Giardia lamblia: Ultrastructural Study of the In Vitro Effect of Benzimidazoles

ABSTRACT Axenically grown Giardia lamblia trophozoites treated with low concentrations of the benzimidazole carbamates albendazole and mebendazole detach from glass culture tubes and lose viability. Scanning electron microscopic observations revealed that these drugs produce grotesque modifications of the cell shape of the parasite and disassembly of the adhesive disc. Transmission electron microscopy showed several stages of the fragmentation of adhesive discs with dispersion of microtubules and microribbons in the cytoplasm. Flagella appeared undamaged. In drug-treated trophozoites electron-dense precipitates were selectively deposited on microtubules and microribbons. The results indicate that the antigiardial effect of benzimidazoles is the result of binding to microtubules and subsequent alterations of the cytoskeleton. The electron microscopic observations also suggest that the drugs may bind to microribbon components of the adhesive disc, possibly giardin proteins.     

Trophozoites of Giardia lamblia may have a Golgi-like structure

Trophozoites of the primitive protozoan Giardia lamblia have been considered as cells which do not present the Golgi complex. Using C(6)-NBD ceramide, which has been shown to label the Golgi complex of mammalian cells, labelling of the perinuclear region of G. lamblia was observed by confocal laser scanning microscopy. Transmission electron microscopy of thin sections and of replicas of freeze-fractured cells revealed the presence of concentric perinuclear membranes resembling the Golgi complex.     

How nuclei of Giardia pass through cell differentiation: semi-open mitosis followed by nuclear interconnection

Differentiation into infectious cysts (encystation) and multiplication of pathogenic trophozoites after hatching from the cyst (excystation) are fundamental processes in the life cycle of the human intestinal parasite Giardia intestinalis. During encystation, a bi-nucleated trophozoite transforms to a dormant tetra-nucleated cyst enveloped by a protective cyst wall. Nuclear division during encystation is not followed by cytokinesis. In contrast to the well-studied mechanism of cyst wall formation, information on nuclei behavior is incomplete and basic cytological data are lacking. Here we present evidence that (1) the nuclei divide by semi-open mitosis during early encystment; (2) the daughter nuclei coming from different parent nuclei are always arranged in pairs; (3) in both pairs, the nuclei are interconnected via bridges formed by fusion of their nuclear envelopes; (4) each interconnected nuclear pair is associated with one basal body tetrad of the undivided diplomonad mastigont; and (5) the interconnection between nuclei persists through the cyst stage being a characteristic feature of encysted Giardia. Based on the presented results, a model of nuclei behavior during Giardia differentiation is proposed.     

The ultrastructure and ultracytochemistry of the basement membrane of the Galleria mellonella fat body

Summary The fat body lobes of Galleria mellonella are surrounded by basement membrane — a fine granular layer of connective tissue. This membrane has an affinity for ruthenium red. The results obtained after treatment of the fat body with neuraminidase, hyaluronidase, phospholipase C and proteolytic enzymes suggest that glycoproteins and phospholipoproteins are constituents of this basement membrane. The basement membrane also has the ability to bind concanavalin A-peroxidase, which is associated with the presence of mannoside residues.The preliminary results of these studies were presented at the IX Conference of Electron Microscopy, Gdask, Poland (Dutkowski, 1975)I am greatly indebted to Professor A. Przececka for her encouragement to undertake this study. The excellent technical assistance of Mrs. K. Mroziska, Mrs. Z. Kamiska and the engineering staff of the Laboratory is gratefully acknowledged     

