Wound healing in the trematode Schistosoma

The ability of adult Schistosoma mansoni to effect wound healing over an exposed surface has been demonstrated. In transected worm segments a new external plasma membrane formed over the exposed tegumental cytoplasm. An elevated leading edge of tegument developed around the margin of the wound; the surface of this region was highly convoluted and there was a proliferation of membranous bodies within its cytoplasm. Inward migration of the leading edge over the exposed internal tissues took place. The resulting new tegument lacked spines and sensory endings. There was no regeneration of basal lamina or tegumentary cytons. In vitro maintenance of worm segments for 3 weeks did not give rise to any major ultrastructural changes in the tissues away from the wound.     

Ultrastructure of the cercomere of the cysticercoid, Trichocephaloides megalocephala

The cercomere of the larvae of Trichocephaloides megalocephala is formed exclusively by tegumentary tissues. The cytons of the tegument contain a great number of ribosomes, developed Golgi complex and numerous mitochondria that is indicative of the intensive synthesis, protein substances first of all. A distinct secretion of multivesicular membranes into subtegumentary space and further secretion of vesicles through the distal portion of the tegument into the external cyst cavity was observed. The main function of the cercomere is a production of the ground substance which apparently fulfils a protective function as it is suggested for glycocalix.     

The effect of colchicine on translocation of incorporated [3H] proline in Hymenolepis diminuta

Portions of adult Hymenolepis diminuta were exposed to a fixed concentration of colchicine (5 X 10(-4) M) in order to determine its effect upon incorporation of [3H] L-proline. Additional studies of the effect of colchicine upon tegumental morphology were performed. Autoradiographs showed a significant decrease in amount of incorporated label in the distal tegument of colchicine tissue and a heavy accumulation of label in the parenchyma. Radioassays indicated that the effect of colchicine on proline-incorporated protein was qualitative rather than quantitative suggesting that colchicine inhibits translocation in the tegument. It was hypothesized that microtubules within the internuncial processes facilitate movement of cell products from tegumentary cytons to the body surface.     

The antigen of bile canaliculi of the mouse hepatocyte: identification and ultrastructural localization

Summary The AgB10 antigen of bile canaliculi of the mouse hepatocyte was identified using monoclonal antibodies. The M_r value of 116000 for AgB10 was measured by immunoblotting. The tissue localization of AgB10 was studied by light and electron microscopy using the immunoperoxidase technique. AgB10 was predominantly present on the microvillus membrane of bile canaliculi, the brush border of intestinal mucosa and apical surfaces of the epithelial cells in some other organs. A small amount of AgB10 was detected on the basolateral domain of the hepatocytes. AgB10 was specific for hepatocytes and was not found in the other cell types of the liver. In primary hepatocyte culture, AgB10 was localized on the surface of cells during the first 24 h, predominantly at the sites of cell-cell and cell-substratum contacts. After 48 h of culture AgB10 gradually disappeared from contracting cell surfaces and became concentrated only in the reconstituted bile canaliculi.     

The antigen of bile canaliculi of the mouse hepatocyte: identification and ultrastructural localization

The AgB10 antigen of bile canaliculi of the mouse hepatocyte was identified using monoclonal antibodies. The Mr value of 116000 for AgB10 was measured by immunoblotting. The tissue localization of AgB10 was studied by light and electron microscopy using the immunoperoxidase technique. AgB10 was predominantly present on the microvillus membrane of bile canaliculi, the brush border of intestinal mucosa and apical surfaces of the epithelial cells in some other organs. A small amount of AgB10 was detected on the basolateral domain of the hepatocytes. AgB10 was specific for hepatocytes and was not found in the other cell types of the liver. In primary hepatocyte culture, AgB10 was localized on the surface of cells during the first 24 h, predominantly at the sites of cell-cell and cell-substratum contacts. After 48 h of culture AgB10 gradually disappeared from contacting cell surfaces and became concentrated only in the reconstituted bile canaliculi.     

Crystals of virus-like particles in the metacestodes of Taenia solium and T. crassiceps

Crystals of virus-like particles (VLP) are described as occurring in the nuclei of damaged tegumentary cytons from carcasses of Taenia solium metacestodes that had been stripped of their teguments. The VLP are grouped as parallel lines of round particles in an hexagonal packaging of spheroids forming small or large crystals. The individual particles have an external diameter of 36-37 nm and a wall of 5-6 nm thick, which surround a cavity of lower electron density. As identical crystals were also observed in normal tissues of T. solium and of T. crassiceps, it is suggested that both species of cysticerci are normal carriers of a similar species of virus. The possible biological implications of this condition are discussed.     

