Schistosoma mansoni: histological localization of gelatinase in the preacetabular glands of cercariae

Proteolytic activity was demonstrated in secretions from the preacetabular glands of cercariae of Schistosoma mansoni. Thin gelatin film substrates were lysed by living cercariae stimulated to penetrate by application on the films of skin surface lipid. Lysis was directly related to number of cercariae, time, and temperature of incubation and pH of the medium. Gelatinase activity in unfixed frozen sections of cercariae incubated on the gelatin films was in the preacetabular glands which are the source of the secretion emptied into skin during penetration. Protease activity, therefore, appears to be related to penetration. The schistosome larvae which made the penetration attempt satisfied the accepted criteria for schistosomules, and therefore appeared to have transformed into schistosomules even though they did not successfully penetrate anything.     

Schistosoma mansoni: localization of calcium-detecting reagents in electron-lucent areas of specific preacetabular gland granules

In an attempt to establish the exact location of calcium within the preacetabular glands of cercariae of Schistosoma mansoni, these larvae were exposed to reagents (potassium oxalate, potassium pyroantimonate, chloranilic acid, and silver nitrate) useful in the detection of calcium, and were subsequently observed with the aid of light and electron microscopes. Cercariae incubated in potassium oxalate and viewed in polarized light showed birefringence only in the preacetabular gland funduses. At the ultrastructural level, the preacetabular glands of potassium oxalate-treated cercariae had no electron-dense precipitate, but instead had translucent, irregularly shaped inclusions, similar to spaces left by volatilized calcium oxalate as described by others. Pyroantimonate treatment, on the other hand, localized the reaction in the electron-lucent areas of the light-spotted granules. The von Kossa silver nitrate procedure destroyed the secretory granules; therefore, an electron-dense precipitate was distributed throughout the gland. However, pretreatment with chloranilic acid before fixation preserved the granules, and subsequent exposure to the von Kossa silver nitrate gave a reaction identical to that obtained with the pyroantimonate alone. When viewed in polarized light, chloranilic acid-incubated cercariae showed birefringence in the fundus and duct areas.     

Differential proteomic analysis of laser-microdissected penetration glands of avian schistosome cercariae with a focus on proteins involved in host invasion

Schistosome invasive stages, cercariae, leave intermediate snail hosts, penetrate the skin of definitive hosts, and transform to schistosomula which migrate to the final location. During invasion, cercariae employ histolytic and other bioactive products of specialized holocrine secretory cells – postacetabular (PA) and circumacetabular (CA) penetration glands. Although several studies attempted to characterize protein composition of the in vitro-induced gland secretions in Schistosoma mansoni and Schistosoma japonicum, the results were somewhat inconsistent and dependent on the method of sample collection and processing. Products of both gland types mixed during their secretion did not allow localization of identified proteins to a particular gland. Here we compared proteomes of separately isolated cercarial gland cells of the avian schistosome Trichobilharzia szidati, employing laser-assisted microdissection and shotgun LC-MS/MS, thus obtaining the largest dataset so far of the representation and localization of cercarial penetration gland proteins. We optimized the methods of sample processing with cercarial bodies (heads) first. Alizarin-pre-stained, chemically non-fixed samples provided optimal results of MS analyses, and enabled us to distinguish PA and CA glands for microdissection. Using 7.5 × 10⁶ μm³ sample volume per gland replicate, we identified 3347 peptides assigned to 792 proteins, from which 461 occurred in at least two of three replicates in either gland type (PA = 455, 40 exclusive; CA = 421, six exclusive; 60 proteins differed significantly in their abundance between the glands). Peptidases of five catalytic types accounted for ca. 8% and 6% of reliably identified proteins in PA and CA glands, respectively. Invadolysin, nardilysin, cathepsins B2 and L3, and elastase 2b orthologs were the major gland endopeptidases. Two cystatins and a serpin were highly abundant peptidase inhibitors in the glands. While PA glands generally had rich enzymatic equipment, CA glands were conspicuously abundant in venom allergen-like proteins.     

