Production and characterization of a monoclonal antibody against 28.5 kDa tegument antigen of Fasciola gigantica

A monoclonal antibody (MoAb) against the 28.5 kDa tegumental antigen of Fasciola gigantica was produced by the hybridoma technique using spleen cells from BALB/c mice immunized with the tegumental extract from adult F. gigantica. This MoAb was found to be of the isotype IgG(1), kappa-light chain, and shown by immunoblotting to specifically react with the 28.5 kDa antigen present in the tegument, excretion-secretion material of the adult, whole-body extracts of newly excysted juveniles, 5-week-old juvenile and adult parasites. It did not cross-react with antigens from other trematode parasites, including Schistosoma mansoni, Eurytrema pancreaticum and Paramphistomum spp. Immunolocalization of this antigen by indirect immunofluorescence indicated that it was present as a major component of the adult tegument, particularly in its outer rim, tegumental cells, and their processes. Furthermore, the epithelium linings of the oral sucker, buccal tube, pharynx, caecal bifurcation, both male and female genital canals, which were the continuation of the tegumental-type epithelium, were also positively stained with this MoAb. A similar pattern of immunolocalization, but with weaker staining intensity, was observed in newly excysted, 5- and 7-week-old juveniles. Thus this antigen is expressed in all developmental stages of the parasite, and it could be a strong candidate for immunodiagnosis and vaccine development.     

The existence of gonadotropin-releasing hormone-like peptides in the neural ganglia and ovary of the abalone, Haliotis asinina L.

Gonadotropin-releasing hormone (GnRH) is a neuropeptide that is conserved in both vertebrate and invertebrate species. In this study, we have demonstrated the presence and distribution of two isoforms of GnRH-like peptides in neural ganglia and ovary of reproductively mature female abalone, Haliotis asinina, using immunohistochemistry. We found significant immunoreactivities (ir) of anti-lamprey(l) GnRH-III and anti-tunicate(t) GnRH, but with variation of labeling intensity by each anti-GnRH type. lGnRH-III-ir was detected in numerous type 1 neurosecretory cells (NS1) throughout the cerebral and pleuropedal ganglia, whereas tGnRH-I-ir was detected in only a few NS1 cells in the dorsal region of cerebral and pleuropedal ganglia. In addition, a small number of type 2 neurosecretory cells (NS2) in cerebral ganglion showed lGnRH-III-ir. Long nerve fibers in the neuropil of ventral regions of the cerebral and pluropedal ganglia showed strong tGnRH-I-ir. In the ovary, lGnRH-III-ir was found primarily in oogonia and stage I oocytes, whereas tGnRH-ir was observed in stage I oocytes and some stage II oocytes. These results indicate that GnRH produced in neural ganglia may act in neural signaling. Alternatively, GnRH may also be synthesized locally in the ovary where it could induce oocyte development.     

Existence of an egg-laying hormone-like peptide in male reproductive system of the giant freshwater prawn, Macrobrachium rosenbergii

The giant freshwater prawn, Macrobrachium rosenbergii, is an important aquaculture species. A better understanding of the molecular components of reproduction in this species would help to advance the prawn production. In the present study, we demonstrated the presence of an egg laying hormone (ELH)-like peptide in the male reproductive system. First, an antibody to the abalone (a)ELH was generated, and by Western blot it was shown to specifically bound to a protein from the male M. rosenbergii reproductive tissues with a similar size to molluscan ELH. This aELH-like peptide was localized in spermatogonia in the testes of all three male morphotypes: blue claw, orange claw and small males. Moreover, the aELH-like peptide was detected in the epithelium of the spermatic duct and its associated smooth muscle cell layers and on the outer surface of spermatozoa. As well, the aELH-like peptide was detected in the spermatophore located in the female thelycum at 4–6?h post-mating, indicating that it was transferred to the female during copulation. Taken together, we suggest that this aELH-like peptide could be as a male inducing factor that helped to accelerate female spawning. Liquid chromatography of crude extracts and immunoblot analysis suggested that the aELH-like peptide could be further purified for ultimate characterization.     