Structural observations on the alimentary canal of Paranthessius anemoniae, a copepod associate of the snakelocks anemone Ammonia sulcata

Light and electron microscopy has shown the alimentary canal of Paranthessius to be composed of clearly defined foregut, midgut and hindgut regions. The spacious foregut is cuticle-lined and separated from the midgut by a valve. The midgut epithelium is composed of columnar cells with an apparent secretary/absorptive rôle, and amoeboid cells thought to engulf material from the lumen. The amoeboid cells have large electron-dense central vacuoles containing carbohydrate-and protein-staining material. These cells appear to be sloughed off into the lumen to form part of a faecal pellet. Apart from their digestive rôle the midgut cells store lipid and it is considered possible that they have an osmoregulatory function. The hindgut epithelium cell type, lacks a cuticular layer and is thought to be mainly concerned with absorption. The alimentary canal is surrounded by strands of longitudinal and circular muscle.     

Identification of critical residues of an immunodominant region of Echinococcus granulosus antigen B

Immune evasion strategies often shape the immunogenicity of parasite components. We recently found that the N-terminal extension of the major subunit of Echinococcus granulosus antigen B (AgB), the causative agent of hydatid disease, concentrates the immunoreactive B cell epitopes of the native molecule. The nature of this immunodominance was analyzed using four monoclonal antibodies (mAbs) defining overlapping epitopes in this region of the AgB molecule. The minimal epitope requirements of these mAbs were determined using phage display peptide libraries. The consensus sequences isolated with the mAbs, and alanine replacement analysis with synthetic peptides mapped the relevant molecular contacts within a short stretch corresponding to residues 17-24 of the AgB major subunit. Substitution of two critical residues within this stretch produced a dramatic loss of antigenicity, as determined by using patient sera. The circular dichroism spectra of the antigen, together with the distribution of the contact residues, suggest that this region adopts an amphipathic alpha-helix structure that clusters the contact residues on its polar side. To provide further insight in the interpretation of the structure activity relationships for this immunoreactive region of E. granulosus AgB, we developed a model for the N-terminal extension of the AgB major subunit, which helps to rationalize our data.     

Monoclonal Antibodies Identify a Subset of Dense Granules in Cryptosporidium parvum Zoites and Gamonts

ABSTRACT. Two monoclonal antibodies raised against purified oocysts and excysted sporozoites of Cryptosporidium parvum identified antigens located in the anterior half of sporozoites by indirect immunofluorescence microscopic assay. The monoclonal antibodies also reacted with Triton X-100-insoluble antigens of asexual and sexual stage parasites developing in epithelial cells in vitro and identified a 110 kilodalton antigen on immunoblots of sodium dodecyl sulfate-extracted oocysts. Immunoblotting reactivity was abolished by prior treatment of blotted antigen with periodic acid suggesting that the monoclonal antibodies recognize a carbohydrate or carbohydrate-dependent epitope(s). By immunoelectron microscopy, the antibodies reacted with a family of small, electron-dense granules located predominantly in the central region of merozoites and also with a population of cytoplasmic inclusions in macrogamonts. In addition, the monoclonal antibodies prominently labeled the parasitophorous vacuole membrane of all intracellular stages examined suggesting that the corresponding antigen(s) may be exocytosed from the granules to become associated with Triton X-100-insoluble components of the vacuolar membrane or cytoskeleton.     

Zinc Toxicity and Xylem Vessel Wall Alterations in White Beans

When white beans are exposed to excess zinc, reddish brown patchesappear along the leaf veins. Ultrastructural observations ofthe xylem vessels in the discoloured zones show several modificationsof the vessel walls including gelation of the pit membranes,coating of the lumen surface with an abnormal layer and depositionof electron-dense material in the secondary vessel walls. Histochemicalstudies indicate that the altered pit membranes and the coatinglayer stain positively for lipid, while the secondary wall depositsstain positively for phenolic compounds. Phaseolus vulgaris L., white bean, xylem vessels, zinc toxicity     

Scanning and transmission electron microscopical observations on the tegument of excysted metacercariae and adults of Zygocotyle lunata.

Scanning and transmission electron microscopical observations were made on the tegument of excysted metacercariae and adults of the paramphistome, Zygocotyle lunata (Digenea: Trematoda). In accord with other paramphistomes studied, this species lacks spines and mitochondria in the tegumentary syncytium and associated cytons. The newly excysted metacercarie, which possessed relatively few tegumental papillae, were cylindrical in comparison to adults which were distinctly flat. The adults had large numbers of tegumental papillae in the region of the oral sucker and acetabulum.     