Schistosoma mansoni: histochemical analysis of the postacetabular gland secretion of cercariae

Contents of the funduses and ducts of the postacetabular glands of Schistosoma mansoni cercariae, the secreted deposits, and the surface film were compared by their histochemical reactions. Techniques for carbohydrate-containing substances, neutral and acid mucosubstance, proteins and amino acids, and enzymes were used. The secretion reacted differently before (within the glands) and after (in secreted deposits) emission.Before emission, the postacetabular gland contents reacted as a neutral mucosubstance containing periodate-engendered and periodate-reactive aldehydes rich in vic-glycols or their substituted amines, probably hexoses other than glucose, such as fucose or galactose. No reactions of significance were observed for acid groups or for glycogen or lipids. In this state, the secretion is termed mucigen.After emission, the secretion stained not only as mucigen, but also as acid mucosubstance, apparently sialomucin. After emission, it is termed mucin.It is probable that acid radicals were present in mucigen but were masked stearically by the presence of adjacent neutral radicals or basic proteins. The surface film reacted as both a neutral and an acid mucosubstance. Evidence suggested that the film itself was neutral and that the reaction for acid mucosubstance was from an overlay of mucin secreted from the postacetabular glands.Proteins and amino acids, especially arginine, and some tyrosine and tryptophan were indicated in mucigen and in mucin by the histochemical tests. There was no histochemical evidence of enzymes. Secretion of the postacetabular glands is concluded from histochemical reactions, as from earlier chromatographic data (Stirewalt and Evans 1960), to be a carbohydrate-protein-lipid complex.     

Schistosoma mansoni: fine structure of cercarial acetabular glands

Emerged cercariae of Schistosoma mansoni were studied with the electron microscope for the purpose of describing the acetabular (penetration) glands and their cellular investment. The two pre- and three postacetabular unicellular glands consist of enlarged aboral areas (funduses) and their oral extensions as ducts. The glands were morphologically similar except for their shape and secretory globules. In the funduses of the postacetabular glands the globules were of a single type, spheroidal to irregular in shape, with numerous electron-dense areas. Preacetabular secretory globules appeared to be of several types, varying in size, shape, and homogeneity. Some were of uniform density; others showed electron-lucid areas. The fine structure of both pre- and postacetabular globules changed as they were compared in the funduses, in progressively oral areas of the ducts and after extrusion. These changes were thought to be transitional. Microtubules and close cellular investiture of the glands by muscle, nerve, and unclassified cells with extended interwoven processes appeared to provide structural support.     

日本血吸虫尾蚴发育的超微结构观察：Ⅱ.腺体

在透射电镜下观察不同发育期日本血吸虫尾蚴的腺体。结果：胚球期（Ｓ２）主要出现体细胞分裂与分化,首次证明分泌细胞及其小体在Ｓ２出现,雏体期（Ｓ３）见到前钻腺细胞体与钻腺管束及分泌小体。成熟前期（Ｓ４）钻腺分泌小体基质分Ａ／Ｂ为主两型,分别演变为成熟期（Ｓ）５的后／前钻腺分泌小体。头腺分泌小体亦在Ｓ４出现。从前钻腺体排至头器远端腺管的分泌小体,显眼透明球消失成为致密同质性小体,已为大家共识。而在后钻腺     

日本血吸虫皮肤型童虫透射电镜观察

本文报道应用透射电镜观察并比较0.5、3和12小时龄的日本血吸虫皮肤型童虫的超徽结构特征。结果表明,除了外质膜外,其他的超微结构,如体被、肌层、体被下细胞、胞质桥、头腺、钻腺和食道等结构在尾蚴感染后3小时均未见再有明显的变化。     

Plantar epidermis of the guinea pig and characteristics of the stratum corneum.

