Neuropeptide schistosomin inhibits hormonally-induced ovulation in the freshwater snail Lymnaea stagnalis.

This study examines the interaction between the caudodorsal cell hormone (CDCH) and schistosomin, a peptide secreted by the central nervous system of the snail (Lymnaea stagnalis) infected with the avian schistosome Trichobilharzia ocellata. Non-infected snails were injected with synthetic as well as native CDCH in the absence or presence of purified schistosomin. The response to 2 pmol of synthetic CDCH was blocked for 90% by coinjection with 3.5 pmol of schistosomin. The ovulation-inducing activity of extracts of cerebral commissures (the storage area of native CDCH) was also blocked by schistosomin. The degree of inhibition (65%), however, was less than that observed with synthetic CDCH. These results show that schistosomin inhibits ovulation and egg laying in Lymnaea. This explains the decrease or absence of egg laying in schistosome-infected freshwater snails.     

Effects of snail size and age on the prevalence and intensity of avian schistosome infection: relating laboratory to field studies

Both the prevalence and intensity of patent infection by avian schistosomes (Trichobilharzia ocellata) increase with increasing size of lymnaeid snails (Stagnicola elrodi) collected in Flathead Lake, Montana. Because the size and age of a snail are positively correlated, snails of different sizes may have experienced differential duration of exposure to and development of infection. Another possibility is that infection itself induces snail gigantism. Each of these possibilities could lead to increased prevalence and intensity of infection among the oldest-largest snails. To decouple size variation from many correlated effects of age and to test for parasite-induced gigantism, laboratory experiments standardized snail size-age-at-infection, exposure history, inoculating dose, and duration of infection. The positive relationship between size and prevalence was eliminated in the laboratory, but the relationship between size and infection intensity remained. Laboratory results thus suggest that infection intensity is related to snail size per se, whereas prevalence in the field is related to snail size only through the correlation between size and age. In addition, under these experimental conditions, infected snails were no larger than uninfected snails, so the patterns observed in the field might not be attributable to gigantism.     

Detection of bird schistosomes in lakes by PCR and filter-hybridization

Many lakes around the world are contaminated with bird schistosome cercariae, which penetrate into human skin, causing an itching dermatitis called "swimmers' itch." Bathers could be forewarned from exposure to the larvae and ecological examinations could be performed, when a sensitive method to detect the parasites in aquatic systems, where lots of organisms hinder microscopic examinations, would be available. For this purpose we cloned, sequenced, and analyzed a 396 bp tandem repeated DNA sequence from Trichobilharzia ocellata (ToSau3A), and employed it for developing molecular detection assays. It hybridized with less than 100 pg DNA from different Trichobilharzia species (T. ocellata, Trichobilharzia franki, and Trichobilharzia regenti), but not with 10 ng DNA from other related or sympatric trematodes. A PCR assay, amplifying this sequence with the same specificity, detected 100 fg T. ocellata DNA, 1 cercaria in 0.5 g plankton, and 2 cercariae in 0.5 g host snail tissues.     

Morphological heterogeneity among fractionated alveolar macrophages in their release of lysosomal enzymes

When coupled with separation of alveolar macrophages (AM) into four different density fractions (I, II, III and IV) by discontinuous Percoll gradient centrifugation, ultrastructural heterogeneity was evident in secreting process of lysosomal enzymes. Lower dense AM (I and II) released high levels of acid phosphatase and cathepsin B, whereas higher dense ones (III and IV) did not. Ultrastructurally, there were multiple ruffling and active extension of long cytoplasmic processes from one pole or around the cell surface of AM obtained from the higher density fractions. In contrast, AM from lower dense fractions had much less cytoplasmic processes and contained more cytoplasmic vacuoles showing positive reactions of acid phosphatase. These cells featured more frequently round or ovoid knobs with acid phosphatase activity along and from the tips of the cytoplasmic processes, suggestive of exocytosis. It was suggested that these ultrastructural changes linked to the maturation process and release of lysosomal enzymes from differentiated AM.     