Patterns of immunoreactivity specific for gustducin and for NCAM differ in developing rat circumvallate papillae and their taste buds

α-Gustducin and neural cell adhesion molecule (NCAM) are molecules previously found to be expressed in different cell types of mammalian taste buds. We examined the expression of α-gustducin and NCAM during the morphogenesis of circumvallate papillae and the formation of their taste buds by immunofluorescence staining and laser-scanning microscopy of semi-ultrathin sections of fetal and juvenile rat tongues. Images obtained by confocal laser scanning microscopy in transmission mode were also examined to provide outlines of histology and cell morphology. Morphogenesis of circumvallate papillae had already started on embryonic day 13 (E13) and was evident as the formation of placode. By contrast, taste buds in the circumvallate papillae started to appear between postnatal day 0 (P0) and P7. Although no cells with immunoreactivity specific for α-gustducin were detected in fetuses from E13 to E19, cells with NCAM-specific immunoreactivity were clearly apparent in the entire epithelium of the circumvallate papillary placode, the rudiment of each circumvallate papilla and the developing circumvallate papilla itself from E13 to E19. However, postnatally, both α-gustducin and NCAM became concentrated within taste cells as the formation of taste buds advanced. After P14, neither NCAM nor α-gustducin was detectable in the epithelium around the taste buds. In conclusion, α-gustducin appeared in the cytoplasm of taste cells during their formation after birth, while NCAM appeared in the epithelium of the circumvallate papilla-forming area. However, these two markers of taste cells were similarly distributed within mature taste cells.     

Immunohistochemical expression of type II collagen in the lingual mucosa of rats during organogenesis of the tongue

OBJECTIVES: We examined the timing of the appearance and distribution of type II collagen as a possible component of the extracellular matrix that is involved in the morphogenesis of the rat tongue. METHODS: We examined the immunofluorescence of type II collagen, differential interference contrast (DIC) images, and images recorded in transmission mode after toluidine blue staining by laser-scanning microscopy (LSM) during the morphogenesis of filiform papillae and the keratinization of the lingual epithelium of rats on semi-ultrathin sections of epoxy resin-embedded samples. RESULTS: Immunoreactivity specific for type II collagen was scattered on cells over a wide area of the mesenchymal connective tissue of the fetal tongue on day 15 after conception (E15), when the lingual epithelium was composed of one or two layers of cuboidal cells. Immunoreactivity specific for type II collagen was recognisable on cells of the lamina propria of the lingual mucosa and around the developing lingual muscle of fetuses at E17 and E19. On E19, the epithelium was clearly of the stratified squamous type. At postnatal stages after birth (P0), immunoreactivity became more and more significant in the connective tissue of the lamina propria with the advancing of morphogenesis of the filiform papillae. In addition, immunoreactivity was widely distributed in the connective tissue around the lingual muscle, as myogenesis in the tongue advanced. The lingual epithelium was composed of stratified squamous cells, and keratinization of the lingual epithelium proceeded gradually as morphogenesis of filiform papillae continued during postnatal development. CONCLUSION: Type II collagen appeared not only in the connective tissue of the lamina propria as the morphogenesis of filiform papillae occurred and the lingual epithelium became keratinized but also in the endomysium and perimysium around the lingual muscle after myogenesis of the tongue is complete at P0.     

Morphological changes induced by cyclophosphamide in crypt epithelium of the small intestine in mice: Light and electron microscopic studies

When a single sublethal dose (300 mg/kg body weight) of cyclophosphamide was injected intraperitoneally into experimental mice, several morphological alterations were detected in the crypt epithelium within a few hours. These were (a) mitotic activity of epithelial cells decreased, reaching the lowest level at 16 hours; (b) an abnormally high number of lymphocytes and cells with eosinophilic granules appeared in the epithelium; later these cells degenerated and the maximum number of dead cells was observed at six hours after the drug injection. At four days post-injection the morphology of crypt epithelium had returned to normal. Electron microscopic examination revealed that although the mitotic activity of crypt epithelial cells was depressed, there was little change in their morphology following the drug administration. However, many intermediate stages of degeneration of lymphocytes and eosinophilic cells were observed in the intercellular spaces as well as apparently inside the cytoplasm of crypt epithelial cells. The eosinophilic cells are characterized by the presence of large crystal-containing granules in the cytoplasm, and are believed to be equivalent to “globular leucocytes” described by many investigators. Evidence from this study suggests that lymphocytes and globular leucocytes are more sensitive to cyclophosphamide than crypt epithelial cells, and that they degenerate in crypt epithelium.     