The guinea pig plantar epidermis was examined by light-microscopical histochemical methods and by transmission electron microscopy. Autolysis of cell structure was much less complete in guinea pig plantar horny layer than in the back, and stainable cytoplasm was retained in keratinized cells but organelles were lost except for some degraded ultrastructural remnants. By light microscopy the whole thickness of the horny layer showed bound phospholipid and bound cysteine, and there was a weak cystine reaction at the peripheries of the keratinized cells. In ultrastructure the keratohyalin contained slightly larger subparticles than in the back skin. The horny layer was not divisible into basal, intermediate and superficial regions as in hairy skin. The stratum lucidum of light microscopy was not defined in electron micrographs. Osmium-stained cytoplasmic material was retained in horny cells about to be desquamated, in contrast to the empty appearance of these cells in hairy skin. Epidermal cells in plantar skin have ultrastructural cytoplasmic processes which are longer than they are broad. In the horny layer these interdigitate with those of neighbouring cells and are held together by lateral demonsomal junctions. Probably this gives mechanical strength against shearing forces experienced by the plantar horny layer.     

Skin penetration of infective hookworm larvae. I. The path of migration of infective larvae of Ancylostoma braziliense in canine skin

Migratory behaviour of Ancylostoma braziliense was studied in relation to the structure of the skin in dogs after primary infections. Data were obtained studying serial sections of lateral skin areas 6 mm in diameter, which had been exposed to larvae. The sections were stained either with Harris' haematoxylin and eosin or with P.A.S. or as outlined by Crossmon. Most of the larvae managed to penetrate the skin within 1/2 hr after the application. Hairs did not seem to constitute sites of entry. The larvae moved into the horny layer where edges of keratinized cells provide uneven spots. They migrated approximately parallel to the surface from the horny layer into the living epidermis and continued into an external root sheath of a hair follicle. They could only leave this site via sebaceous glands for the dermis or via apocrine sweat glands for the hypodermis. Tunnels from the epidermis into the dermis, however, suggested that a direct trans-epidermal migration had occurred. The vessels invaded by larvae were hypodermal lymphatic vessels. The first ones were found in these structures 1/2 h after the onset of the exposure.     

Schistosoma mansoni: biochemical evidence for morphogenetic change from cercaria to schistosomule

Conditions for obtaining in vitro the transformation of cercariae in larvae similar to schistosomules are described. They consist of either centrifuging cercarial suspension or packed cercariae at room temperature, or incubating at 30 C packed cercariae. The product obtained was a mixture of cercariae, tails, cercarial bodies, and schistosomule-like larvae as revealed by stereomicroscopy. Tails and cercariae were mechanically separated.Analysis of samples of schistosomule-like preparations obtained by centrifuging cercariae at room temperature revealed that many of the organisms were PAS negative and water sensitive, and showed no localized staining with alizarin and no tendency to form Cercarienhüllen Reaktion (envelopes) in immune human sera. Elimination of the contents of the preacetabular glands was followed during centrifugation or incubation by assaying the proteolytic and/or esterolytic activities in the pellets obtained at different times. The specific activity of the secretion collected increased as a function of time. Electrophoresis of extracts of organisms before and after centrifugation at room temperature showed decrease of PAS stained bands.     

Schistosoma mansoni: Cercarial degradation of a radioactively labeled collagen gel

The apparent penetration activity of Schistosoma mansoni cercariae was quantified by means of an in vitro assay with a radioactively labeled Type I collagen gel. Both live cercariae and cercarial preacetabular gland secretions degraded the collagen. The addition of skin lipid or linoleic acid to the gel surface enhanced the degradation by live cercariae.     

Schistosoma mansoni: Development of acetabular glands of cercaria at ultrastructural level

Cercariae of Schistosoma mansoni in daughter sporocysts in Biomphalaria glabrata were studied with the electron microscope to observe the maturing process of their acetabular glands. The undifferentiated acetabular gland displays its enlarged basal area (fundus) and extended narrow process (duct) before other organ systems are recognized. Its fundus contains a prominent nucleus and subcellular organelles typical of active secretory cells.The secretory granules of the postacetabular glands are formed in a milieu of dilated rough endoplasmic reticulum and Golgi. Two morphologically different secretory granules are produced: (1) homogeneously granular ones, and (2) other granular ones with electron dense bodies in their matrices. Mostly, the homogeneously granular ones are produced first in the fundus and are forced into the ducts as the other type is formed.The secretory granules of preacetabular glands are formed from translucent vacuoles which arise from an environment of endoplasmic reticulum and Golgi. Two morphologically different secretory granules are produced: (1) one type has a homogeneous dense matrix, and (2) the other type has a less dense matrix containing electron-lucid bodies.The duct of an undifferentiated acetabular gland has either filamentous material or microtubules dispersed in its cytoplasm. Once microtubules are formed, they persist during the life of the cercaria. The microtubules are believed to have possibly two functions: (1) to support the long duct, and (2) to assist the movement of the secretory granules into the channels of the ducts where they remain until released during host penetration.Few of the subcellular organelles associated with secretory granules formation are seen in the duct except the area in close proximity to the fundus; thus, the few secretory granules produced in the duct are in this region.     