Leishmania tropica: characterization of a lipophosphoglycan-like antigen recognized by species-specific monoclonal antibodies.

Species-specific monoclonal antibodies to Leishmania tropica, T11 and T13-15, recognize membranal and secreted antigens. The membrane form of the antigen migrates on sodium dodecyl sulfate-polyacrylamide gels with a diffuse molecular weight from 15 to 50 kDa and can be labeled with palmitic acid, myoinositol, galactose, glucosamine, and inorganic phosphate. Both phosphate and sugar-labeled material were isolated from metabolically labeled promastigotes by affinity chromatography on antibodies coupled to Sepharose 4B. No binding to Ricinus communis agglutinin was observed. This material behaves like lipophosphoglycans from other Leishmania but contains unique species-specific epitopes. It is susceptible to cleavage by phospholipase C and after digestion no longer partitions into the detergent phase following a Triton X-114 extraction. All four monoclonal antibodies appear to recognize a carbohydrate epitope on the lipophosphoglycan since periodate treatment of this material bound to nitrocellulose essentially eliminated antibody binding. In addition, T15 binding could be blocked by 5 mM mannose-6-PO4 and fructose-1- or 6-PO4, but not by mannose, glucose, fructose, or the additional PO4 derivatives examined. The antibodies recognize a similar but not identical epitope, as demonstrated by a competitive radioimmunoassay using 125I-labeled T11, T13, and T15. Expression of surface antigen is elevated during the promastigote stationary phase.     

Negative staining and immunoelectron microscopy of adhesion-deficient mutants of Streptococcus salivarius reveal that the adhesive protein antigens are separate classes of cell surface fibril

The subcellular distribution of the cell wall-associated protein antigens of Streptococcus salivarius HB, which are involved in specific adhesive properties of the cells, was studied. Mutants which had lost the adhesive properties and lacked the antigens at the cell surface were compared with the parent strain. Immunoelectron microscopy of cryosections of cells labeled with affinity-purified, specific antisera and colloidal gold-protein A complexes was used to locate the antigens. Antigen C (AgC), a glycoprotein involved in attachment to host surfaces, was mainly located in the fibrillar layer outside the cell wall. A smaller amount of label was also found throughout the cytoplasmic area in the form of small clusters of gold particles, which suggests a macromolecular association. Mutant HB-7, which lacks the wall-associated AgC, accumulated AgC reactivity intracellularly. Intracellular AgC was often found associated with isolated areas of increased electron density, but sometimes seemed to fill the entire interior of the cell. Antigen B (AgB), a protein responsible for interbacterial coaggregation, was also located in the fibrillar layer, although its distribution differed from that of the wall-associated AgC since AgB was found predominantly in the peripheral areas. A very small amount of label was also found in the cytoplasmic area as discrete gold particles. Mutant HB-V5, which lacks wall-associated AgB, was not labeled in the fibrillar coat, but showed the same weak intracellular label as the parent strain. Immunolabeling with serum against AgD, another wall-associated protein but of unknown function, demonstrated its presence in the fibrillar layer of strain HB. Negatively stained preparations of whole cells of wild-type S. salivarius and mutants that had lost wall-associated AgB or AgC revealed that two classes of short fibrils are carried on the cell surface at the same time. AgB and AgC are probably located on separate classes of short, protease-sensitive fibrils 91 and 72 nm in length, respectively. A third class of only very sparsely distributed short fibrils (63 nm) was observed on mutant HB-V51, which lacks both wall-associated AgB and AgC antigens. The identity of these fibrils and whether they are present on the wild type are not clear. The function of long, protease-resistant fibrils of 178 nm, which are also present on the wild-type strain, remains unknown.     