Flow cytometric measurements of cytoplasmic calcium changes in human platelets

Thrombin-induced stimulation of human platelets is accompanied by a dramatic increase in cytoplasmic calcium concentrations followed by a slow decrease. These changes are very rapid, are maximal by 10-15 s, and can be detected with probes such as Indo-1. Suspension studies using spectrofluorometry, which reflect a value which is the average of 3 x 10(7) cells per ml, indicate a thrombin dose-dependent increase in cytoplasmic calcium at doses up to 0.025 units per ml. We show here, using flow cytometry, that at less than half-saturating thrombin doses only subpopulations of platelets rather than the entire sample are responding. The extent of these responses, however, still depends on thrombin concentration. When the thrombin doses are between half and fully saturating, one subpopulation responds fully (i.e., its extent of increase in cytoplasmic calcium concentration, [Ca++]in is 100% of that seen at saturating thrombin concentrations) while the remaining platelets respond partially or not at all. There is thus evidence of positive cooperativity leading to disproportionate thrombin receptor occupancy on different subpopulations when platelets are subjected to subsaturating doses of thrombin. The existence of responding subpopulations may explain how the reported multiple stimulations of the same suspension of platelets at low thrombin doses occur.     

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.     

Heterogeneity of immunohistochemical staining with pulmonary surfactant protein A among fractionated alveolar macrophages which involves metabolism of pulmonary surfactant.

Alveolar macrophages (AM) which are separated into four fractionated subpopulations (I, II, III and IV), represented differential immunohistochemical staining with antibody against pulmonary surfactant protein A (SP-A). In light microscopy, the least dense AM (fraction I) were intensely stained with antibody to SP-A in numerous granules of the cytoplasm, whereas the most dense cells (fraction IV) showed immuno-reactivity with the antibody in several granules distributed in the spreading and elongating cytosol. By Western blot analysis, antibody to SP-A recognized a triplet of nature molecules of SP-A in AM lysate. However, the antigen of the AM lysate almost disappeared when the cells were cultured for more than two days, which indicate that AM do not synthesize SP-A and have digested intracellular SP-A during the cultivation. Immunoelectron microscopically, AM of fraction IV sometimes had very large vacuoles including lamellar body-like structures, probably pulmonary surfactant immediately after taken up from the alveolar lumen by them, which were heavily deposited with gold particles indicating antigenic site of SP-A. Whereas cells of fraction I contained numerous cytoplasmic vacuoles that were frequently labelled with the immuno-gold particles and were not associated with lamellar body-like structures, which may indicate that the materials in the vacuoles are digesting. The results of this experiments suggest that pulmonary surfactant, layered on the alveolar epithelium, is in part taken up by higher dense AM and is digested during a process of their maturation in the direction of lower dense cells, which undergo an important role of metabolism of pulmonary surfactant by AM subpopulations.     

Cell spreading factor. Occurrence and specificity of action.

A factor required for spreading of substratum-attached baby hamster kidney cells (BHK), Chinese hamster ovary (CHO) cells, HeLa cells, and L cells has been isolated and purified from fetal calf serum. A similar factor has also been found in calf, porcine, human, rabbit, and chicken sera. The spreading factor was active when adsorbed to the substratum and prior adsorption of other proteins prevented cell spreading, regardless of the addition of spreading factor or unfractionated serum to the incubation medium. Antibody against the fetal calf spreading factor inhibited the spreading activity associated with unfractionated fetal calf serum and also the spreading activity associated with calf serum and porcine serum. In model system studies it was found that antibody against BHK cell surfaces induced cell spreading when the antibody was adsorbed to the substratum; when it was present in the incubation medium as well as on the substratum, cell spreading was not observed. The data are discussed in terms of the hypothesis that there is a specific serum factor which adsorbs to the substratum surface and is thereby activated, and which then forms the target for certain cell surface receptors. Interaction between adsorbed-activated factor and cell surface receptors leads to cell spreading.     

Comparative studies on the internal defense system of schistosome-resistant and -susceptible amphibious snail Oncomelania nosophora 1. Comparative morphological and functional studies on hemocytes from both snails

Two morphologically distinct blood cell types (hemocytes), Type I and Type II were found coexisting in hemolymph from two kinds of snails, Oncomelania nosophora strain, viz. from the Nirasaki strain (schistosome-resistant snail) and the Kisarazu strain (schistosome-susceptible snail). Ten min after inoculation of SRBC, the majority of Type I cells from Nirasaki strain flattened and spread over the surface of the glass plate by extending pseudopodia. In the Kisarazu strain, Type I cells adhered to the surface of substrate with spike-like filopodia, but did not form spreading lamellipodia. Type I cell from the Nirasaki strain phagocytosed SRBC but that from the Kisarazu strain did not. The starting time of recognition of foreign materials was slightly different in the Type I hemocytes from the two strains. Type II cells from both strains were round and lymphocyte-like. Ten or sixty min after incubation, Type II cells from neither strain adhered to the surface of substrate or SRBC, and did not phagocytose SRBC. Type II cells from the Nirasaki strain were quite similar to those from the Kisarazu strain. We concluded that Type I cells from the schistosome-resistant snail, Nirasaki strain, possessed higher phagocytic activity than those from the susceptible snail, Kisarazu strain, despite the morphological similarities of the hemocytes from both strains.     