Immunohistochemical analysis of type III collagen expression in the lingual mucosa of rats during organogenesis of the tongue

We examined the distribution of immunofluorescence due to immunostaining of type III collagen, differential interference contrast (DIC) images and images obtained in the transmission mode after toluidine blue staining by laser-scanning microscopy of semi-ultrathin sections of epoxy resin-embedded samples, during morphogenesis of the filiform papillae, keratinization of the lingual epithelium, and myogenesis of the rat tongue. Immunoreactivity specific for type III collagen was distributed widely in the mesenchymal connective tissue in fetuses on day 15 after conception (E15), at which time the lingual epithelium was composed of one or two layers of cuboidal cells and the lingual muscle was barely recognizable. Immunoreactivity specific for type III collagen was clearly detected on the lamina propria in fetuses on E17 and E19, and it was relatively distinct just beneath the lingual epithelium. Immunoreactivity specific for type III collagen was sparsely distributed on the connective tissue around the developing lingual muscle. In fetuses on E19, the epithelium became clearly stratified and squamous. At postnatal stages from newborn (P0) to postnatal day 14 (P14), keratinization of the lingual epithelium advanced gradually with the development of filiform papillae. On P0, myogenesis of the tongue was almost completed. The intensity of the fluorescence immunoreactivity specific for type III collagen at postnatal stages was almost same as that on E19. The immunoreactivity around the fully mature muscle was relatively distinct between P0 and P14. Thus, type III collagen appeared in conjunction with the morphogenesis of filiform papillae and the keratinization of the lingual epithelium as well as in the connective tissue that surrounded the lingual muscle during myogenesis of the rat tongue.     

Ultrastructural Changes in the Intestinal Absorptive Cells of the Mouse Induced by Capsaicin in Relation to Thiamine Absorption

The correlation between an inhibition of thiamine absorption and ultrastructural changes in the intestinal absorptive cells of the mouse jejunum induced by capsaicin was examined. In mice fed with capsaicin 1 and 2 mg/kg BW/day for 12 weeks, thiamine absorption was inhibited by 2.7% and 12.5% ( p <0.001) respectively. Ultrastructural alterations were observed in villus absorptive cells of jejunum in mice fed with capsaicin 1 and 2 mg/kg BW/day for 12 weeks. The most striking ultrastructural changes occurred in mice fed with capsaicin 2 mg/kg BW/day for 12 weeks. The organelle most affected by capsaicin was the mitochondria. The result shows that the ultrastructural changes of intestinal absorptive cells may be associated with the inhibition of thiamine absorption.     

Distribution of 28.5 kDa antigen in the tegument of adult Fasciola gigantica

Monoclonal antibody (MoAb) specific to 28.5 kDa tegumental antigen (TA) was used to localize this antigen in various tissues of adult Fasciola gigantica by means of indirect immunofluorescence, immunoperoxidase and immunogold techniques. The indirect immunofluorescence and immunoperoxidase detections revealed that this antigen was concentrated in the tegument particularly in its outer rim, tegumental cells and their processes, epithelial linings of the oral sucker and the proximal part of digestive tract. It was also detected at a moderate concentration in spermatogenic cells in the testes, cells of Mehlis' gland, oocytes within the ovary, and ovum within the egg of adult parasites. At TEM level, the immunogold detection showed deposit of gold particles specifically in G(2) tegumental granules and on the surface membrane. Thus, this antigen is expressed in the tegument and associated structures of adult parasites, and it could be a major component of the G(2) granules which are shown to fuse with the surface membrane and contribute material to replace the casted-off membrane. This process is a part of membrane turnover that prevents the parasite from being attacked by the host immune effector cells.