The median body of Giardia lamblia: an ultrastructural study

Giardia lamblia is an intestinal parasite of several mammals. The most striking feature of Giardia is the presence of a complex and unique cytoskeleton, and among its components the median body (MB) is the least defined microtubular structure. In the present study, we used a technique that allowed the removal of the plasma membrane and observation of cytoskeletal structures by both routine scanning electron microscopy (SEM) and field emission high resolution SEM. This technique permitted new observations such as details and insights of the median bodies, not previously described or controversial in the literature. Light microscopy after Panotic staining, immunofluorescence microscopy using several antibodies, and thin sections were also used to better characterized the Giardia MB. The new observations concerning the median bodies were : (1) they are not one or two structures, but varied in number, shape and position ; (2) they were found in mitotic and interphasic trophozoites, in disagreement with previous works ; (3) they were present in about 80 % of the cells, and not in 50 % of the cells, as previously described ; (4) they could be connected either to the plasma membrane, to the adhesive disc, and caudal flagella, and thus they are not completely free in the cells, as published before ; (5) they can protrude the cell surface ; (6) their microtubules react with several anti-tubulin and -beta giardin antibodies. These observations add new data on the scarce literature and to this largely understudied cell structure.     

The single dynamin family protein in the primitive protozoan Giardia lamblia is essential for stage conversion and endocytic transport

Dynamins are universally conserved large guanosine triphosphatases, which function as mechanoenzymes in membrane scission. The primitive protozoan Giardia lamblia has a single dynamin-related protein (GlDRP) with an unusual domain structure. Giardia lacks a Golgi apparatus but generates transient Golgi-like delay compartments dubbed encystation-specific vesicles (ESVs), which serve to accumulate and mature cyst wall proteins during differentiation to infectious cyst forms. Here, we analyze the function of GlDRP during growth and encystation and demonstrate that it relocalizes from peripheral endosomal-lysosomal compartments to nascent ESVs. We show that GlDRP is necessary for secretion of the cyst wall material and ESV homeostasis. Expression of a dominant-negative GlDRP variant does not interfere with ESV formation but blocks cyst formation completely prior to regulated exocytosis. GlDRP colocalizes with clathrin at the cell periphery and is necessary for endocytosis of surface proteins to endosomal-lysosomal organelles in trophozoites. Electron microscopy and live cell imaging reveal gross morphological changes as well as functional impairment of the endocytic system in cells expressing the dominant-negative GlDRP. Thus, giardial DRP plays a key role in two distinct trafficking pathways and in organelle homeostasis, both essential functions for the proliferation of the parasite in the gut and its transmission to a new host.     

In vivo determination of the gap2 gene promoter activity in Giardia lamblia

A shuttle vector for Escherichia coli and Giardia lamblia was modified to produce a reporter plasmid, which monitors the expression of prescribed gene in G. lamblia by measuring its luciferase activity. Promoter regions of the gap2 gene, one of the genes induced during encystation, were cloned into this plasmid, and the resultant constructs were then transfected into trophozoites of G. lamblia. Transgenic trophozoites containing one of the 3 gap2-luc reporters were induced to encystation, and characterized with respect to gap2 gene expression by measuring their luciferase activities. Giardia containing a gap2-luc fusion of 112-bp upstream region showed full induction of luciferase activity during encystation.     

The activity of a developmentally regulated cysteine proteinase is required for cyst wall formation in the primitive eukaryote Giardia lamblia.

Giardia is an intestinal parasite that belongs to the earliest diverging branch of the eukaryotic lineage of descent. Giardia undergoes adaptation for survival outside the host's intestine by differentiating into infective cysts. Encystation involves the synthesis and transport of cyst wall constituents to the plasma membrane for release and extracellular organization. Nevertheless, little is known about the molecular events related to cyst wall biogenesis in Giardia. Among the components of the cyst wall there are two proteins that we have previously identified and characterized: CWP1 (26 kDa) and CWP2 (39 kDa). Expression of these proteins is coordinately induced, and both concentrated within encystation-specific secretory vesicles before their extracellular polymerization. Although highly similar to each other at the amino terminus, CWP2 includes a COOH-terminal 121-amino acid extension. Here, we show that this extension, rich in basic residues, is cleaved from CWP2 before cyst wall formation by an intracellular cysteine proteinase activity, which is induced during encystation like CWPs. Specific inhibitors prevent release of cyst wall materials, abolishing cyst wall formation. We also report the purification, cloning, and characterization of the encystation-specific cysteine proteinase responsible for the proteolytic processing of CWP2, which is homologue to lysosomal cathepsin C. Encystation-specific cysteine proteinase ESCP possesses unique characteristics compared with cathepsins from higher eukaryotes, such as a transmembrane domain and a short cytoplasmic tail. These features make this enzyme the most divergent cathepsin C identified to date and provide new insights regarding cyst wall formation in Giardia.     