Motile behavior and protrusive activity of migratory mesoderm cells from the Xenopus gastrula.

During Xenopus gastrulation, the mesoderm migrates across a fibronectin (FN)-containing substrate, the inner surface of the blastocoel roof (BCR). A possible role for FN is to promote the extension of cytoplasmic processes which serve as locomotory organelles for mesoderm cells. To test this idea, the interaction of prospective head mesoderm (HM) cells with FN was examined in vitro. Nonattached HM cells extend filiform processes from an active region of the cell surface. This spontaneous activity is modulated by cell attachment to FN. Additional active regions appear, and cytoplasmic lamellae extend from these sites, leading to cell spreading and translocation. Thus, although FN seems not to induce processes de novo, it modulates a spontaneous protrusive activity to yield the extension of lamellae along the substrate surface. As putative locomotory organelles, HM cell protrusions were characterized functionally. They adhere rapidly and selectively to in situ substrates, preferentially to FN, and retract upon attachment. During translocation, the passive cell body is moved by the activity of the protrusions. Lamellae continuously extend, retract, or split into parts. This leads to an intermittent, nonpersistent mode of translocation. The polarity of HM cells, as expressed in the arrangement of protrusions, bears no constant relationship to the orientation of the cell body, and a cell can change its direction of movement without a corresponding rotation of the cell body. This may be relevant with respect to the mechanism by which mesoderm cells translate guidance cues of the BCR into a polarized, oriented cell structure during directional migration in situ.     

Human monocyte spreading in vitro--inducers and effects on Fc and C3 receptors.

Human peripheral blood monocytes undergo cytoplasmic spreading following attachment to a glass surface. The extent of spreading is greater in the presence of antigen-antibody complexes, manganese, subtilisin and dithiothreitol. The human blood monocyte spreads more rapidly than the mouse peritoneal macrophage and is not inhibited by serum. Fc receptor activity is diminished when spreading is induced by antigen-antibody complexes and is not affected by other inducers. The binding of erythrocytes coated with C3 and the ingestion of latex particles are not inhibited during cytoplasmic spreading.     

Navigation within host tissues: Schistosoma mansoni and Trichobilharzia ocellata schistosomula respond to chemical gradients

After penetration of human or duck host's skin schistosomula of Schistosoma mansoni and Trichobilharzia ocellata migrate parallel to the surface in the epidermis, then they enter the dermis and venules prior to further migration. This study focuses on potential behavioural mechanisms and host cues which may enable this navigation within host tissues. We stimulated cercariae to penetrate into agar substrates and to transform to schistosomula, and analysed their orientation behaviour within chemical concentration gradients. Both species were chemotactically attracted by low molecular weight fractions of their host's serum (human, duck) and D-glucose and L-arginine were identified as attractive components in serum. They responded to gradients, which established after addition of very low concentrations of D-glucose (1 microM in T. ocellata and 2 microM in S. mansoni) and L-arginine (0.025 microM in T. ocellata and 1.0 microM in S. mansoni). The response to D-glucose was specific as other saccharides had no stimulatory activity. L-Arginine stimulated chemotactic orientation both when free and bound in peptides. However, the two species responded differently to the position of L-arginine within the peptide (terminal or subterminal), and only S. mansoni, not T. ocellata, responded to peptides occurring in serum and endothelial cells: fibronectin (1 microM), bradykinin (25 pM) and its fragment 1-5 (2.5 microM). Both species adjusted their body axis with the ventral side towards the higher concentrations of D-glucose and of L-arginine. We argue that the chemotactic orientation and the alignment of the body axis enable the parasites (i) to orientate towards deeper skin layers and avoid accidental perforation of the covering skin surface layers, (ii) to determine their position during their surface-parallel migration within the epidermis, (iii) to locate blood vessels.     