The comparative fine structure and surface glycoconjugate expression of three life stages of Leishmania major

The cellular ultrastructure and surface glycoconjugate expression of three life stages of Leishmania major were compared. Noninfective logarithmic phase promastigotes (LP) are immature cells bearing a thin cell coat, short flagellum, small and empty flagellar pocket, and a loose cytoplasm filled with profiles of ER and large Golgi complex. LP also contain subpopulations of maturing cells containing less ER and Golgi and synthesizing cytoplasmic granules of different size, number, and electron-density. Infective or metacyclic promastigotes (MP) are fully differentiated nondividing forms with a thickened, prominent cell coat, long flagellum, distended flagellar pocket filled with secretory material, and few cytoplasmic organelles other than abundant electron-dense granules. Tissue amastigotes also contain electron-dense cytoplasmic granules, their flagellar pockets are also enlarged and contain secretory material, but they lack a discernable cell coat. Immunogold labeling of GP63 on the cell surface was extensive only on amastigotes. Promastigote GP63 appeared to be masked by the presence of a densely packed lipophosphoglycan (LPG) coat which was extensively labeled on the entire surface of MP and LP. An elongated, developmentally modified form of LPG was abundantly labeled only on MP. LPG was poorly labeled on amastigotes, arguing that the promastigote cell coat is a stage-specific structure which is lost during intracellular transformation.     

Comparison of antigens recognized by xenogeneic and allogeneic anti-Ia antibodies: Evidence for two classes of Ia antigens

Previously published data suggest that both xenogeneic and allogeneic anti-Ia sera can recognize carbohydrate-defined antigenic determinants on the surface of lymphocytes. There is also evidence, based on studies with allogeneic anti-Ia sera, that protein-defined Ia antigens exist. In this paper the relationship between these two types of Ia antigen was examined. It was found that in capping studies, the allogeneic anti-Ia serum could cap off the antigens recognized by the xenogeneic antiserum, whereas the xenogeneic antibodies could, at least partially, clear the surface of lymphocytes of Ia antigens detected by the allogeneic antibodies. On the other hand, when immunoprecipitates of radioiodinated cell-surface antigens were examined by SDS-polyacrylamide-gel electrophoresis, it was found that the xenogeneic anti-Ia serum did not immunoprecipitate any labeled material. In contrast, the allogeneic antiserum immunoprecipitated a labeled molecule which corresponded to the protein-defined Ia antigen described by others. Finally, it was shown that serum Ia antigens could be bound by either mouse or rabbit anti-Ia antibody, and this binding blocked any further reactivity with either serum. These results were interpreted as suggesting that two separate classes of Ia antigen molecule appear on the lymphocyte surface-one class has carbohydrate-defined antigenic specificities and the other has protein-defined determinants. Allogeneic anti-Ia sera contain antibodies against both these antigenic systems, whereas xenogeneic sera recognize only the carbohydratedefined series. The genetic implications of this interpretation are discussed.     

Antibody purification-independent microarrays (PIM) by direct bacteria spotting on TiO2-treated slides

The preparation of effective conventional antibody microarrays depends on the availability of high quality material and on the correct accessibility of the antibody active moieties following their immobilization on the support slide. We show that spotting bacteria that expose recombinant antibodies on their external surface directly on nanostructured-TiO(2) or epoxy slides (purification-independent microarray - PIM) is a simple and reliable alternative for preparing sensitive and specific microarrays for antigen detection. Variable domains of single heavy-chain antibodies (VHHs) against fibroblast growth factor receptor 1 (FGFR1) were used to capture the antigen diluted in serum or BSA solution. The FGFR1 detection was performed by either direct antigen labeling or using a sandwich system in which FGFR1 was first bound to its antibody and successively identified using a labeled FGF. In both cases the signal distribution within each spot was uniform and spot morphology regular. The signal-to-noise ratio of the signal was extremely elevated and the specificity of the system was proved statistically. The LOD of the system for the antigen was calculated being 0.4ng/mL and the dynamic range between 0.4ng/mL and 10μg/mL. The microarrays prepared with bacteria exposing antibodies remain fully functional for at least 31 days after spotting. We finally demonstrated that the method is suitable for other antigen-antibody pairs and expect that it could be easily adapted to further applications such as the display of scFv and IgG antibodies or the autoantibody detection using protein PIMs.     

Immunoelectron microscopic demonstration of the exocytosis of dense granule contents into the secondary parasitophorous vacuole of Sarcocystis muris (Protozoa, Apicomplexa)

Merozoites of the parasitic protozoon Sarcocystis muris (Apicomplexa) possess three types of characteristic organelles with electron dense contents named rhoptries, micronemes, and dense granules, which are supposed to be involved in the parasite-host cell interactions during and after invasion. Dense granules were purified from a merozoite homogenate by centrifugation on a sucrose density gradient. It was shown by SDS polyacrylamide gel electrophoresis that they contain a major protein of 21 kDa. Polyclonal antibodies raised against this protein were applied to ultrathin frozen and Lowicryl-K4M-embedded sections of the parasite before and after host cell invasion. Dense granules were distinctly labeled by immunogold before and after invasion. After host cell invasion the parasite is enclosed in a secondary parasitophorous vacuole which contains an electron-dense material. This deposition was heavily labeled by anti 21 kDa antibodies which clearly demonstrated that the dense granule contents is released into the secondary parasitophorous vacuole.     