Sorting of cyst wall proteins to a regulated secretory pathway during differentiation of the primitive eukaryote, Giardia lamblia

Giardia lamblia, which belongs to the earliest identified lineage to diverge from the eukaryotic line of descent, is one of many protists reported to lack a Golgi apparatus. Our recent finding of a developmentally regulated secretory pathway in G. lamblia makes it an ideal organism with which to test the hypothesis that the Golgi may be more readily demonstrated in actively secreting cells. These ultrastructural studies now show that a regulated pathway of transport and secretion of cyst wall antigens via a novel class of large, osmiophilic secretory vesicles, the encystation-specific vesicles (ESV), is assembled during encystation of G. lamblia. Early in encystation, cyst antigens are localized in simple Golgi membrane stacks and concentrated within enlarged Golgi cisternae which appear to be precursors of ESV. This would represent an unusual mechanism of secretory vesicle biogenesis. Later in differentiation, cyst antigens are localized within ESV, which transport them to the plasma membrane and release them by exocytosis to the nascent cell wall. ESV are not observed after completion of the cyst wall. In contrast to the regulated transport of cyst wall proteins, we demonstrate a distinct constitutive lysosomal pathway. During encystation, acid phosphatase activity is localized in endoplasmic reticulum, Golgi, and small constitutive peripheral vacuoles which function as lysosomes. However, acid phosphatase activity is not detectable in ESV. These studies show that G. lamblia, an early eukaryote, is capable of carrying out Golgi-mediated sorting of proteins to distinct regulated secretory and constitutive lysosomal pathways.     

The ultrastructure of the human fetal pineal gland

Summary The ultrastructure of the pineal gland of 18 human fetuses (crown-rump lengths 30–178 mm) was investigated.The pineal gland exhibits a pyramidal shape and consists of an anterior and posterior lobe. Only one parenchymal cell type, the pinealocyte, was observed. Few neuroblasts were seen between the pinealocytes and in the extended perivascular space. The pinealocytes possess all the organelles necessary for hormone synthesis. No specific secretory granule could be observed. The organ is abundantly vascularized and richly innervated. The morphology of the capillaries indicates the existence of a blood-brain barrier.The ultrastructure of the human fetal pineal gland suggests that the gland has a secretory function in early intrauterine life. Acknowledgements. The author is grateful to Mrs. Yael Balslev and Miss Inger Ægidius for their able technical assistance. This investigation was supported in part by The Carl and Ellen Hertz's foundation and the Johann and Hanne Weimann foundation.     

The ultrastructure of the accessory sex organs of the male rat

Summary The fine structure of the nuclei of epithelial cells of the dorsal lobe of the rat prostate were studied after administration of three different antiandrogenic compounds.The nucleolus appears to undergo a progressive disorganisation with partial fragmentation and dispersion of its normal components. Different types of intranuclear inclusions were found.The various alterations observed were often encountered within the same section. This may indicate that the nuclear alterations occur in the same compartment of the cell, and represent a dysfunction of integrated biochemical events occurring within this compartment.The findings support a view that the stimulatory secretory effect of androgens is mediated via a secretory center, located within the nucleolusassociated chromatin. Within this secretory center, the initial steps of the secretory process, the binding of the DHT receptor complex to DNA is assumed to occur.     