Nucellar projection transfer cells in the developing wheat grain

Summary Transfer cells in the nucellar projection of wheat grains at 25 ±3 days after anthesis have been examined using light and electron microscopy. Within the nucellar tissue, a sequential increase in non-polarized wall ingrowth differentiation and cytoplasmic density was evident. Cells located near the pigment strand were the least differentiated. The degree of differentiation increased progressively in cells further removed from the pigment strand and the cells bordering the endosperm cavity had degenerated. Four stages of transfer cell development were identified at the light microscope level. Wall ingrowth differentiation followed a sequence from a papillate form through increased branching (antler-shaped ingrowths) which ultimately anastomosed to form a complex labyrinth. The final stage of wall ingrowth differentiation was compression which resulted in massive ingrowths. In parallel with wall ingrowth deposition cytoplasmic density increased. During wall deposition, paramural and multivesicular bodies were prominent and were in close association with the wall ingrowths. The degeneration phase involved infilling of cytoplasmic islets within the wall ingrowths. This was accompanied by complete loss of the protoplast. The significance of this transfer cell development for sucrose efflux to the endosperm cavity was assessed by computing potential sucrose fluxes across the plasma membrane surface areas of the nucellar projection cells. Transfer cell development amplified the total plasma membrane surface area by 22 fold. The potential sucrose flux, when compared with maximal rates of facilitated membrane transport of sugars, indicated spare capacity for sucrose efflux to the endosperm cavity. Indeed, when the total flux was partitioned between the nucellar projection cells at the three stages of transfer cell development, the fully differentiated stage III cells located proximally to the endosperm cavity alone exhibited spare transport capacity. Stage II cells could accommodate the total rate of sucrose transfer, but stage I cells could not. It is concluded that the nucellar projection tissue of wheat provides a unique opportunity to study transfer cell development and the functional role of these cells in supporting sucrose transport.Abbreviations CSPMSA cross sectional plasma membrane surface area - LPMSA longitudinal plasma membrane surface area - PTS tri-sodium 3-hydroxy-5,8,10-pyrenetrisulfonate     

Leukocyte biophysics. An invited review.

The biophysical properties of leukocytes in the passive and active state are discussed. In the passive unstressed state, leukocytes are spherical with numerous membrane folds. Passive leukocytes exhibit viscoelastic properties, and the stress is carried largely by the cell cytoplasm and the nucleus. The membrane is highly deformable in shearing and bending, but resists area expansion. Membrane tension can usually be neglected but plays a role in cases of large deformation when the membrane becomes unfolded. The constant membrane area constraint is a determinant of phagocytic capacity, spreading of cells, and passage through narrow pores. In the active state, leukocytes undergo large internal cytoplasmic deformation, pseudopod projection, and granule redistribution. Several different measurements for assessment of biophysical properties and the internal cytoplasmic deformation in form of strain and strain rate tensors are presented. The current theoretical models for active cytoplasmic motion in leukocytes are discussed in terms of specific macromolecular reactions.     

Leukocyte biophysics

The biophysical properties of leukocytes in the passive and active state are discussed. In the passive unstressed state, leukocytes are spherical with numerous membrane folds. Passive leukocytes exhibit viscoelastic properties, and the stress is carried largely by the cell cytoplasm and the nucleus. The membrane is highly deformable in shearing and bending, but resists area expansion. Membrane tension can usually be neglected but plays a role in cases of large deformation when the membrane becomes unfolded. The constant membrane area constraint is a determinant of phagocytic capacity, spreading of cells, and passage through narrow pores. In the active state, leukocytes undergo large internal cytoplasmic deformation, pseudopod projection, and granule redistribution. Several different measurements for assessment of biophysical properties and the internal cytoplasmic deformation in form of strain and strain rate tensors are presented. The current theoretical models for active cytoplasmic motion in leukocytes are discussed in terms of specific macromolecular reactions.     

Differential staining of neutrophils and monocytes: Surface and cytoplasmic iron-binding proteins