Schistosoma mansoni: mechanism of cercarial tail loss and its significance to host penetration

The mechanism of tail loss from cercariae of Schistosoma mansoni was investigated under experimental conditions. Tail loss from 90% or more of cercariae occurred in less than 10 min when packed organisms were incubated at 30 C in a minimal volume of water. Proteolytic secretions from the acetabular glands did not play a significant part in this process since the addition of known protease inhibitors nor the addition of secretions collected from other cercariae influenced the rate of the process. Tail loss was inhibited when the packed cercariae were incubated in saline at concentrations of 0.05 M or above though glandular secretion occurred at an equal rate in both water and saline. Tail loss was also inhibited by the chelating agents ethylenediaminotetraacetic acid (EDTA) and ethyleneglycoltetraacetic acid (EGTA). A marked feature of cercariae packed in water was their clumping and adhesion to the walls of the containing vessel by secretions from the postacetabular glands. Such clumping did not occur in saline or chelant solutions. It is suggested that the most probable mechanism for tail loss is a simple mechanical trauma effected by the movement of the tail acting against the resistance of the secretion-fixed body.During incubation under conditions which remove the “coat” from the body of the cercaria, the “coat” of the tail remains intact.     

日本血吸虫尾蚴人工方法转变的童虫超微结构的观察

本文报道日本血吸虫尾蚴经注射器推压和血清孵育两种人工方法转变的童虫与载体皮肤型童虫的透射及扫描电镜的观察结果。描述了三种童虫在转变后3小时至12小时其糖膜、外质膜、体被内包含体及腺体的超微结构的变化。     

Secretory and basal cells of the epithelium of the tubular glands in the male Mullerian gland of the caecilian Uraeotyphlus narayani (Amphibia: Gymnophiona)

Caecilians are exceptional among the vertebrates in that males retain the Mullerian duct as a functional glandular structure. The Mullerian gland on each side is formed from a large number of tubular glands connecting to a central duct, which either connects to the urogenital duct or opens directly into the cloaca. The Mullerian gland is believed to secrete a substance to be added to the sperm during ejaculation. Thus, the Mullerian gland could function as a male accessory reproductive gland. Recently, we described the male Mullerian gland of Uraeotyphlus narayani using light and transmission electron microscopy (TEM) and histochemistry. The present TEM study reports that the secretory cells of both the tubular and basal portions of the tubular glands of the male Mullerian gland of this caecilian produce secretion granules in the same manner as do other glandular epithelial cells. The secretion granules are released in the form of structured granules into the lumen of the tubular glands, and such granules are traceable to the lumen of the central duct of the Mullerian gland. This is comparable to the situation prevailing in the epididymal epithelium of several reptiles. In the secretory cells of the basal portion of the tubular glands, mitochondria are intimately associated with fabrication of the secretion granules. The structural and functional organization of the epithelium of the basal portion of the tubular glands is complicated by the presence of basal cells. This study suggests the origin of the basal cells from peritubular tissue leukocytes. The study also indicates a role for the basal cells in acquiring secretion granules from the neighboring secretory cells and processing them into lipofuscin material in the context of regression of the Mullerian gland during the period of reproductive quiescence. In these respects the basal cells match those in the epithelial lining of the epididymis of amniotes.     