Comparison of the local immune response against Giardia lamblia cyst wall protein 2 induced by recombinant Lactococcus lactis and Streptococcus gordonii

Lactococcus lactis and Streptococcus gordonii are lactic acid bacteria (LAB) currently being advocated for use as live antigen delivery vehicles to mucosal sites. Since both vehicles differ in their capability to persist within the small intestine and in their mode of antigen delivery, we sought to compare them to determine which one was superior. In this study, we compared the efficacy of recombinant L. lactis and S. gordonii to stimulate intestinal immune responses against Giardia lamblia cyst wall protein-2 in BALB/c mice. Oral administration of either vector significantly increased the number of CD4(+) T helper and B-cells in the mesenteric lymph nodes (MLN) and Peyer's patches (PP) of immunized animals. Delivery of recombinant CWP2 (rCWP2) by L. lactis stimulated a balanced IFN-gamma/IL-4 response (MLN and PP cells) and a CWP2-specific intestinal IgA antibody response. Alternatively, delivery of rCWP2 by S. gordonii stimulated a higher frequency of IFN-gamma secreting MLN and PP cells, as well as doubling the amount of CWP2-specific intestinal IgA. In challenge studies, L. lactis and S. gordonii reduced cyst output by 71 and 90%, respectively. When compared to each other, S. gordonii-immunized animals shed 65% fewer cysts than their L. lactis-immunized counterparts. Based on these findings, we concluded that S. gordonii was superior to L. lactis as an intestinal vaccine delivery vehicle.     

The ultrastructure of the accessory sex organs of the male rat

Summary The fine structure of the nuclei of epithelial cells of the dorsal lobe of the rat prostate were studied 2, 3, 5, 7 and 21 days after castration. The nucleolus appears to undergo a progressive disorganisation with partial fragmentation and dispersion of its normal components.Changes in the nucleoplasm were primarily reflected by a condensation of chromatin, particularly along the nuclear membrane and adjacent to the nucleolus. Later, different types of intranuclear inclusions were observed.After 21 days, the nuclei were characterized by an irregular outline with large indentation. Within the nucleoplasm aggregates of coarse granular chromatin were found. No cell necrosis was observed, indicating that androgen deprivation results in a remodeling of the cell to a less active state with marked cellular alterations and cessation of secretion, but apparently with some of their basic functions still intact.Injections of testosterone completely reverse the castrated-induced alterations.The changes observed are assumed to be due to the withdrawal of the androgenic stimulus, with a direct influence on the secretory function of the cell. The findings support the view that the stimulating secretory effect of androgen is mediated via an intranuclear androgen receptor, probably located in the nucleolus-associated-chromatin. It is also proposed that the secretory function of the epithelial cells of the prostatic complex, initiated by androgens, may be regulated by an intranuclear secretory center.     

The ultrastructure of the accessory sex organs of the male rat

Summary The seminal vesicles and the coagulating gland of the rat were studied 2, 3, 5, 7 and 21 days after castration. The major changes within the seminal vesicles were primarily formation of whorls of the rough endoplasmic reticulum (RER), followed by a general atrophy with a numerical reduction of the RER-profiles, and with general simplification of the cytoplasm due to loss of the organelles. It was a gradually reduction of secretion granules, diminution of the Golgi apparatus, formation of pigment bodies and autophagic vacuoles. Lipid droplets were observed in the basal cytoplasm of the epithelial cells. In the coagulating gland, similar changes occurred within the Golgi area and the lysosome complex. On the other hand, cisternae of the basal endoplasmic reticulum tended to persist in many cells. The similarity in response strongly suggests that the pathogenetic mechanisms are similar in both organs, i.e. atrophy due to deprivation of the androgenic stimulus. The deprivation of androgen gave rise to an inflammatory-like process with infiltration of lymphocytes and macrophages. The increased number of macrophages may indicate that they contribute in some way to the involution of the prostate by removing the material in the autophagic vacuoles.     