Summary Lactoferrin, transferrin, and ferritin were systematically visualized and semiquantified in neutrophils and monocytes/macrophages using indirect immunofluorescence and functional cytochemical techniques. They localized on cell surfaces and within the cytoplasm at the light and electron microscopical levels. In normal subjects, subpopulations of blood neutrophils and monocytes had surface lactoferrin, but little surface transferrin or ferritin was observed on these cells. Most neutrophils had brilliant granular cytoplasmic positivity for lactoferrin; variable fractions of monocytes had weak to moderate diffuse cytoplasmic lactoferrin staining localized most prominently to the cytoplasmic matrix. Most neutrophils had cytoplasmic ferritin, but few had cytoplasmic transferrin, whereas larger subpopulations of monocytes had cytoplasmic staining reactions for both proteins. To analyse maturing cells, the iron nitrilotriacetate-acid ferrocyanide method was adapted for the light microscopical analaysis of neutrophils and monocytes/macrophages in soft agar culture. Further, a combined stain that visualizes iron nitrilotriacetate-acid ferrocyanide reactivity and -naphthyl butyrate esterase activity in cells in blood and marrow smears was developed. The relative quantities and subcellular distribution of iron-binding proteins in neutrophils and monocytes/macrophages defined by the present methods can be correlated with biochemical, maturational, and functional properties of these cells.     

Spreading of cells on various substrates evaluated by Fourier analysis of shape

In order to evaluate in mathematical terms the morphological changes occurring in the course of cell spreading, Fourier analysis of shape was applied. Human urothelial Hu 961 b cells plated on type IV collagen, fibronectin, laminin, glass and bovine serum albumin (BSA) were studied. Fourier parameters describing cell shape as well as surface areas covered by the cells on the substrate were subjected to statistical analysis. Using analysis of variance and discriminant analysis it was found that parameters describing cell shape (both gross shape of cells and their fine scale contour foldings) possessed a higher power of discrimination between the cells spread on various substrates than the differences in cell surface areas. In the course of observation (75 and 150 min) the highest number of attached cells and highest degree of spreading were found when cells were plated on type IV collagen. Moderate alterations in cell shape and moderate increase of surface area were seen in the group of cells seeded on fibronectin, whereas the cells plated on laminin, glass and BSA revealed a moderate increase of surface area, but no changes in their shape were observed. The differences in attachment of cells and in the degree of their spreading might be due to the variation in expression of plasma membrane receptors for various substrates. The Fourier analysis of cell shape coupled with measurement of surface area is a good tool for quantitative evaluation of cell spreading and can be used for discrimination between cells spread on different substrates.     

Spreading of cells on various substrates evaluated by fourier analysis of shape

Summary In order to evaluate in mathematical terms the morphological changes occurring in the course of cell spreading, Fourier analysis of shape was applied. Human urothelial Hu 961 b cells plated on type IV collagen, fibronectin, laminin, glass and bovine serum albumine (BSA) were studied. Fourier parameters describing cell shape as well as surface areas covered by the cells on the substrate were subjected to statistical analysis. Using analysis of variance and discriminant analysis it was found that parameters describing cell shape (both gross shape of cells and their fine scale contour foldings) possessed a higher power of discrinunation between the cells spread on various substrates than the differences in cell surface areas. In the course of observation (75 and 150 min) the highest number of attached cells and highest degree of spreading were found when cells were plated on type IV collagen. Moderate alterations in cell shape and moderate increase of surface area were seen in the group of cells seeded on fibronectin, whereas the cells plated on laminin, glass and BSA revealed a moderate increase of surface area, but no changes in their shape were observed. The differences in attachment of cells and in the degree of their spreading might be due to the variation in expression of plasma membrane receptors for various substrates. The Fourier analysis of cell shape coupled with measurement of surface area is a good tool for quantitative evaluation of cell spreading and can be used for discrimination between cells spread on different substrates.Supported in part by a grant (MZ-XIV) from the Polish Ministry of Health and Welfare     

Ultrastructure of the ovarian medulla in the newly hatched chick treated with human chorionic gonadotropin

Summary The ultrastructural characteristics of the ovarian medulla of the newly hatched white leghorn chick were studied in control animals and compared with chicks that were treated with human chorionic gonadotropin during embryonic development. The ovarian medulla was mainly occupied by epithelial cells which formed cords or islets surrounded by a basal lamina. Within this epithelial compartment, steroidogenic cells, poorly differentiated cells and a lacunary system could be recognized. When chicks were treated with human chorionic gonadotropin, steroidogenic cells became discernible; there was an increment in the amount of cytoplasm and the area of mitochondria. Poorly differentiated cells exhibited signs of stimulation, and transitional images suggested the transformation of these cells into steroidogenic cells. The epithelial cells of the lacunar system also displayed stimulated cytoplasmic organelles. Evidence was supplied suggesting that relatively undifferentiated cells persist in the ovarian medulla until hatching and can develop into steroidogenic cells under gonadotropic stimulation.