记中国南方广东省南雄盆地晚白垩世一新的窃蛋龙类化石

文中记述的窃蛋龙类一新属新种：遗忘始兴龙Shixinggia oblita gen.et sp.nov.是由北京自然博物馆于1995年采自中国广东省南雄盆地的始兴县。它具有以下特征：相对短的肠骨前突,肠骨高度与长度之比率大,并且肠骨的前突及后突的腹边缘均高于髋臼的背边缘。遗忘始兴龙还显示另一特征为股骨近端转子嵴内表面具有一大的气孔,胫骨近端也有类似的相对小的孔,而这些孔在其他已知的窃蛋龙类中均没有报道。这是继2003年报道的黄氏河源龙之后,中国南方第二个新的窃蛋龙类化石。     

Schistosoma mansoni: partial characterization of enzyme(s) secreted from the preacetabular glands of cercariae.

Secretions from the preacetabular glands of cercariae of Schistosoma mansoni were collected over skin surface lipid on a warmed glass surface in a system providing a temperature gradient. Proteolytic activity of these secretions was linear with respect to the numbers of cercariae secreting. The enzyme was relatively storage stable in water or glycine-NaOH buffer. It was highly sensitive to the buffer system, glycine-NaOH buffer providing the highest proteolytic activity against Azocoll and gelatin substrates. In the glycine buffer, the pH optimum for the enzyme was 8.5–8.8; the temperature optimum was 51 C with the Q₁₀ for Azocoll hydrolysis in the range of 30–50 C approximately 2.2. Of the activity 0.4% was lost at 5 C, 1.7% at 35 C, and 100% at 60 C within 10–15 min. Two Sephadex column chromatography fractions showed proteolytic activity against Azocoll. The fraction with greatest activity was not associated with either of the two peaks of high absorbancy at 280 nm, but the lower proteolytic peak coincided with the lower absorbancy peak in the area of peptides below 10,000 MW. Calibration of the Sephadex column with proteins of known molecular weight showed the MW of the main proteolytic fraction of the secretion to be approximately 25,000–27,000.     

Leaf glands act as nectaries in Diplopterys pubipetala (Malpighiaceae)

Leaf glands of Diplopterys pubipetala were studied with light and electron microscopy. Aspects of their secretion, visitors and phenology were also recorded. Glands occur along the margin, at the apex and at the base of the leaf blade. All the glands begin secretion when the leaf is still very young, and secretion continues during leaf expansion. The highest proportion of young leaves coincides with the beginning of flowering. The glucose‐rich secretion is collected by Camponotus ants, which patrol the newly formed vegetative and reproductive branches. All the glands are sessile, partially set into the mesophyll, and present uniseriate epidermis subtended by nonvascularised parenchyma. The glands at the apex and base are larger and also consist of vascularised subjacent parenchyma. The cytoplasm of epidermal and parenchyma cells has abundant mitochondria, polymorphic plastids filled with oil droplets and a few starch grains. Golgi bodies and endoplasmic reticulum are more abundant in the epidermal cells. The parenchyma cells of the subjacent region contain chloroplasts and large vacuoles. Plasmodesmata connect all the nectary cells. The zinc iodide–osmium tetroxide (ZIO) method revealed differences in the population of organelles between epidermal cells, as well as between epidermal cells and parenchyma cells. Ultrastructural results indicate that leaf glands of D. pubipetala can be classified as mixed secretory glands. However, the secretion released by these glands is basically hydrophilic and composed primarily of sugars, hence these glands function as nectaries.     

日本血吸虫尾蚴钻穿宿主皮肤的方式

本文报道了日本血吸虫尾蚴钻穿宿主皮肤的全过程和早期童虫在8种动物皮肤中动态分布的结果。阐明了血吸虫尾蚴钻穿宿主皮肤是依靠其体内头腺及/或钻腺分泌物的酶促作用、头器伸缩的探查作用及全身肌肉运动的机械作用而协同完成的。指出了尾蚴入侵皮肤界面和童虫在皮肤内移行常以倾斜角度前进,并非完全呈垂直方向或沿毛囊皮脂腺的通道。观察到童虫钻破皮肤血管壁进入血管腔的情景,这有力地提供了血吸虫童虫从皮肤进入血液循环系统的直接证据。