Closure of the micropyle during embryonic development of some pelagic fish eggs

The fine structure of the egg envelope and micropyle was studied in unfertilized and developing eggs of the flounder Paralichthys olivaceus (Temminck & Schlegel), the Alaska pollack Theragra chalcogramma (Pallas), the Japanese tilefish Branchiostegus japonicus (Houttuyn) and the porgy Pagrus major (Temminck & Schlegel). The outer envelope surface of the unfertilized egg was wrinkled, while the inner surface was folded. The micropyle of the unfertilized egg consisted of a shallow vestibule and a distinct canal. The micropylar region of the inner surface of the envelope had a conical- or bowl-shaped protrusion. In developing eggs, the thickness of the envelope decreased and showed smooth outer and inner surfaces which indicated that it had been stretched tangentially at the time of the perivitelline space formation. The lumen of the micropylar canal was invariably occupied with envelope material. We postulate that the blockage of the micropylar canal is a result of the stretching of the envelope. The closure of the micropyle inhibits sperm and external pathogens from penetrating into the perivitelline space and seems to be involved in both the permanent prevention of polyspermy and the protection of the developing embryo from bacterial infection.     

Identification of a synaptic vesicle-specific membrane protein with a wide distribution in neuronal and neurosecretory tissue

Two different monoclonal antibodies, characterized initially as binding synaptic terminal regions of rat brain, bind a 65,000-dalton protein, which is exposed on the outer surface of brain synaptic vesicles. Immunocytochemical experiments at the electron microscope level demonstrate that these antibodies bind the vesicles in many different types of nerve terminals. The antibodies have been used successfully to purify synaptic vesicles from crude brain homogenates by immunoprecipitation onto the surface of polyacrylamide beads. The profiles of the structures precipitated by these beads are almost exclusively vesicular, confirming the vesicle-specificity of the antibodies. In SDS gels, the antibodies bind a single protein of 65,000 daltons. The two antibodies are not identical, but compete for binding sites on this protein. Immune competition experiments also demonstrate that the antigenic components on the 65,000-dalton protein are widely distributed in neuronal and neural secretory tissues. Detectable antigen is not found in uninnervated tissue--blood cells and extrajunctional muscle. Low levels are found in nonneural secretory tissues; it is not certain whether this reflects the presence of low amounts of the antigen on all the exocytotic vesicles in these tissues or whether the antigen is found only in neuronal fibers within these tissues. The molecular weight and at least two antigenic determinants of the 65,000-dalton protein are highly conserved throughout vertebrate phylogeny. The two antibodies recognize a 65,000-dalton protein present in shark, amphibia, birds, and mammals. The highly conserved nature of the determinants on this protein and their specific localization on secretory vesicles of many different types suggest that this protein may be essential for the normal function of neuronal secretory vesicles.     

Morphological changes in erythrocytes induced by malarial parasites

Host cell alterations induced by Plasmodium falciparum, P. brasilianum, P. vivax and P. malariae were described by electron microscopy and post-embedding immunoelectron microscopy. P. falciparum infection induces knobs, electron-dense material and clefts in the erythrocyte. Clefts are involved in exporting P. falciparum antigen from the parasite to the erythrocyte membrane. P. falciparum antigen is present in knobs which adhere to endothelial cells causing the blockage of cerebral capillaries and ensuing pathological changes in cerebral tissues. P. brasilianum infection induces knobs, short and long clefts and electron-dense material. These structures appear to contain different P. brasilianum antigens. This indicates that each structure functions independently in trafficking P. brasilianum protein to the erythrocyte surface. P. vivax infection induces caveola-vesicle complexes and clefts in the erythrocyte. These structures are also involved in trafficking P. vivax protein from the parasite to the erythrocyte membrane. P. malariae induces caveolae, electron-dense material, vesicles, clefts and knobs in the erythrocyte. Although vesicles and caveolae are seen in the erythrocyte cytoplasm, they do not form caveola-vesicle complexes as seen in P. vivax-infected erythrocytes. They also appear to be involved in trafficking of malaria antigens. These studies, therefore, indicate that host cell changes occur in order to facilitate the transport of malarial antigens to the host cell membrane. The significance of these phenomena is still not clear.