Recent observations on the ultrastructure of human urothelium

Summary An electron microscopic study of normal bladder urothelium of elderly patients ranging in age from 61 to 82 years has shown the occurrence of unusually thin regions consisting of either one or two layers of undifferentiated cells interspersed between 3–4 cell layers thick regions. A morphometric study has confirmed the existence of a pattern of cytodifferentiation in cells of the thick region. The generally microvillous nature of the luminal surface is attributed to incompletely differentiated cells that have come to occupy the superficial layer. The lack of thickened and/or asymmetric membrane plaques in luminal plasma as well as the dearth of characteristic precursor vesicles in the cytoplasm are also explicable in terms of a failure of normal cell differentiation. It is suggested that the unusual features noted are consequences of tissue ageing rather than prognostic of cancer. There are indications that the aged urothelium may be prone to increased leakiness and the bladder tissues may therefore be at greater risk from urine-borne chemicals and carcinogens.     

Ruthenium red staining and tannic acid fixation of dental basement membrane

Summary Ruthenium red staining and tannic acid fixation were used to analyse the fine structure of embryonic mouse dental basement membrane in intact first mandibular molars or in EDTA-isolated dental papillae. Preameloblasts are separated from extracellular matrix proper by a basal lamina that contains regularly arranged proteoglycan granules of about 10 nm in diameter. This distribution pattern is particularly evident in the inner and outer lamina rara of the basal lamina associated with EDTA-isolated dental papillae. The plasmalemma of preameloblasts demonstrates electron dense plaques on the inner leaflet. Ruthenium red positive granules (50 nm in diameter) coat non-striated and striated fibrils of the matrix. Hyaluronidase treatment digested the ruthenium red positive granules. Tannic acid fixation allowed the demonstration of filaments within the lamina rara interna, connecting the lamina densa with plasmalemma of preameloblasts. These observations are discussed in the context of the terminal differentiation of odontoblasts.These studies were supported by INSERM, grant n 537785 and DGRST     

Studies on the ultrastructure of the integument of the rotifer Habrotrocha rosa Donner (Aschelminthes)

Summary The integument of the rotifer Habrotrocha rosa Donner is provided with pores and formed by an extrasyncytial cuticle and a syncytial hypodermis. The hypodermis peripherally contains 3 layers of dense cytoplasm and borders the cuticle by an asymmetric cell membrane. The wall of the pores is stiffened proximally like an annulus. The pores lead into cytoplasmic invaginations which are surrounded by vesicles. Close to and also beneath the condensed cytoplasmic layers microbodies are found, which are interpreted as microperoxisomes. Subhypodermal layers of muscles are connected with the cytoplasm of the hypodermis by desmosome-like structures.I am indebted to Dr. H. Breucker, Anatomisches Institut Hamburg, and Prof. Dr. W. Becker, Zoologisches Institut Hamburg, under whose direction this work was carried out. Supported by the Deutsche Forschungsgemeinschaft (Be 464/10)     

The ultrastructure of the cell types in the endocrine pancreas of the horse

Summary The islets of Langerhans of the equine pancreas were examined with the electron microscope after immersion or perfusion fixation. Five cell types could be distinguished after fixation by either technique: 1. A-cells, situated at the center of the islets, 2. B-cells, containing mostly pale granules and constituting the principal cell type of the periphery of the islets, 3. D-cells, also located mainly at the periphery of the islets, 4. G-cells, found at the edge of the islets and in the exocrine pancreas, and 5. S-cells, (small granule cells), which are relatively few in number and occur only in the islets. The function and age-dependent modifications of these cells are discussed. The formation of light and dark cells and of mixed cells are regarded as artifact, since cells of this type occur only under the condition of immersion fixation.Dedicated to Professor Dr. Drs. h.c. W. Bargmann on the occasion of his 70th birthday.