R. Wolff, Max-Planck Institute for Biophysical Chemistry, Göttingen, for stimulating discussions, to Ursula König, Christa Mahlmeister and Renate Steffens for skilful technical assistance, and to Heidi Waluk for the photographic workSupported by grants from Deutsche Forschungsgemeinschaft (Br 634/1, Dr 91/1, Un 34/4, Ste 105/19)Dedicated to Prof. Dr. med. H. Leonhardt on the occasion of his 60. birthday     

Vascular permeability to proteins and peptides in the mouse pineal gland

Summary The permeability of fenestrated capillaries in the mouse pineal gland to proteins and peptides was demonstrated by means of ultrastructural tracers. Horseradish peroxidase (HRP) and microperoxidase (MP) were injected intravenously and allowed to circulate for approximately 30 s, 1 min, 5 min, 1 or 2h. The tissue was then fixed by vascular perfusion or by immersion with aldehydes. In all experiments a pronounced extravasation of HRP and MP occurred. Transendothelial vesicular transport seemed to have occurred across the fenestrated capillaries. The most pronounced tracer labeling of vesicles was found after 1 min of MP- or HRP-circulation. The vesicles were uncoated and more than 70 % of the HRP-and MP-containing vesicles exhibited diameters between 50 and 110 nm. Furthermore, three other transcapillary pathways taken by the tracers are suggested: 1) via intercellular junctions, 2) through fenestrae and 3) via channels formed by fusion of vesicles with the luminal and abluminal cell membranes. Based on these results, it is assumed that the capillaries in the mouse pineal gland are also permeable to peptides synthesized and secreted by the pineal gland.Part of this study was presented at the EMCELL-76 meeting, Copenhagen, 1976     

Immunofluorescence of somatostatin-producing sites in the hypothalamus of the tadpole,Alytes obstetricans Laur.

Summary Three sites of somatostatin-synthesizing perikarya, or a related antigen, were determined by immunofluorescence in the hypothalamus of the tadpole, Alytes obstetricans (Amphibia, Anura). Two sites of neurosecretory perikarya were localized in the preoptic nuclei of the anterior hypothalamus; the axons extended either to the anterior diencephalon or to the median eminence and the pituitary. The third site was found in the posterior hypothalamus. These neurosecretory cells showed a strong immunofluorescent reaction; their axons all terminated at the level of the median eminence. Somatostatin cells were only found in intact or hypophysectomized tadpoles given somatotropin (STH). The strong reaction observed in hypophysectomized tadpoles was possibly due to the loss of the terminal portion of the neurosecretory pathway (median eminence and pituitary) by which the agent is transported to the site of discharge.     

A direct observation technique for studies on rhizoplane and rhizosphere colonization

Summary A slide incubation chamber was described which allowed small plants to be grown from seed and the root systems to be observed microscopically. A fluorescence stain, the ammonium salt of 8-anilino-1-naphthalene sulfonic acid, was applied to the soil in which the roots were growing and the stained microorganisms on the roots and in the rhizosphere were counted. A statistical pattern analysis technique, the two-within-four randomization test, was used to analyze the data obtained from quadrats on the roots. Distinct colonization patterns and colony growth, especially of bacteria, were easily distinguished with the technique.     

Immunocytochemical identification of proctolinlike immunoreactivity in the terminal ganglion and hindgut of the cockroach Periplaneta americana (L.)

Summary In the American cockroach, the distribution and connections of neuronal elements of the terminal ganglion-proctodeal nerve-hindgut system were investigated by means of immunohistochemical methods and axonal CoCl₂ iontophoresis. Proctolinlike immunoreactivity was localized within neurons of the terminal ganglion projecting into the proctodeal nerve on the one hand, and in nerve cells without a direct connection to this system on the other. Immunohistochemically, in whole mount preparations fibres of the proctodeal nerve and terminal structures in the hindgut musculature exhibit strong proctolinlike immunoreactivity. At the light- and electron-microscopic levels the pathways of about 30 somata of the proctodeal neural system were characterized by cobalt chloride iontophoresis. The relationships of cobalt filled and immunoreactive neuronal structures are discussed.For the preparation of tritiated proctolin we thank Dr. S. Reißmann, WB Biochemie, Sektion Biologie, FSU JenaThe authors wish to thank G. Schörlitz, Film- und Bildstelle, FSU Jena, for photographs of whole mount preparations and Ms. A. Zinßer and Mrs. B. Cosack for excellent technical assistance.     

Distribution of intermediate filaments in amphibian oxyntic cells

Summary Intermediate filaments of toad oxyntic cells were isolated and analysed by SDS-PAGE. The major proteins of the residue were identified as actin and a 51,000 dalton polypeptide. Immunological crossreactivity between toad oxyntic cell intermediate filament components and anti-prekeratin, was shown by double immunodiffusion tests and indirect immunofluorescence. The immunofluorescent decoration of oxyntic cells and the electron microscope images are coincident in locating the intermediate filaments mainly at the cortical and perinuclear basal zones. Furthermore, the cortical zone appears especially rich in prekeratin-like material at its adluminal third. This results in a cup-like structure that encloses the cell portion occupied by the tubulovesicular system, which does not contain intermediate filaments. The translocation of membranes occurring during the secretory cycle of the oxyntic cell, has been attributed to a system of contractile proteins. The disposition of the prekeratin-like material suggests a role for intermediate filaments in the generation of movement, produced by actin and myosin interaction, by providing a fixed plane for the anchoring of actin microfilaments.     

Identification of a secretomotor centre in the brain of Locusta migratoria,controlling the secretory activity of the adipokinetic hormone producing cells of the corpus cardiacum

Summary After retrograde filling of axons terminating in the glandular lobe of the corpus cardiacum (CC) of Locusta migratoria with cobalt chloride, a paired group of about 15 cobalt containing cells was demonstrated in the lateral area of the protocerebrum. The axons of these cells run via the NCC II into the glandular lobe of the CC. These small neurons have the characteristics of secretory cells; they contain secretory granules of about 1000 Å in diameter. The axon terminals in the glandular lobe, making synaptic contacts with the glandular cells, contain secretory granules of the same size. It is therefore concluded that the cell groups in the protocerebrum control the activity of the glandular cells which produce an adipokinetic hormone. Arborizations of fibers of the lateral secretomotor cells are present in the dorsal neuropile of the protocerebrum, ventral of the mushroom bodies and along the tracts of the NCC I within the brain. It is proposed that these arborizations are sites of synaptic input. It is discussed that the axons of these cells might receive additional synaptic input in the storage lobe of the CC.The localization of cell bodies, the axons of which enter the storage part of the CC is described. The course of the axon tracts of the various cell groups in the protocerebrum and their connections with the NCC I and NCC II are demonstrated.Supported by the Foundation for Fundamental Biological Research (BION) which is subsidized by the Netherlands Organization for the Advancement of Pure Research (ZWO). The electron microscopical investigations were performed at the EM-unit of the Faculty of Biology, State University of Utrecht (Director: Prof. Dr. J.C. van de Kamer)The author is greatly indebted to Dr. A.M.Th. Beenakkers and Dr. H.H. Boer for their active interest and helpful advise. Thanks are also due to Mr. H. van Kooten and his staff for making the macro- and microphotographs, to Mr. L.W. van Veenendaal for preparing the electron micrographs and final assistance in the preparation of the photo pages and to Mr. D. Smit, who made the drawings